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Functional genomics of the A600 locus in Leishmania mexicana Murray, Angus Stewart 2005

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F U N C T I O N A L GENOMICS OF T H E A600 L O C U S IN LEISHMANIA MEXICAN A  by A N G U S STEWART M U R R A Y B . S c , M c G i l l University, 1999  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPY in THE F A C U L T Y OF G R A D U A T E STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH C O L U M B I A October, 2005  © Angus Stewart Murray, 2005  Abstract Abstract Protozoan parasites of the genus Leishmania are the causative agent of a spectrum of important human diseases collectively referred to as leishmaniasis. Leishmania has a digenetic lifecycle alternating between the promastigote and amastigote stages. In the amastigote stage, Leishmania are obligate intracellular parasites that replicate actively in the macrophage phagolysosome. The mechanisms used by amastigotes to survive within the acidic and hydrolytic environment of the phagolysosome and to suppress macrophage activation remain to be determined. The identification and characterization of genes preferentially expressed in the amastigote stage should elucidate novel parasite mechanisms used to establish a persistent infection. The amastigote-specific L. mexicana cDNA, A600, was cloned previously in this laboratory using a suppression-subtraction PCR (SS-PCR) approach. The A600 gene did not share sequence identity with any known genes, although expression of the mRNA transcript was seven-fold higher in amastigotes. Southern blot analysis indicated that multiple A600 coding sequences existed in the L. mexicana genome. In the present study, the multi-gene A600 locus was cloned and restriction mapping identified four open reading frames: A600-1, A600-2.1, A6002.2, and A600-3. The A600-J and A600-3 genes shared 78% D N A sequence identity. A cross-species comparison of the A600 genes using L. mexicana (New World) and L. major (Old World) revealed that divergence of the A600-1 and A600-3 genes occurred in an ancestral Leishmania species. A targeted gene deletion approach was used to determine the cellular function of the A600 genes. A n /ftfOO-deficient mutant (A600~ ) of L. mexicana was generated using two A  rounds of homologous recombination. A600~ promastigotes differentiated to amastigotes in A  response to temperature shift and acidification of culture media, but showed significant ii  Abstract growth inhibition. During in vitro infection studies, A6Q0~'~ promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600~ amastigotes to proliferate was restored by re-introduction of the A600-1 A  gene, but not the A600-3 gene. Finally, B A L B / c mice infected with L. mexicana A600-'- cells did not produce lesions, while infection with wildtype cells caused progressive cutaneous lesions. The results from these experiments show that the A600-1 gene was essential for continued proliferation of amastigotes,  and potentially for development  of chronic  leishmaniasis. Leishmania gene expression is regulated post-transcriptionally via m R N A stability or translational control mechanism, usually via regulatory elements in the 3'UTR. This study used a luciferase reporter construct to show that stage-specific expression of the A600-3 mRNA transcript was mediated by the 3'UTR. Progressive deletions generated in the A600-3 3'UTR identified a 250 bp region, RD2, that negatively regulated luciferase protein expression in promastigotes. A 15 nt sequence in the RD2 region was conserved in the divergent A600-1 3'UTR and may constitute a novel regulatory element. It is proposed that /raws-acting factors interact with this regulatory element to inhibit translation of the A600 proteins in promastigotes.  iii  Table of Contents Table of Contents Abstract  ii  Table of Contents  iv  List of Figures  vii  List of Abreviations  x  Acknowledgements  xii  1.  2.  INTRODUCTION  1  1.1  Leishmania  1  1.2  Leishmania Biology  7  1.3  Leishmania Surface Molecules  12  1.4  Parasite-Vector Interactions  13  1.5  Mammalian Host-Parasite Interactions  15  1.6  Innate Immunity to Leishmania Infection  21  1.7  Adaptive Immune Response to Leishmania Infection  27  1.8  Cytokine-mediated Modulation of the Immune Response  1.9  Leishmania Genome & Gene Expression  .<•.  30 33  1.10 The A600 Gene  40  1.11 Thesis Objectives  42  METHODS A N D MATERIALS  44  2.1  Leishmania Culture  44  2.2  Bacterial Strains and Vectors  44  2.3  Nucleic Acid Isolation  45  2.3.1  Isolation of Leishmania Genomic DNA  2.3.2  Isolation ofPlasmid DNA  2.3.3  Isolation of Leishmania RNA  45 46  iv  46  Table of Contents 2.4  Protein Isolation 2.4.1  2.5  2.6  2.7  2.8  2.9  3.  47  Protein Isolation from Leishmania  47  Gel Electrophoresis  48  2.5.1  Non-Denaturing Agarose Gel Electrophoresis  48  2.5.2  Southern Blot Analysis  48  2.5.3  Northern Blot Analysis  49  2.5.4  Western Blot Analysis  50  Generation and Purification of Polyclonal Antibody  50  2.6.1  Conjugation of an A600 Peptide to KLH Carrier Protein  50  2.6.2  Preparation of A600-peptide Conjugated Affinity Column  2.6.3  Affinity Purification of a-A600 Antibody  51 51  Molecular Biology Techniques  52  2.7.1  Restriction Enzyme Digestion  52  2.7.2  DNA Ligation and Transformation of Bacteria  53  2.7.3  Polymerase chain reaction  2.7.4  Labeling DNA Probes  2.7.5  Exonuclease(III) Deletions  2.7.6  DNA Sequencing  2.7.7  Leishmania Transfection and Cloning  54 54 54 55 56  Generation of Constructs for Transfection  57  2.8.1  Targeted Deletion Constructs  57  2.8.2  Luciferase Reporter Constructs  58  Macrophage Techniques  58  2.9.1  Peritoneal Macrophage Isolation  2.9.2  Infection of Peritoneal Macrophages with Leishmania  2.9.3  BALB/c Mouse Infections with Leishmania  2.9.4  Isolation of Lesion Amastigotes  58 ;  ; 59 60 60  C H A R A C T E R I Z A T I O N OF THE A600 L O C U S 3.1  Results  62 •  3.1.1  Genomic Arrangement of Related A600 Genes  3.1.2  Cloning the A600 Locus  •  62 62 64  v  Table of Contents  3.2 4.  3.1.3  Restriction Map of the A600 Locus  3.1.4  Sequencing the Complete A600-1 and A600-3 Genes  3.1.5  Expression Profiles of the A600-1 andA600-3 Genes  Discussion  85 91  Results 4.1.1  4.2  97  Generation of A600 Deletion Mutant Parasites by Homologous Recombination  5.2  97  4.1.2  A600 Knockout Confirmation  4.1.4  Knockout Reconstitution with the A600-1 andA600-3 Genes  4.1.6  TheA600-l Gene is required for Replciation in Amastigotes  4.1.7  A600 genes are required for the development of murine leishmaniasis  Discussion  103 108 112 114  120  R O L E OF T H E A600-3 3'UTR FOR STAGE-SPECIFIC G E N E EXPRESSION 5.1  6.  78  G E N E R A T I O N A N D C H A R A C T E R I Z A T I O N OF A600 K N O C K O U T M U T A N T S 97 4.1  5.  71  Results  129 129  5.1.1  Luciferase Reporter Constructs  5.1.2  TheA600-3 3 'UTR Regulates Amastigote-Specific Gene Expression  5.1.3  Regulatory Element Exists at Position 1500 - 2500 of the A600-3 3 'UTR. 136  5.1.4  Analysis of Luciferase Expression in Transfected Leishmania  5.1.5  Fine Mapping the Regulatory Sequence in the RD Region  Discussion  129  137 143 149  SUMMARY  157  REFERENCES  159  vi  131  List of Figures List of Figures Figure 1.  Clinical Outcomes of Leishmaniasis  Figure 2.  Images of Leishmania and the Sandfly Vector  5  Figure 3.  The Leishmania Lifecycle  6  Figure 4.  Arrangement of Genes of L. major Chromosome 1  Figure 5.  Model for Polycistronic Transcription in Leishmania  Figure 6.  Southern blot analysis of the L. mexicana A600 genes  Figure 7.  Southern blot analysis to restriction map the L. mexicana A600 locus  Figure 8.  Enrichment for Xholrestriction fragments that encode the A600 genes  Figure 9.  The pBluescript-E8 and -G2 plasmids contained large genomic D N A inserts  Figure 10.  2  36 38 63 65 68  69  Comparison of L. mexicana genomic D N A and the pBST-E8 plasmid DNA  70  Figure 11. Restriction Digests of the pBST-E8, -G2, and - G 6 Plasmids  72  Figure 12. Restriction Map of the A600 Locus  74  Figure 13. Southern Blot Analysis of the A600-3 Downstream Sequence  77  Figure 14. Exonuclease (III) Deletion Panel of the pBST-A600yC Plasmid Figure 15.  79  Alignment of the L. mexicana A600-1 (LmxA6001) and A600-3 (LmxA6003) Coding Sequences  81  Figure 16. Alignment of the L. mexicana A6001 (LmxA6001) and A6003 (LmxA6003) Protein Sequences  82  Figure 17. Alignment of L. mexicana A600-1 (LmxA6001) and L. major A600-1 (LmjA6001) Coding Sequences 83 Figure 18. Alignment of the L. mexicana A600-3 (LmxA6003) and L. major A600-3 (LmjA6003) Coding Sequences  84  Figure 19. Northern Blot Analysis for A600-3 Gene Expression  86  Figure 20. A600-3 Expression During Axenic Amastigote Differentiation  89  vii  List of Figures Figure 21.  Comparison of L. mexicana A600-1 and A600-3 Gene Expression  Figure 22.  Schematic for Knockout Constructs and Targeted Deletion of the A600 Locus  90  98  Figure 23.  Targeted Replacement of the A600 Locus in L. mexicana Wildtype Cells  100  Figure 24.  Targeted Replacement of the Remaining at the A600 Locus  Figure 25.  Confirmation for Targeted Deletion of the L. mexicana A600 Locus  Figure 26.  Absence of A600-1 and A600-3 Expression in L. mexicana A600-'- (KO)  102 105  Parasites  106  Figure 27.  Western Blot Analysis for A600 Protein Expression  107  Figure 28.  Reconstitution of the A600~'~ Strain with the A600-1 or A600-3 Genes  Figure 29.  Expression of the A600-1 and A600-3 Genes in the L. mexicana Wildtype, A600''\ KO+A6001.1, and KO+A6003.1 Clones  Figure 30. Leishmania Growth Curves  110  111 113  Figure 31.  72 hour Infection of B A L B / c Peritoneal Macrophages  116  Figure 32.  120 Hour Infection of B A L B / c Peritoneal Macrophages.  117  Figure 33.  Quantitation of Parasite Burden in Pertional Macrophages  118  Figure 34.  Lesion Development in Leishmania-mfected B A L B / c Mice  Figure 35.  Plasmid Maps of the Luciferase Reporter Constructs  ;  Figure 36. L. mexicana Clones Transfected with the Luciferase Reporter Plasmids  119 130 132  Figure 37. Luciferase Gene Expression as a Chimeric Transcript with the A600-3 3'UTR  134  Figure 38.  Effects of the A600-3 3'UTR for Luciferase Protein Expression  135  Figure 39.  Schematic of PCR-based A600-3 3'UTR Deletion Strategy  138  Figure 40.  Targeted Integration of Luciferase Reporter Constructs at the A600 Locus Figure 41. Effects of A600-3 3'UTR Deletions on Luciferase Expression viii  139 141  List of Figures Figure 42.  Figure 43.  Effects of the A600-3 3'UTR Expression  Deletions  On Luciferase  Protein  Targeted Deletions Across the Regulatory Domain (RD) in the A600-3 3'UTR  142  145  Figure 44.  Effects of A600-3 3'UTR Deletions for Expression of the Luciferase Gene 146  Figure 45.  Effects of A600-3 3'UTR Deletions for Expression of the Luciferase Protein  Figure 46.  Conserved 15 nt Sequence in the RD2 Region of the A600-3 3'UTR  ix  147 148  Abreviations List of Abreviations  Amp  ampicillin  APC  antigen presenting cell  BSA  bovine serum albumin  cDNA  complementary deoxyribonucleic acid  dATP  deoxyadenosine triphosphate  dCTP  deoxycytidine triphosphate  DEPC  diethyl pyrocarbonate  dGTP  deoxyguanosine triphosphate  DMF  dimethyl formamide  DNA  deoyyribonucleic acid  DTT  dithiothreitol  dTTP  deoxythymidine triphosphate  EDTA  efhylenediamine tetraacetate  FBS  fetal bovine serum  GPI  glycophosphoinositol  GuHCl  guanidine hydrochloride  HEPES  N-(hydroxyethyl)piperazine-N' -(2-ethanesulfonic acid)  IFNy  interferon gamma  IL  interleukin  IPTG  isopropyl (3-D-1 -thiogalactopyranoside  IR  intergenic region  kb  kilobase  X  Abreviations kbp  kilobase pair  kDa  kilodalton  KLH  keyhole limpet hemocyanin  LPG  lipophosphoglycan  LmxA6001  Leishmania mexicana A6001  LmxA6003  Leishmania mexicana A6003  LmjA6001  Leishmania major A6001  LmjA6003  Leishmania major A6003  LUC  luciferase  MHC  major histocompatibility  MOPS  3-(N-Morpholino)propanesulfonic acid  ORF  open reading frame  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PCR  polymerase chain reaction  RNA  ribonucleic acid  SDS  sodium dodecyl sulfate  SMCC  succinimidyl 4-(N-Maleimidomethyl) cyclohexane-l-carboxylate  TBS  tris buffered saline  TNFct  tumor necrosis factor alpha  Tris  tris(hydroxymethyl)aminomethane  UTR  untranslated region  xi  Acknowledgements Acknowledgements  I dedicate this thesis to my parents, Donald and Rohays Murray, who offered their encouragement, support, and love throughout my life. Also, I dedicate this thesis to my sister Fiona, whom I admire so much for her accomplishments and great sense of humour.  I would like to thank my supervisor and mentor, Dr. Robert McMaster, for his continued encouragement, guidance and wisdom. It has been a great experience and pleasure studying under his supervision. I thank Golareh Habibi for her friendship, encouragement, and contributions to this study. I am grateful to Christine Fu for help with the gene expression studies. Also, I am indebted to Dr. Corinna Warburton for teaching me the molecular biology techniques, critically reading my thesis, and many cinnamon buns!  I am grateful to the members of my supervisory committee, Dr. Alice Mui, Dr. Dixie Mager, and Dr. Fumio Takei, for their valuable insight throughout my thesis. I also extend my thanks to members of the McMaster lab, past and present: Dr. Tanya Nelson, Dr. Suzie Hingley-Wilson, Dr. Laura Sly, Dr. Celia Almeida Tate, Dr. Nisha Dogra, Kirk Leifso, Esther Tang, and Catherine Chambers for their help and friendship. Finally, A l i Ghanipour, Ivan Waissbluth, and Pooran Qasimi have been great friends, whom I enjoyed working with each day.  xii  Chapter 1 - Introduction 1.  1.1  Introduction  Leishmania Leishmania spp.  are  an  intracellular,  protozoan  parasite  belonging  to  Trypanosomatidae family and is the causative agent of leishmaniasis disease. Leishmania currently infects 15 million people worldwide. 350 million people live in tropical and subtropical regions where leishmaniasis is endemic (Modabber, 1993). During the past decade there has been a steady increase in the rate of new infections and the co-infection of Leishmania and H I V is emerging as a new clinical entity in some regions (Desjeux et al, 2003). There are 3 broad clinical types of leishmaniasis. Cutaneous disease, also referred to as Oriental Sore, is the most common form of leishmaniasis, accounting for 50 to 75% of new cases. The disease, which primarily affects children, manifests as a skin ulcer and persists for several months to more than one year. Lesions can be severely disfiguring and leave long-lasting scarring on visible areas of the body (Figure la-c).  Mucocutaneous  disease is seen almost exclusively in South America. It results from reactivation of a primary cutaneous lesion and causes extensive inflammation of the mucosal tissues of the nose and mouth (Figure Id). Visceral leishmaniasis, or kala-azar disease, involves infection of the liver, spleen, and bone marrow resulting in hepatosplenomegaly (Figure le). It is fatal unless treated with anti-leishmanial drugs (Alexander et al, 1992). The current treatment uses pentavalent antimonial drugs, which have adverse side effects for patients. Unfortunately, the majority of leishmaniasis cases occur in developing countries where anti-leishmanial drugs are not readily available. Attempts to produce a successful vaccine, which confers long term immunity, have been unsuccessful.  1  Chapter 1 - Introduction  a  b  http://wvvw.kolping.de/kf/kolping_solidargemeinschaft/ media/afghanistan-leishmania-350.jpg  c  d  http://www.webescuela.edu.py/ Imagenes/lesma3.jpg  Figure 1.  http://www.erin.utoronto.ca/~w3env100y/env/ENV100/ sci/eco_gifs/leishmania2.jpg  e  http://www.mpibpc.gwdg.de/abteilungen/ 293/PR/00_01/leish.html  Clinical Outcomes of Leishmaniasis.  Photographs of patients with various forms of leishmaniasis, (a-c) Cutaneous leishmaniasis presents as skin lesions at the site of infection, (d) Mucocutaneous causes massive inflammation in mucosal tissues of the nose and mouth, (e) Visceral leishmaniasis results from infection of the liver, spleen, and bone marrow. The enlarged liver and spleen are outlined on the abdomen. 2  Chapter 1 - Introduction Over twenty, morphologically indistinguishable species of Leishmania have been identified. Each species can be characterized by molecular methods including restriction analysis of kinetoplastid D N A , nuclear D N A hybridization, isoenzyme patterns, and serological testing. Leishmania species can be grouped on the basis of geographical location (New World or Old World) and the clinical disease that they cause. Cutaneous leishmaniasis is caused primarily by the following Leishmania species: L. tropica, L. major, L. mexicana, and L. pifanoi. Mucocutaneous disease is caused almost exclusively by infection with L. braziliensis, but also by L. guyanensis and L. panamensis. Visceral disease is caused by the L. donovani, L. infantum, andZ. tropica, and£. chagasi (Markell, 1999). There are two distinct morphological stages in the Leishmania lifecycle, the promastigote and the amastigote (Figure 2a,b and Figure 3).  The promastigote is the  flagellated, motile stage that resides extracellularly in sandfly vectors of the genus Phlebotomus or Lutzomyia (Figure 2c). Interestingly, there is a species-specific requirement for the parasite to be transmitted by certain species of the Phlebotomus sandfly vector (Sacks et al,  1994). Shortly after entering the sandfly vector, the non-infectious procyclic  promastigote attaches to the sandfly midgut and multiplies rapidly. Procyclic parasites differentiate into non-dividing, virulent metacyclic promastigotes, which involves extensive modifications to the carbohydrate side chains on the major surface glycolipid (McConville et al, 1992). These alterations to the parasite membrane result in release of the developing promastigote  from the midgut epithelium and allow for migration of metacyclic  promastigotes to the foregut and pharynx of the sandfly vector (Pimenta et al, 1992; Saraiva et al, 1995; Soares et al, 2002). Metacyclic promastigotes remain in the sandfly pharynx and are innolculated into the skin of the mammalian host during the sandfly blood meal. Macrophages rapildly internalize 3  Chapter 1 - Introduction Leishmania, via 'zipper' and 'coiling' phagocytosis, into an endosomal compartment that is commonly referred to as the phagosome. According to these phagocytosis models, initial macrophage binding to the parasite recruits additional receptors; the complement receptors CR1 and CR3 have important roles for both Leishmania binding and uptake (Rittig et al., 1998; Rittig et al, 2000). The Zez's/jmaw'a-containing phagosome fuses with a lysosome to form the phagolysosome, or parasitophorous vacuole; this acid environment is rich in hydrolytic enzymes, including cathepsins (Courret et al, 2002). Promastigote differentiation to the amastigotes is triggered by acidic pH and elevated temperature (Bangs et al, 1993). The hydrolytic conditions inside the phagolysosome are lethal for almost all infectious organisms. However, successful obligate, intracellular pathogens have evolved various strategies to ameliorate these conditions. Mycobacterium tuberculosis prevents acidification of the phagosome compartment by exclusion of a proton-ATPase transporter from the phagosome membrane (Sturgill-Koszycki et al, 1994; Clemens et al, 1995). Legionella pneumophila, Salmonella typhimurium, and Toxoplasma spp. are all intracellular pathogens that abrogate phagosome maturation by inhibition of phagosome-lysosome fusion (Ishibashi et al, 1990; Buchmeier et al, 1991; Mordue et al, 1999; Clemens et al, 2000). Phagosome maturation is delayed by infection with L. donovani promastigotes, while L. major and L. amazonensis promastigotes do not delay this process (Antoine et al, 1998; Courret et al,  2002). Leishmania survival within the parasitophorous  vacuole is  accomplished by differentiation of promastigotes to the amastigote stage, a process that involves major morphological and biochemical changes in the parasite. In addition to being resistant to the hydrolytic environment of the parasitophorous vacuole, amastigotes also modulate the anti-microbial and immunological strategies that macrophages normally utilize to  clear  an  infection  (Handman, 4  1999).  Amastigotes  Chapter 1 - Introduction  J lacrophage nucleus  !  H. Zaima an  R.L. Iacobson©1996  Figure 2.  1  Images of Leishmania and the Sandfly Vector.  (a) Promastigotes in culture medium, (b) Geimsa stained Leishmania-mfectcd macrophage. The macrophage nucleus and intracellular amastigotes are indicated with black arrows, (c) The sandfly vector that inoculates promastigotes into a mammalian host during bloodmeals. 5  Chapter 1 - Introduction  Intracellular amastigote  Proliferation in the midgut www.wehi.edu.au/research/ overview/inf.html  Figure 3.  The Leishmania Lifecycle.  Leishmania alternate between the promastigote and amastigote stages. Promastigotes reside extracellularly in the sandfly vector. Amastigotes are the obligate intracellular stage that replicates within parasitophorous vacuoles of host macrophage cells. 6  Chapter 1 - Introduction eventually cause lysis of the host cell and the liberated amastigotes subsequently infect neighbouring macrophages. The lifecycle is completed when the sandfly takes a blood meal from a Leishmania-infected host and ingests Leishmania-infected macrophages or free amastigotes.  1.2  Leishmania Biology Leishmania spp. contain specialized organelles, not found in most organisms, that are  involved in metabolic and endocytic pathways. A brief description of these organelles is provided with particular emphasis placed on the flagellar pocket, a region of the membrane specialized for endocytic and secretory function.  i)  Kinetoplast: Organisms belonging to the phylogenetic order Kinetoplastida are  classified on the basis of a unique mitochondrial organelle known as the kinetoplast. Each cell contains a single kinetoplast where oxidative phosphorylation takes place. In addition, a process called R N A editing alters the actual mRNA sequence from the kinetoplast D N A (kDNA) encoded sequence (Stuart et al., 2005).  ii)  Glycosome: Leishmania and  Trypanosoma spp.  contain  membrane-bound  cytoplasmic organelles called glycosomes (de Souza, 1984). Glycosomes resemble mammalian peroxisomes, which are broadly defined as organelles that contain enzymes required for catalase activity, peroxidase metabolism, P-oxidation of fatty acids, and ether phospholipid synthesis. However, in Leishmania and Trypanosoma spp. catalase activity has not been demonstrated in the glycosomes. Similar findings have been made in other  7  Chapter 1 - Introduction protozoan parasites, such as Toxoplasma gondii, where it has been suggested that the catalase enzyme may be located in the cytosol (Ding et al, 2000). The discovery of glycosomes in trypanosomatids was elucidated initially in T. brucei and then shown to exist in different species of Leishmania (de Souza, 2002). Biochemical analysis of peroxisome-like organelles isolated from Trypanosoma and Leishmania showed that enzymes involved in several metabolic pathways, including glycolysis, are located in these organelles. Furthermore, enzymes involved in carbon dioxide fixation, purine salvage, and de novo pyrimidine synthesis, which are found in the cytosol of mammalian cells, are localized to glycosome organelles of typanosomatid parasites (Hart et al, 1984; Hassan et al, 1985; Mottram et al, 1985).  Hi)  Acidocalcisome:  It has been known for many years that trypanosomatid parasites  contain electron dense granules in their cytoplasm. Previous studies in L. amazonensis indicated that dense vacuoles in amastigotes were acidic (Antoine et al,  1988). The  biological structure and function of these granules, termed acidocalcisomes, was a mystery until studies in T. brucei demonstrated that calcium ions are concentrated in these vesicles by a C a / H translocating ATPase activity (Vercesi et al, 1994). Subsequently, the existence of 2+  +  acidocalcisomes has been shown in other trypanosomatids, including Leishmania, and inhibition of calcium release from the acidocalcisome was shown to cause reduced virulence of L. mexicana amastigotes (Docampo et al, 1995; L u et al, 1997). Acidocalcisomes were shown to concentrate phosphorus, calcium, magnesium, and zinc at high concentrations relative to the cytoplasm. The phosphorus stored in acidocalcisomes of T. cruzi, T. brucei, and L. major is present as PPi, as well as long-chain and short-chain polyphosphates. PPi is the predominant high-energy phosphate present in these organisms (LeFurgey et al, 1990; 8  Chapter 1 - Introduction Moreno et al., 2000). Recently, the identification of a vacuolar H -ATPase, a C a / H +  2+  +  translocating ATPase, and a vacuolar proton-translocating pyrophosphatase have provided molecular evidence for the biochemical features of the acidocalcisome (de Souza, 2002).  iv)  Megasome: Leishmania mexicana contain spherical, large membrane-bound vesicles  that have been designated as megasomes (Alexander et al., 1975). Interestingly, megasomes are only observed in the amastigote stage of the lifecycle and during differentiation of promastigotes into amastigotes (Pral et al., 1993). These organelles are more abundant in lesion-derived amastigotes than axenically cultivated amastigotes, but megasomes have not been described in log phase or metacyclic promastigotes. Megasomes vary in number, accounting for 3-15 % of the amastigote total cell volume (Ueda-Nakamura et al., 2001). Acid phosphatases and cysteine proteases are localized to the megasome suggesting that this organelle is similar to lysosomes found in other eukaryotic cells. Knockout studies on the Leishmania mexicana cysteine proteases gene families determined that these genes, and therefore the megasome organelle, are important for parasite virulence and modulation of the host immune response (Bart et al., 1997; Alexander et al., 1998). The molecular mechanisms for targeting proteins to megasomes have remained elusive. Lysosomal-targeting of proteins in mammalian cells often occurs via mannose-6-phosphate receptors, however this lysosomal trafficking mechanism is not involved in Leishmania (Boukai et al., 2000). Another study has shown that a short amino acid sequence in the pro-domain of the Leishmania Cathepsin L like proteases, which is conserved in the pre-domain of mammalian procathepsin L , is involved in targeting these proteins to the megasome in Leishmania (Huete-Perez et al., 1999). The L. mexicana cysteine protease B (CPB) proteins are transported to the flagellar pocket and subsequently to the megasome, with either direct or indirect processing and 9  Chapter 1 - Introduction trafficking by cysteine protease A (CPA) proteins (Brooks et al, 2000). Fusion of endocytic vesicles with megasomes occurs at the late-stages of the endocytic pathway (Pupkis et al, 1986; Russell et al, 1992).  iv)  Flagellar Pocket: A l l trypanosomatids have a region known as the flagellar pocket,  which is located at the anterior region of Leishmania cells. The plasma membrane invaginates where the flagella emerges from the cell body and forms a continuous structure with the flagellar membrane. Invagination of the plasma membrane produces a unique, extracellular compartment that is isolated from the surrounding environment. There is a substantial body of evidence that the flagellar pocket is a specialized region of the cell for secretory and endocytic activities. Endocytosis of extracellular macromolecules occurs exclusively at the flagellar pocket. Two distinct mechanisms have been described: non-specific fluid phase endocytosis and receptor-mediated endocytosis. In T. brucei, it was shown that gold-labeled proteins bound specifically to the flagellar pocket, and not to other regions of the plasma membrane (Coppens et al, 1987). In mammalian cells, endocytosis occurs via membrane invagination and subsequent budding into vesicles coated with clathrin protein. A clathrin-like protein has been isolated from endocytic vesicles of T. brucei and coated vesicles have been identified in endocytic vesicles of L. mexicana promastigotes (Webster et al, 1990; Weise et al, 2000). A large tubule extending the entire length of Leishmania promastigotes,  named  the  multivesicular tube (MVT), is filled with small vesicles that label with internalized particles. The L. mexicana M V T was initially described as an extension of the endoplasmic reticulum because enzymes involved in glycophosphoinositiol synthesis were detected within this compartment (Ilgoutz et al, 1999). However, more recent studies in Leishmania using fluid10  Chapter 1 - Introduction phase markers showed accumulation of these markers in the M V T . Glycophosphoinositol (GPI)-anchored and transmembrane  green fluorescent  protein (GFP) proteins  were  transported, via the golgi apparatus, to the flagellar pocket and subsequently internalized into the M V T compartment (Ghedin et al., 2001). These data provided convincing evidence that the M V T had biological properties comparable to the endosomes of higher eukaryotes and may therefore be an integral part of the endocytic processes in trypanosomatids. The secretory pathway in Leishmania occurs via the classical membrane trafficking and secretory pathways of higher eukaryotes. Rough endoplasmid reticulum (ER) has been described as a tubular network throughout the cytoplasm, while the golgi apparatus consists of 4-6 flattened tubules. Numerous vesicles exist between the trans-golgi and flagellar pocket that are believed to function as secretory vesicles (Bangs et al, 1993; Weise et al, 2000). The majority of surface proteins and complex glycolipids on Leishmania are tethered to the membrane by a GPI-anchor that is attached in the ER. Further modification of macromolecules by the addition of carbohydrate side chains to these surface macromolecules occurs in the golgi apparatus. The secretory pathway of Leishmania is unique in that membrane associated and secreted proteins are all initially transported to the flagellar pocket. Secreted macromolecules are translocated to the lumen of the flagellar pocket and membrane associated macromolecules are distributed across the plasma membrane. The most abundant secreted molecules from Leishmania promastigotes are a filamentous, secreted acid phosphatase (sAP) and a fibrous network of proteoglycans.(Ilg et al, 1991) It is proposed that the filamentous sAP is produced by monomelic subunits, which are secreted into the lumen of the flagellar pocket. Only the monomelic subunits of these large polymers have been detected in the cytosol (Stierhof et al., 1994).  11  Chapter 1 - Introduction 1.3  Leishmania Surface Molecules Leishmania promastigotes are inoculated into the blood of the mammalian host and  rapidly phagocytosed by various phagocytic cells at the site of infection, including Langerhans cells, neutrophils, monocytes and macrophages (Moll, 1993; Handman, 1999; Laskay et al, 2003). The virulence of metacyclic promastigotes is associated with changes in the glycosylation state of surface molecules (Sacks et al, 1984; Saraiva et al, 1995; Soares et al, 2005). The two major Leishmania surface molecules, leishmanolysin (gp63) and lipophosphoglycan (LPG), are specific ligands for macrophage receptors and provide protection from host serum factors. GP63, a zinc-metaloprotease, is the major surface protein on Leishmania (Fong et al, 1982). The precursor protein undergoes post-translational modifications, including N glycosylation and the addition of a C-terminal glycosylphosphatidylinisotol (GPI) anchor predicted to function in membrane attachment (Voth et al, 1998). The gene encoding GP63 from L. major was cloned and it was later reported that the gp63 genes are tandemly repeated (Button et al, 1988; Button et al, 1989). Subsequent studies have shown that gp63 gene copy number and chromosomal arrangement varies between different Leishmania species (Webb et al, 1991; Medina-Acosta et al, 1993; Roberts et al, 1993). Expression of L. major gp63 genes 1-7 is developmentally regulated; genes 1 to 5 are expressed only in log phase promastigotes, gene 6 is constitutively expressed, and gene 7 is metacyclic-specific (Joshi et al, 1998). L P G is a complex glycolipid and represents the most abundant surface molecule on promastigotes. Surface L P G forms a dense glycocalyx covering the entire surface of promastigotes and the flagellum. The structure of L P G has been extensively studied and consists of four distinct domains: (1) a lipid anchor, (2) a glycan core, (3) a disaccharide 12  Chapter 1 - Introduction backbone, and (4) a neutral oligosaccharide cap (McConville et al,  1990).  There is  significant interspecies variation in both the cap structure and the amount of glycosylation of the disaccharide backbone. Importantly, developmental changes occur in the structure of LPG  as Leishmania mature  from procyclic promastigotes  to virulent metacyclic  promastigotes. These include expansion in the number of disaccharide repeats in the L P G backbone, as well as changes in the actual sugar residues attached to the L P G backbone. Typically, procyclic promastigotes have terminal galactose moieties attached to the disaccharide  backbone,  while  terminal  sugars  of  metacyclic  promastigotes  are  arabinopyranose (McConville et al, 1992). In amastigotes, surface expression of L P G is dramatically decreased and the structure of L P G undergoes further modification. Significant differences in the levels of amastigote surface L P G have been observed among the different species of Leishmania. L. major amastigotes express low levels of L P G , while L. mexicana and L. donavani amastigotes do not appear to express any surface L P G (McConville et al.± 1991;Bahre/a/., 1993).  1.4  Parasite-Vector Interactions There is clear evidence that proteins secreted by promastigotes as well as salivary  proteins of the Lutzomyia sand fly vector are important for successful transmission of Leishmania to its mammalian host. Parasite-derived molecules actively alter sandfly anatomy to ensure metacyclic promastigotes are inoculated into the host. On the other hand, salivary proteins of the sandfly vector enter capillaries of the host during bloodmeals and exert potent biological effects that aid promastigotes during invasion and the early stages of infection of the mammalian host.  13  Chapter 1 - Introduction It has been noted that infected sand flies probe multiple times during feeding (KillickKendrick, 1979). More recently, this has been correlated with Leishmania-derived molecules that damage the cardiac valve of infected sandflies and cause regurgitation of parasites into the mammalian host (Schlein et al., 1992). Metacyclic promastigotes are found mostly in the thoracic midgut and cardia, coincident with the accumulation of a promastigote secretory gel (PSG) plug in these anterior regions. The PSG plug occludes and distorts the midgut, forcing the stomodeal valve open and affecting the feeding success of the sandflies (Rogers et al., 2002). P S G contains a filamentous proteophosphoglycan (fPPG) that is essential for successful parasite infection (Rogers et al., 2004). Salivary gland proteins from the sandfly vector were shown to be important for initiating mammalian infection with Leishmania. In experimental infection studies, coinjection of parasites and salivary gland extract resulted in larger lesions and improved parasite survival, relative to infection with Leishmania alone (Titus et al, 1988). Saliva released during the sandfly bite contains proteins with vaso-dilatory, erythema-inducing properties resulting in recruitment of macrophages to the site of infection. Erythema-inducing factor (EIF) was initially isolated from Lutzomyia salivary gland lysates and shown to have vaso-dilatory properties. Immunoreactivity with antibodies against human calcitonin generelated peptide (CGRP), a potent vaso-dilator, indicated that EIF is a related protein with 10fold higher biological activity that CGRP. Additionally, EIF inhibits macrophage activation by interferon-gamma (IFNy) during in vitro experiments (Ribeiro et al, 1989). A second salivary, Maxadilan, was subsequently isolated from Lutzomyia saliva and shown to be 500times more potent than EIF (Lerner et al, 1991). In addition to vaso-dilatory properties, salivary proteins of Lutzomyia have immunomodulatory function of macrophages that would enhance the ability of promastigotes to establish an infection (Soares et al, 1998; Cavalcante 14  Chapter 1 - Introduction et al, 2003; Norsworthy et al, 2004). Sandfly salivary proteins ammeliorate the ability of Leishmania promastigotes to establish successful infections and vaccination with these proteins had a protective effect during subsequent infections (Morris et al, 2001; Valenzuela et al, 2001; Thiakaki et al, 2005)  1.5  Mammalian Host-Parasite Interactions Leishmania parasites are rapidly engulfed by host macrophage cells at the site of  infection. Macrophage cells are the predominant cell type utilized by the parasite to differentiate to amastigotes, replicate, and complete the lifecycle (Alexander et al, 1992). Interaction with host macrophages has important implications for parasite survival, the host immune response, and the clinical outcome of infection. Dendritic cells, also phagocytic antigen presenting cells, have been described as a host cell for intracellular amastigotes with important implications for mti-Leishmania immunity (Moll, 1993; von Stebut et al, 1998). Interestingly, Leishmania parasites were also found to persist in fibroblast cells after lesions were resolved (Bogdan et al, 2000). Persistence of a few Leishmania cells in fibroblast cells of a "healed" host may provide long term antigen stimulation to the immune system that confers lifelong immunity to subsequent infections. Leishmania species which are the causative agents of cutaneous and visceral leishmaniasis have different tissue tropism for establishing infection. The ubiquitous distribution of macrophages in tissues of the body enables Leishmania to maintain a persistent infection by re-infecting macrophages in the surrounding tissues (Handman, 1999). The recognition and rapid phagocytosis of Leishmania by host macrophages is an important event for survival in the host as it allows the parasite to evade complement-mediated killing. Metacyclic promastigotes are less susceptible to lysis by serum complement factors than procyclic promastigotes (Puentes et al, 1988). The molecular 15  Chapter 1 - Introduction interactions between Leishmania and macrophages have been well characterized. Direct parasite-macrophage recognition occurs through the interaction of macrophage receptors with Leishmania surface molecules. Indirect binding is mediated by opsonization of Leishmania by host serum factors, which facilitates parasite recognition and uptake by macrophages (Mosser et al., 1997). In addition, several macrophage receptors are known to mediate Leishmania recognition by macrophages, which suggests redundancy in the mechanisms used to mediate phagocytosis of Leishmania.  i)  Direct Interaction: The major surface molecules of Leishmania promastigotes, gp63 and L P G , have been  shown to interact directly with macrophage receptors. These experiments were performed in serum-free media to identify host-parasite interactions without opsonization by antibody, complement, C-reactive protein, or mannose-binding lectin. GP63 contains a fibronectin-like Ser-Arg-Tyr-Asp (SRYD) sequence that functionally resembles the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin (Rizvi et al, 1988; Puentes et al, 1999). Leishmania promastigotes neutralized with polyclonal anti-gp63 serum were unable to attach to macrophages, thus demonstrating the involvement of gp63 in macrophages binding. Additional experiments showed that gp63-containing proteoliposomes were efficiently bound and phagocytosed by macrophages; the observed binding was suppressed by greater than 90% when these proteoliposomes were pre-treated with anti-gp63 antibody (Russell et al, 1986). The alpha4/betal fibronectin receptor on macrophages directly binds gp63. This interaction significantly enhances promastigote entry into the macrophage (Brittingham et al, 1999)].  16  Chapter 1 - Introduction L P G , the major glycolipid surface molecule of all Leishmania species, interacts with several carbohydrate recognition receptors on macrophages. L P G is recognized by members of the C D 18 complex of leukocyte integrins, including CR3. Analysis of L P G interaction with CR3 indicated that recognition is mediated by the CR3 carbohydrate-binding site, as opposed to the peptide-binding site (Talamas-Rohana et al, 1990). A subsequent report indicated that CR3 is not sufficient for macrophage binding to non-opsonized Leishmania; however, opsonization with C3 complement confers Leishmania binding capacity. It was concluded that CR3-mediated parasite binding is dependent on complement fixation, whereby C3bi is responsible for binding to CR3. Parasite antigen 2 (PSA-2), a membrane protein with 15 leucine-rich repeats, was also shown to interact directly with murine and human CR3 (Kedzierski et al, 2004). The repeating phosphorylated disaccharide units that form the backbone of L P G were a novel direct ligand for the acute phase protein, C-reactive protein (CRP), which was previously described for recognition of phosphorylcholine residues on bacterial organisms (Culley et al, 1996). Leishmania promastigote binding to macrophages is inhibited by competition with known ligands to the receptor for advanced glycosylation end-products (AGE), mannose-6phosphate receptor, and the mannose-fucose receptor (MFR) (Wilson et al, 1986; Mosser et al, 1987; Saraiva et al, 1987). The major surface proteins and lipids of Leishmania, including gp63 and L P G , are glycosylated molecules, which are proposed to be involved in parasite recognition by these receptors. Interestingly, the mannose-6-phosphate receptor of murine peritoneal macrophages shows significantly lower association with L. mexicana amastigotes relative to promastigotes (Saraiva et al, 1987). Hence, the macrophage M F R is important for L. major promastigote entry into macrophages, whereas the M F R does not enhance lesion amastigote entry into macrophages (Guy et al, 1993). These results suggest 17  Chapter 1 - Introduction that the well characterized glycosylation changes to L P G that occur during parasite differentation may alter the ability of macrophage receptors to recognize amastigotes. Therefore, amastigotes liberated from infected macrophages may utilize a different repertoire of macrophage receptors to re-infect neighbouring macrophage cells during in vivo infection (Saraiva et al, 1987; Davies et al, 1990; Pimenta et al, 1991). L P G surface expression on amastigotes is very low or absent on some Leishmania species, thus suggesting that L P G may be the primary ligand recognized by the macrophage mannose-fructose  receptor  (Pimenta et al, 1991).  ii)  Opsonization by Host Serum Factors and Parasite Survival Leishmania utilize opsonization  and  immune  adherence to  internalization of viable parasites by host phagocytes. promastigotes  promote  rapid  This strategy ensures that  inoculated during the sandfly blood meal avoid being killed by the  complement cascade and establish a successful infection. The innate immune system of most non-immune, vertebrate animals contains natural, mti-Leishmania IgM antibody (Schmunis et al, 1970; Rezai et al, 1975). Natural antibodies are produced by non-classical B - l B cells, which are distinct in terms of development and functional properties from the classical B-2 B cells of the adaptive immune system (Wortis et al, 2001). Anti-Leishmania natural antibodies are present at high concentrations in serum from all vertebrate taxa studied. They broadly recognize trypanosomatid organisms, including Crithidia and Phytomonas species that do not infect vertebrates (Schmunis et al, 1970; Rezai et al, 1975; Dominguez et al, 2003). Natural antibodies play a role in opsonization of Leishmania. Acute phase proteins, including C-Reactive Protein (CRP) and Mannose Binding Lectin (MBL), are released by the liver during an inflammatory immune response and have been shown to opsonize Leishmania 18  Chapter 1 - Introduction by binding to L P G and glycoconjugates, respectively (Green et al, 1994; Culley et al, 1996). However, CRP and M B L are present at more than 100-fold lower concentrations than mti-Leishmania natural antibody and, as such, were proposed to have a less significant role in parasite opsonization than natural antibody (Dominguez et al, 2003). IgG-coated amastigotes attach to macrophages FcyRIIc receptor, which triggers phagocytosis and promotes intracellular parasite survival by stimulating production of the macrophage inhibitory cytokine, IL-10 (Peters et al, 1995; Kima et al, 2000; Kane et al, 2001). Antibody, CRP and M B L bound on the surface of pathogens are major sites for complement deposition and activation of the complement cascade. Leishmania attach to and enter macrophages via CR1- and CR3-mediated phagocytosis (Mosser et al, 1985; Da Silva et al, 1989; Rittig et al, 2000). Promastigotes are rapidly agglutinated and opsonized by natural antibody, which leads to complement activation by the classical pathway (CP) (Pearson et al, 1980; Navin et al, 1989). The rapid kinetics of parasite opsonization by natural antibody and subsequent CP-mediated complement deposition has recently been elucidated through in vitro studies that attempted to reproduce in vivo infection by performing experiments at high serum concentrations. Antibody binding activates rapid CPmediated complement deposition, which occurs within 2-3 minutes of infection, and results in the destruction of greater than 90% of promastigotes (Dominguez et al, 1999; Dominguez et al, 2002). These data contrast previous reports that Leishmania primarily activate the alternative pathway (AP) for complement activation (Mosser et al, 1984). Leishmania have evolved several approaches that improve parasite survival by limiting the lytic effects of complement. Attachment via CR3 not only triggers receptormediated phagocytosis, but also contributes to parasite survival by inhibition of macrophage oxidative burst (Mosser et al, 1987). Metacyclic promastigotes also degrade the C5-C9 19  Chapter 1 - Introduction complement M A C and it has been hypothesized that elongation of surface L P G molecule may be involved in this process (Puentes et al., 1990). Leishmania membrane kinases phosphorylate the C3, C5, and C9 complement components, thus blocking activation of the complement cascade (Hermoso et al, 1991). The Leishmania gp63 membrane protein also converts C3b to C3bi, enabling the parasite to utilize the opsonic properties of C3 while eluding its lytic effects (Brittingham et al, 1995).  iii)  Immune Adherence C3-opsonized metacyclic promastigotes bind to erythrocytes in primates or platelets  in non-primate mammals by an event known as immune adherence (LA) (Mills et al, 1931). Erythrocytes represent the first acceptor cells for inoculated promastigotes and this reaction is complete within 40 seconds (Dominguez et al, 2003). The products of the LA reaction can be visualized with a microscope as rosettes formed by several erythrocytes or platelets surrounding a promastigote. Serum opsonized amastigotes also bind to erythrocytes, which suggests that LA may contribute to amastigote dissemination in an infected host (Dominguez et al, 1999). The human erythrocyte receptor for promastigote-erythrocyte LA complex formation is CR1, which recognizes the C3b, C4b, C3bi, and C l q complement components (Birmingham et al, 2001). Formation of the promastigote-erythrocyte complex enhances Leishmania survival in mammalian hosts by improving the efficiency of promastigote transfer  to  and  internalization by professional  phagocytes,  including neutrophils,  macrophages, and dendritic cells (Dominguez et al, 2003). Considering the rapid kinetics of the early events of a Leishmania infection, Dominguez et al. have proposed that a 60 second time window exists between the LA reaction and the onset of complement-mediated parasite killing. 20  Chapter 1 - Introduction Neutrophils readily engulf but do not kill Leishmania, which suggests that neutrophils may be an early target cell used to evade complement-mediated killing (Laskay et al, 2003). Leishmania-infected neutrophils can be recovered from the skin of infected mice 3 days postinfection and in vitro experiments indicate that Leishmania have an anti-apoptotic effect on neutrophils (Aga et al, 2002). Leishmania infection of neutrophils stimulates secretion of MJJP-ip, a chemokine which recruits macrophages. It has been shown that apoptotic, Leishmania-infected  neutrophils  were  phagocytosed  by  infiltrating  macrophages.  Interestingly, macrophages release the anti-inflammatory cytokine, TGF-P, after ingestion of apoptotic, infected neutrophils (van Zandbergen et al, 2004). In this model of neutrophil involvement during the early stages of infection, infiltrating neutrophils rapidly engulf the Leishmania-erythrocyte IA complex and act as a "Trojan horse" to silently transfer parasites to macrophages, the major target cell for long term Leishmania infection (Alexander et al, 1992; van Zandbergen et al, 2004).  1.6  Innate Immunity to Leishmania Infection Monocytes are bone marrow derived cells that circulate in peripheral blood and  differentiate into macrophages after entering surrounding tissues in response to inflammatory signals (Olivier et al, 2005). These cells are an important component of the innate immune system of vertebrate organisms with potent anti-microbial activities and efficient presentation of antigen to cells of the adaptive immune system. During Leishmania infection, macrophages are activated by parasite recognition via the complement receptors CR1 and CR3, FcyR receptor, scavenger receptor, toll-like receptors (TLRs), and the mannose-fucose receptor (Muraille et al, 2003). IL-12 production by macrophages and dendritic cells during the early stages of Leishmania infection is critical for the development of a protective, cell21  Chapter I - Introduction mediated immune response (Magram et al, 1996; Trinchieri et al, 1996). Macrophages are activated by autocrine tumour necrosis factor-alpha (TNF-ct) production and IL-12dependent interferon-gamma (LFN-y) secretion by natural killer (NK) cells and T cells (Murray et al, 1983; Bogdan et al, 1990; Laskay et al, 1995). Live promastigotes also have the capacity to induce IL-12 independent IFNy secretion by N K cells (Nylen et al, 2003). Natural killer cells are the primary source of LFN-y production during the early stages of Leishmania infection (Scharton et al,  1993). It has been well established that LFN-y  production by T cells is essential for activation of Leishmania-infected macrophages to kill intracellular amastigotes in the later stages of infection (Nacy et al, 1985; Reiner et al, 1990). Containment and killing of Leishmania is largely determined by the ability of host macrophages to limit protein tyrosine phosphatase (PTP)-mediated inhibition of infected macrophages. The LFN-y signalling pathways are critical for macrophage activation and induction of parasite killing. Activated macrophages have several different mechanisms to kill intracellular pathogens, including oxidative burst, nitric oxide (NO) production, iron deprivation, and tryptophan degradation  (Stafford et al,  2002). The most potent  leishmanicidal activities possessed by activated macrophages are oxidative burst and N O production (Bogdan et al, 2000). Persistent infection by Leishmania is associated with macrophage inhibition via suppression of PKC-, Ca2+-, and protein tyrosine kinase (PTK)dependent LFN-yR signaling pathways (Nandan et al, 1995; Olivier et al, 1998; Ray et al, 2000). Rapid activation of the host PTP, SHP-1, has been shown to occur after Leishmania infection and results in dramatic inhibition of LFN-y mediated macrophage activation and persistence of intracellular amastigotes (Blanchette et al,  22  1999; Forget et al, 2001).  Chapter 1 - Introduction Consequently, treatment with the  PTP inhibitor, Peroxovanadium (pV), increases  macrophage responsiveness to IFN-y and stops the progression of cutaneous and visceral leishmaniasis (Olivier et al,  1998). More recently, it was reported that Leishmania  Elongation Factor-1 alpha (EF-lot) is secreted by the parasite and localized to the macrophage cytosol. Leishmania E F - l a directly activated macrophage SHP-1, which resulted in decreased IFN-y responsiveness and blocked N O production (Nandan et al, 2002). i)  Oxidative Burst: Oxidative burst is a potent antimicrobial response of macrophages  and neutrophils that is triggered by phagocytosis. It involves the enzymatic conversion of molecular oxygen (O2) to superoxide anion (O2-) by a 5 subunit enzyme, N A D P H oxidase (Shatwell et al, 1996; Takeya et al, 2003). Superoxide is a precursor for the generation of the following reactive oxygen intermediates (ROIs): hydrogen peroxide, hydroxyl free radical, hyperchlorous acid, and peroxynitrite. The inflammatory cytokines T N F - a and IFN-y initially prime macrophages, while pathogen attachment via the FcyR and the mannosefucose receptors triggers the activation of oxidative burst. receptor-mediated  Conversely, complement  phagocytosis does not trigger oxidative burst.  Internalization of  promastigotes does not stimulate oxidative burst, which correlates with observations that CR1 and CR3 play the most important roles in promastigote phagocytosis (Mosser et al, 1985; Mosser et al, 1987). Internalization of amastigotes does not trigger oxidative burst and is correlated with the fact that amastigote-macrophage interaction does not involve the mannose-fructose receptor (Guy et al, 1993). Phagocytes isolated from patients with chronic granulomatous disease (CGD), characterized by deficiency in oxidative burst, fail to kill Leishmania; however, IFN-y stimulation of these macrophages has been shown to activate non-oxidative mechanisms for killing intracellular parasites (Passwell et al,  1994).  Interestingly, in vivo experiments demonstrated that ROIs were only required during the 23  Chapter 1 - Introduction initial 14 days of infection. L. donovani infection of mice deficient for the gp91phox gene, which encodes one of the subunits of N A D P H oxidase, developed hepatic granulomas and controlled the infection (Murray et al., 1999). Another study with gp91p/jox-knockout mice demonstrated the necessity of N A D P H oxidase for splenic clearance of L. major parasites, although the resolution of cutaneous lesions was not affected by deficiency in oxidative burst (Bios et al., 2003). ii)  Nitric Oxide (NO):  Nitric oxide synthase type 2 (NOS-2), also referred to as  inducible nitric oxide synthase (iNOS), is an enzyme utilized by cells of the immune system to generate the microbicidal product, nitric oxide (NO) (Nathan et al, 1994). In contrast to N A D P H oxidase, the iNOS protein is synthesized de novo in response to ligation of macrophage pathogen recognition receptors (PRRs) and cytokine stimulation (Fonseca et al, 2003). L-arginine as the primary nitrogenous donor in the synthesis of N O by iNOS, which converts N O to the following reactive nitrogen intermediates (RNIs): nitrite, nitrate, and nitrosamines (Bogdan et al, 2000; Fang, 2004). RNIs are highly toxic to pathogens and the cytotoxic effect is mediated by inhibition of iron-dependent enzymes, including P K C , ferritin, and indoleamine 2,3-dioxygenase (LDO) (Reif et al, 1990; Gopalakrishna et al, 1993; Thomases/., 1994). There are several lines of evidence that demonstrate the importance of iNOS expression and N O derivatives for resistance to Leishmania infection. Differences in the level of iNOS expression have been observed in human patients with healing and nonhealing cutaneous leishmaniasis. A large number of iNOS-positive cells are found in small, healing lesions, whereas significantly fewer iNOS-positive cells are detected in larger, nonhealing lesions in patients with localized or disseminating cutaneous leishmaniasis (Qadoumi et al, 2002). Specific inhibitors of the iNOS enzyme, such as the compound L - N M M A , have 24  Chapter 1 - Introduction been an important tool for investigating the role of N O production during an infection. L NMMA-mediated inhibition of the iNOS enzyme prevented macrophage  killing of  intracellular amastigotes; similarly, iNOS-knockout mice were unable to control parasite replication or resolve lesions (Liew et al, 1990; Wei et al, 1995; Diefenbach et al, 1998). Additionally, C57BL/6 mice infected with L. mexicana develop self-limiting lesions that correlate with the high concentrations of circulating N O derivatives, iNOS-positive and IFNy-positive cells. In contrast, B A L B / c mice infected with L. mexicana develop disseminating leishmaniasis characterized by lower levels of iNOS-dependent N O production (Bios et al, 2003). iNOS-dependent N O production also plays a critical role in directing the adaptive immune response to Leishmania infection. iNOS-knockout mice fail to develop an early T h l immune response during Leishmania infection and parasites disseminate to the liver within 24 hours. This lack of iNOS activity correlates with high levels of the inhibitory cytokine,  TGF-P, as well as absent IFN-y and natural killer cell responses (Diefenbach et al, 1998). Subsequent experiments with these iNOS-knockout mice have demonstrated defective IL-12 signaling, thus delaying IFN-y production by natural killer cells and failure to initiate a T h l type CD4+ immune response (Diefenbach et al, 1999; Niedbala et al, 1999). iii)  Iron Deprivation: Macrophages also inhibit the survival of intracellular pathogens by  limiting the availability of free iron. This strategy is accomplished via tight regulation of the proteins involved in iron transport and storage in macrophages. Transferrin protein is bound to circulating iron and intracellular iron stores are bound to ferritin protein. (Jacobs, 1977). The transferrin/iron complex is transported  into macrophages  via  receptor-mediated  endocytosis. In order to limit iron availability to intracellular pathogens, activated macrophages increase expression of transferrin and decrease expression of ferritin and the 25  Chapter 1 - Introduction transferrin receptor (Hirata et al, 1986; Djeha et al, 1995; Mulero et al, 1999). The expression of these genes is regulated by iron response elements (IRE) within the mRNA transcripts. The production of N O by activated macrophages also regulates the availability of intracellular iron to pathogens. N O induces the expression of iron response proteins (IRP), which bind to IREs and regulate the stability or translation initiation of ferritin, transferrin, and the transferrin receptor mRNAs (Drapier et al, 1993; Weiss et al, 1993; Mulero et al, 1999). iv)  Host Genetic Factors: A host resistance locus for intracellular pathogens belonging  to the genera Leishmania, Mycobacterium, and Salmonella was initially characterized as the Ity/Lsh/Bcg locus (O'Brien et al, 1980; Brown et al, 1982; Plant et al, 1982; Skamene et al, 1984). Subsequently, the natural resistance associated macrophage protein 1 {nrampl) gene was cloned by linkage mapping of the Ity/Lsh/Bcg locus in mouse strains with genetic susceptibility to intracellular pathogens (Vidal et al, 1993; Cellier et al, 1994). N R A M P - 1 has been characterized as a membrane transport protein that is specifically expressed in phagocytic cells. A n N R A M P homologue, N R A M P - 2 , is characterized by a ubiquitous tissue expression pattern (Gruenheid et al, 1995). Targeted deletion of the nrampl gene clearly demonstrates the functional role of this gene for host resistance to infection by Leishmania, Mycobacterium, and Salmonella pathogens at early stages of infection (Vidal et al, 1995). Expression of nrampl gene is induced in response to IFN-y and LPS and the protein is expressed on the phagolysosome membrane in phagocytic cells (Govoni et al,  1995;  Gruenheid et al, 1997; Searle et al, 1998). The N R A M P - 1 protein has structural similarities to prokaryotic and eukaryotic ion transporters and sequence homology with the yeast manganese transporter, SMF1 (Cellier et al,  1996; Supek et al,  1996). Biochemical  experiments have shown that NRAMP-1 transports iron and manganese ions from the lumen 26  Chapter 1 - Introduction of the phagolysosomal compartment (Atkinson et al, 1998; Jabado et al, 2000). It is proposed that N R A M P - 1 and N R A M P - 2 , a less selective metal ion transporter, sequester essential divalent ion cofactors away from obligate intracellular pathogens that replicate within the phagolysosomal compartment of phagocytic cells (Forbes et al, 2001).  1.7  Adaptive Immune Response to Leishmania Infection Activation of the adaptive immune response is intimately linked to the activation of  professional antigen presenting cells (APC), including macrophages and dendritic cells. Although macrophages are the definitive host cell for Leishmania, dendritic cells play an important role in determining the type of adaptive immune response to a Leishmania infection (Alexander et al, 1992; Moll et al, 1995; Scott et al, 2002). Macrophages and dendritic cells are crucial for killing intracellular parasites, antigen presentation, and initiation of the adaptive immune response. Natural killer cells are the source of LFN-y production during the early stages of infection (Scharton et al, 1993). However, it is well established that LFN-y production by activated T cells is essential for activation of Leishmania-infected macrophages to kill intracellular amastigotes and initiate a protective immune response (Nacy et al, 1985; Reiner et al, 1990). i)  Antigen Presentation: Macrophages and dendritic cells load peptide antigens from  Leishmania onto M H C class II molecules, which are subsequently transported to the A P C cell surface and presented to T cells (Antoine et al, 2004). Recently, it has been proposed that dendritic cells play an important role in antigen presentation and T cell activation during Leishmania infection. A n elegant experiment by Lemos et al elucidated that antigen presentation by dendritic cells, and not other APCs, was sufficient to induce a protective, Thl-type immune response during Leishmania infection (Lemos et al, 2004). T cell 27  Chapter 1 - Introduction recognition of parasite antigen presented on APCs initiates an immune response that ultimately activates the infected macrophage to kill intracellular amastigotes. Leishmania amastigotes inhibit IFN-y induced M H C class I and class II expression and interfere with antigen presentation by macrophages (Reiner et al, 1987; Fruth et al, 1993; Prina et al, 1993). Live, intracellular amastigotes internalize and degrade macrophage M H C class II molecules, thereby preventing presentation of parasite antigens to T cells during successful infection (De Souza Leao et al,  1995; Wolfram et al,  1995). Promastigote antigens,  however, are efficiently presented to CD4+ T cells on M H C class II molecules during the initial 24 hours of infection; thereafter, there is minimal presentation of parasite antigen by macrophages (Kima et al, 1996). The co-stimulatory molecules CD80, CD86, and CD40 must also be expressed on the A P C cell surface to activate naive T cells (Campbell et al, 1996; Marovich et al, 2000; Brodskyn et al, 2001). Amastigotes interfere with antigen presentation by inhibiting expression of these co-stimulatory molecules in infected macrophages (Kaye et al, 1994; Bogdan et al, 1996). ii)  T cells: The importance of T cells for protective immunity to Leishmania infection  has been demonstrated by adoptive transfer experiments. The adoptive transfer of splenic T cells from B A L B / c mice immunized with irradiated promastigotes conferred protection to naive B A L B / c mice challenged with live parasites (Liew et al, 1984). In particular, adoptive transfer of CD4+ T cells conferred protective immunity to L. major infection in athymic nude mice from a genetically resistant mouse strain. In contrast, the adoptive transfer of CD8+ T cells into athymic nude mice failed to confer protective immunity (Moll et al, 1988). Each naive CD4+ T helper (Th) cell expresses a unique T cell receptor (TCR), which recognizes specific foreign antigens presented in the context of M H C class II. The interaction of a naive CD4+ Th cells with a foreign peptide-MHC class II complex, and a co-stimulatory 28  Chapter 1 - Introduction molecule, results in T cell activation and differentiation. CD4+ Th cells are capable of differentiating into T helper 1 (Thl)-type or T helper 2 (Th2)-type CD4+ T cells, which have dramatically different effects on the resulting immune response (Jankovic et al., 2001). Early immunological studies have demonstrated that, depending on the stimulus, CD4+ T cells produce different cytokine secretion profiles. The T h l subset of CD4+ T cells produce IL-2 and IFN-y cytokines, and initiate a cell-mediated immune response. The Th2 CD4+ subset secretes IL-4 and IL-5 cytokines to stimulates a humoral response (Mosmann et al., 1986). It was subsequently shown that the type of CD4+ T cell response, T h l or Th2, is critical in determining the outcome of Leishmania infection. iii)  Mouse Models for Resistance/Susceptibility: The C57BL/6 and C3H/HeN mouse  strains are well characterized "healing" models for L. major infection, while the B A L B / c mouse strain is the "non-healing" infection model (Kellina, 1973; Nasseri et al, 1979). High concentrations of neutrophils remain at the site of infection for several weeks in B A L B / c mice, but do not in resistant mice (Beil et al, 1992). This observation has led to speculation that the neutrophils at the site of infection may affect Thl/Th2 polarization. Leishmania parasites rapid disseminate to the visceral organs of B A L B / c mice during the initial 24 hours of infection, whereas in resistant mouse strains, parasites are only found at the site of infection and draining lymph nodes. IFN-y production by natural killer cells is required for the prevention of parasite dissemination during the early stages of the infection in resistant mice (Laskay et al, 1995). iv)  Cell-Mediated Immunity (CMI) & Resistance: Thl-type CD4+ T cells, which  produce high levels of IFN-y and IL-12 after stimulation with protective leishmanial antigen, adoptively transfer protective immunity. Conversely, stimulation of CD4+ T cells with nonprotective antigens results in a Th2-type T cell phenotype and fails to transfer protective 29  Chapter 1 - Introduction immunity (Scott et al., 1988). The current model for Leishmania immunity is that a T h l response with LFNy production correlates to healing, while a Th2 response, with high levels of IL-4 and circulating antibody, is associated with non-healing (Heinzel et al., 1989; Scott, 1989; Jankovic et al., 2001). In susceptible B A L B / c mice infected with L. major, lymph node cells secrete high levels of IL-4, IL-5 and low levels of IFN-y; conversely, lymph node cells from resistant C3H/HeN mice secrete high levels of LFN-y and low levels of IL-4 and IL-5 (Scott, 1991). Lnterestingly, both resistant and susceptible mice mount a Th2 response during the first week of infection and produced high levels of IL-4 and IL-10 production. Susceptible B A L B / c mice express high levels of IL-4 and IL-10 throughout the infection, while resistant mouse strains subsequently lower IL-4 and IL-10 expression and induce IL-12 expression (Reiner et al., 1994). Resistance to Leishmania infection is largely determined by the ability of the host immune system to redirect the early induction of a Th2 response towards an IL-12 driven, T h l response with LFN-y dependent killing of intracellular amastigotes (Sacks et al., 2002). Over the past decade, Leishmania immunology research has utilized genetic approaches to define the role of various cytokines in generating a protective immune response.  1.8  Cytokine-mediated Modulation of the Immune Response  i)  IL-4 is widely believed to be the "master switch" for induction of a Th2-type immune  reponse. Knockout experiments have suggested, though, that there may be redundancy in this pathway. IL-4 knockout B A L B / c mice were expected to be resistant to L. major infection and mount an effective T h l response; however, there are conflicting reports on the effect an IL-4 knockout on B A L B / c susceptible mice to L. major infection (Mohrs et al., 1999; NobenTrauth et al., 1999). IL-4Rct knockout B A L B / c mice were resistant to the L. major infection 30  Chapter 1 - Introduction and mounted a protective T h l response (Noben-Trauth et al,  1999). Knockout and  transgenic expression of IL-13, an inhibitory cytokine that also utilizes the IL-4Roc chain, proved that IL-13 was an additional essential factor in determining B A L B / c susceptibility to L. major infection (Matthews et al, 2000). These results provided evidence that the combination of IL-4 and IL-13 was required for induction of a strong Th2 response. It was proposed that the immunodominant L A C K antigen stimulated a large number of L A C K reactive CD4+ Th2 cells in L. major infected B A L B / c mice and therefore played a critical role in determining susceptibility (Julia et al, 1996; Launois et al, 1997). More recent reports have undermined this hypothesis with data indicating that the intensity of the L A C K dependent response and the number of LACK-reactive CD4+ T cells was equivalent in both susceptible and resistant mouse strains (Julia et al, 1999; Stetson et al, 2002). ii)  The severity of human leishmaniasis strongly correlates with increased levels  of IL-10 (Ghalib et al, 1993; Karp et al, 1993). There is accumulating evidence that CD4+CD25+ regulatory T cells, rather than APCs, are the cellular source of IL-10 during in vivo infection. These cells are known to accumulate within the lesion and regulate the antiLeishmania inflammatory response in both susceptible and resistant mouse strains (Powrie et al, 1994; Belkaid et al, 2002). IL-10 abrogates the antigen presentation capacity of macrophages and dendritic cells, which is associated with suppression of M H C and co-stimulatory molecule expression (Chang et al, 1994; Koch et al, 1996). In addition, IL-10 stimulates humoral Th2 responses, while suppressing Thl-type cell-mediated immunity (Fiorentino et al, 1991; Mosmann et al, 1991; Chang et al, 1994). IL-10 has potent inhibitory effects for macrophages and plays an integral role in the susceptibility of B A L B / c mice to L. major infection. A dramatic increase in IL-10 production by Leishmania-mfected macrophages 31  has been correlated with  Chapter I - Introduction suppression of IL-12 and T N F - a secretion, in combination with inhibition of N O production (Gazzinelli et al, 1992; Kane et al, 2001). Furthermore, ligation of FcyRII by IgG-coated amastigotes induces IL-10 and suppresses IL-12 production (Kane et al, 2001). IL-10 knockout B A L B / c mice are resistant to L. major infection, and transgenic expression of IL10 in a resistant mouse strain or immunized B A L B / c mice abrogates resistance to L. major infection (Groux et al, 1999; Kane et al, 2001; Yamakami et al, 2002). iii)  TGF-p is an anti-inflammatory cytokine with similar effects to IL-10 on macrophage  activation; however, TGF-P also inhibits development of a Thl-type immune response (Ding et al, 1990; Bogdan et al, 1993; Gorelik et al, 2002). There is abundant evidence that activation of TGF-P contributes to susceptibility to Leishmania infection (Barral-Netto et al, 1992; Barral et al, 1993; Gantt et al, 2003). Higher levels of TGF-p, which correlate with lower levels of iNOS, have been detected in progressive lesions of infected B A L B / c mice relative to healing lesions in resistant C57BL/6 mice (Stenger et al, 1994). Furthermore, liver granuloma cells from L. chagasi-infected B A L B / c mice secrete large amounts of TGFP, which inhibits IFN-y production (Wilson et al, 1998). Treatment of Leishmania-mfected mice with anti-TGF-P antibody promotes a healing, T h l immune response (Li et al, 1999). These observations  suggest that Leishmania-induced TGF-P production by infected  macrophages is an important parasite pro-survival mechanism. iv)  IL-12 is a cytokine secreted by activated macrophages and dendritic cells, which is  required for CD4+ T h l cell differentiation, proliferation, and IFN-y production (Manetti et al, 1993). IL-12 production and signaling through the IL-12 receptor is critical for the initiation of a healing Thl-type immune response during Leishmania infection (Mattner et al, 1996; Stamm et al, 1999). It is a well established fact that Leishmania infection leads to  32  Chapter 1 - Introduction sustained suppression of IL-12 production by infected macrophages (Reiner et al, 1994; Carrera et al, 1996; Belkaid et al, 1998; Weinheber et al, 1998). Furthermore, macrophages isolated from lesions produced low levels of IL-12 (von Stebut et al, 1998). Recombinant IL-12 (rIL-12) treatment has been shown to cure L. major infected B A L B / c mice and promote a cell-mediated, Thl-type immune response with high levels of IFN-y and low IL-4 levels (Heinzel et al, 1993; Scott, 1993). IL-12 is required for activation of natural killer cells during the early stages of infection and to promote a Thl-type response in murine leishmaniasis (Scharton-Kersten et al, 1995). Resistant CH3 mice injected with rIL-4 or anti-IL-12 antibody at the time of infection mount an early Th2-type response, as expected, but maintain a resistant phenotype with resolution of the lesions (Chatelain et al, 1992; Hondowicz et al, 1997). IL-12 knockout C57BL/6 mice are susceptible to L. major infection and mount a Th2 response (Mattner et al, 1996).  1.9  Leishmania Genome & Gene Expression  i)  Leishmania Genome: The Leishmania genome contains approximately 33 M b of  D N A distributed across more than 36 chromosomes. The genomic D N A is not capable of chromatin condensation and, as such, it has not been possible to obtain a Giemsa stained karyotype for Leishmania. Pulse-field gel electrophoresis has been employed successfully to separate chromosomes and estimate chromosome number (Britto et al, 1998). There is a slight variation in the number of chromosomes between Leishmania species with L. major having 36 chromosomes, while L. donovani and L. mexicana have 35 and 34 chromosomes, respectively. Annotation of the completed genomes of L. major and L. infantum predicted approximately 8,200 genes (Ivens et al, 2005; Worthey et al, 2005). A high degree of synteny was detected when genome sequences of different Leishmania species were 33  Chapter 1 - Introduction compared. Interestingly, the L. major and L. donovani completed genomes differed by fewer than 20 genes. The genome of L. brazilensis, the causative agent of muco-cutaneous leishmaniasis, is currently being sequenced with 5x coverage at the Sanger Centre by a whole genome shotgun sequencing approach and is eagerly awaited. The vast majority of Leishmania genes do not contain cw-spliced introns, although complex post-transcriptional R N A processing mechanisms  exist in trypanosomatid  organisms. The average size of open reading frames (ORFs) in the L. major genome is 1.7 kb with intergenic regions averaging 1.4 kb in length. The average ORF length was comparable with observations in Trypanosoma, but the 600-700 bp intergenic sequence length in these organisms was shorter (El-Sayed et al, 2005). Analysis of the annotated genome also provided some interesting features in terms of the abundance and homology of Leishmania genes relative to other organisms that have been sequenced. 45% of the annotated L. major genes encoded hypothetical proteins of unknown function, but these genes were predicted in the genomes of other organisms suggesting that gene prediction was accurate. Furthermore, 29% of annotated genes were unique to trypanosomatid parasites (Leishmania and Trypanosoma), while 6 % were unique to Leishmania (El-Sayed et al, 2005). Comparison of genome organization across trypansomatids showed a large variation in chromosome numbers. L. major had 36 chromosomes, while T. brucei and T. cruzi had 11 and 55 chromosomes, respectively. Despite large differences in chromosome numbers, the genomes of Leishmania and Trypanosoma parasites share 70% identity with large blocks of synteny. The Leishmania genome is thought to represent an ancestral genome and a series of chromosome fusion events resulted in reduction of chromosome number in the T. brucei genome (Ghedin et al, 2004). Chromosome regions that had loss of synteny were often 34  Chapter 1 - Introduction associated repetitive D N A (rRNA gene clusters, multigene families, and retroelements) or strand switch regions of polycistronic transcription units (see section on gene expression). The trypanosomatid genomes did not undergo any major gene loss events and the core proteome is highly conserved with few novel domains or gene types. It has been observed that, in general, T. brucei proteins contained a higher abundance of signalling and protein-protein interaction domains than L. major, although the significance of this finding is unknown (El-Sayed et al., 2005). The genome of L. major Friedlin was aneuploid at chromosome 1 as shown by over representation of chromosome 1 B A C clones in a B A C library and higher abundance of this chromosome on C H E F pulse field agarose gels (Dr. Peter Myler, 2004 TriTryp Genome Meeting). Aneuploidy has been observed also in the highly polymorphic, T. cruzi C L Brenner strain. Upregulation of gene expression in a large region of L. major chromosome 30 has been observed in a drug resistant strain of L. major. This suggests that whole chromosome amplification may be a parasite strategy for mediating drug-resistance through elevated expression of genes involved in drug inactivation or efflux from cells (Dr. Marc Ouellette, 2004 TriTryp Genome Meeting).  ii)  Leishmania Gene Expression: The physical arrangement of genes on Leishmania  chromosomes correlated with the complex molecular events associated with mRNA processing proposed in many of the seminal publications on Leishmania gene regulation. The Leishmania chromosome 1 sequence was the first complete chromosome sequence published by the Leishmania genome sequencing consortium (Myler et al, 1999). The surprising result of sequence annotation was the tandem, head-to-tail arrangement of protein coding genes on this chromosome. The 29 protein coding genes most proximal to the left telomere were encoded on one chromosome strand, whereas the remaining 50 protein coding genes on this 35  Chapter 1 - Introduction  MylerPJ.PJV>i£ 1999  Figure 4.  Arrangement of Genes of L. major Chromosome 1.  36  Chapter I - Introduction chromosome were situated in a head-to-tail arrangement on the other chromosome strand (Figure 4). Completion of the L. major, T. brucei and T. cruzi genome sequencing projects corroborated this result as genes were tandemly arranged on a single chromosome strand with strand-switch events occurring at varying intervals along chromosomes. The strandswitch regions were also associated with synteny breaks and chromosome fusion events in the ancestral trypanosomatid genome (El-Sayed et al., 2005). Promoter elements for R N A polymerase II have not been identified upstream of any protein coding genes in Leishmania. The genome contains only a few of the basal transcription factors required for transcription initiation in higher eukaryotes. It has been proposed that entire chromosomes are transcribed into polycistronic R N A transcription units, which are subsequently trans-spliced to form mature, monocistronic m R N A (Clayton, 2002). Polycistronic gene expression is a well defined process for prokaryotic operons, but eukaryotic Leishmania transcription units display major differences. First, bacterial operons are used to transcribe functionally related genes induced in response to an environmental stress or stimulus. Leishmania genes encoded within a polycistronic transcription unit encompass genes involved in a wide variety of cellular processes. Second, bacterial operons are transcribed into a stable, multigenic R N A transcript that is subsequently translated into the various proteins. In contrast, the Leishmania polycistronic transcription unit is rapidly spliced and processed to produce individual m R N A transcripts. The polycistronic transcription unit is rapidly processed into mature, monocistronic m R N A transcripts (Figure 5). In the current model for gene expression, polycistronic R N A transcripts are trans-spliced to allow for the addition of a 39 nucleotide miniexon onto the 5' end of each mRNA. Trans-splicing occurs 100 to 400 nt upstream of the A T G start codon at an  AG  dinucleotide  immediately  downstream 37  of  a  polypyrimidine  tract  Chapter 1 - Introduction  H  P G K coding region  •  Insertion, |  ryi  PGKA  Extension, P G K C S p l i c e d leader with 5' cap 3'-untranslated regions  Clayton C E . E M B O J., 2002.  Figure 5.  Model for Polycistronic Transcription in Leishmania. 38  Chapter 1 - Introduction (LeBowitz et al, 1993; Matthews et al, 1994). Polyadenylation of each m R N A is coupled to the trans-splicing for the downstream transcript. The enzyme or enzyme complex that catalyzes polyadenylation of mRNA precursors has not been identified. However, it has been proposed that either an endonuclease cleaves the R N A at the poly(A) addition site or an exonuclease digests the mRNA in a 3'-5' direction with subsequent polyadenylation (LeBowitz et al, 1993). The A A T A A A sequence that is strongly associated with initiation of polyadenylation in many organisms was absent from the intergenic sequences in Leishmania. Experiments using a primer extension approach indicate that polyadenylation is initiated at a non-specific site approximately 200 bp upstream of the trans-splicing site. This observation suggests that a secondary R N A structure forms to facilitate binding of an endonuclease or termination of exonuclease activity.  iii)  Stage-specific Gene Expression in Leishmania: The promastigote and amastigote  stages of the Leishmania parasite lifecycle survive and replicate in vastly different environments: the midgut of the sandfly and the phagolysosome of mammalian macrophage cells, respectively. It was widely believed that parasite differentiation and survival in these different environments would be accompanied by a large change in gene expression (Handman, 1999). Genes that are preferentially expressed in the amastigote stage could be essential for parasite survival within the phagolysosome or modulation of the host immune response. Therefore, genes with an amastigote-specific expression profile have been sought after as potential drug targets or candidate vaccine components. The advent of genomic approaches to analyze global gene expression profiles has enabled researchers to quantify changes in gene expression accurately during the Leishmania lifecycle. Comparison of global gene and protein expression profiles from promastigotes and 39  Chapter 1 - Introduction amastigotes indicated that less than 3% of genes had greater than 2-fold changes in expression (Akopyants et al, 2004; Nugent et al, 2004). In Leishmania, it has been shown that stage-specific gene expression is regulated post-transcriptionally, often by sequence elements in the 3' untranslated region (UTR) that affect R N A stability or translation initiation (discussed further in Chapter 5).  1.10  The A600 Gene One of the primary research focuses has been to identify and characterize Leishmania  genes preferentially expressed in amastigotes. The rationale for this research focus was that large changes in global gene expression should be required to mediate the significant morphological and biochemical differences between promastigotes and amastigotes. A suppression-subtraction P C R (SS-PCR) approach was employed initially to identify genes preferentially expressed in the amastigote stage. Three amastigote-specific c D N A fragments (A600, A800, and A850) were isolated in an SS-PCR experiment using L. mexicana promastigote and axenic amastigote cDNA. The amastigote-specific A600, A800, and A850 c D N A fragments corresponded to 3'UTR sequences, as c D N A was produced from the 3' end of m R N A transcripts using an oligo-dT primer. The ORF for the gene corresponding to each c D N A fragment was determined to identify the corresponding differentially expressed gene. The A850 c D N A fragment corresponded to 3'UTR sequence of a B-tubulin gene. The A600 and A800 c D N A fragments were arranged contiguously in the 3'UTR of a novel gene, which was named A600. The fact that these two c D N A fragments, A600 and A800, were isolated from the same m R N A transcript provided convincing evidence that expression of A600 gene was upregulated in amastigotes and validated the ability of the SS-PCR approach to identify differentially expressed genes. The complete sequence for the proximal 2.3 kb of the A600 40  Chapter 1 - Introduction c D N A did not share sequence identity to known genes, although four putative ORFs were predicted. A small open ORF of 282 bp, which encoded a 10.6 kDa predicted protein, was selected on the basis of % G C content, Leishmania codon usage bias, and proximity to an upstream trans-splicing site. The predicted A600 protein also did not share identity with known protein, but a hydrophobic sequence in the N-terminus predicted that the A600 protein contained either a signal peptide for secretion or a transmembrane domain. This work was performed by a previous graduate student (Bellatin et al., 2002). Northern blot analysis determined that the A600 gene encoded a 3.3 kb mRNA transcript, which was 8-fold more abundant in axenic amastigotes than log phase or stationary phase promastigotes. The size of the m R N A transcript implied that the A600 gene consisted of a 105 bp 5'UTR sequence, a 282 bp ORF, and a 2,900 bp 3'UTR sequence. It was interesting to note that the A600 ORF encoded a small protein, while the mRNA transcript had an unusually large 3'UTR. Recent annotation of the L. major genome determined that the average 3'UTR sequence was 1.5 kb in length, so the A600 3'UTR was almost two-fold larger than the average Leishmania gene. The genomic arrangement of the A600 gene was analyzed by Southern blot analysis of genomic D N A digested with a panel of restriction enzymes. A single band was detected in each lane when the blot was hybridized with a probe for the A800 sequence of the A600 3'UTR, which suggested that a single copy of the A600 gene may be present in the L. mexicana genome. Multiple bands were observed in each lane when the blot was stripped and hybridized with a probe for the A600 coding sequence, although this probe only hybridized to one high molecular weight band in Xhol digested D N A . This result indicated that the L. mexicana genome contained multiple copies of the A600 gene and that these genes may be tandemly repeated on an Xhol restriction fragment. 41  Chapter 1 - Introduction 1.11  Thesis Objectives The overall objectives of this thesis were to elucidate a function for the A600 gene  and to determine the mechanisms that regulate stage-specific A600 gene expression. Although clues pertaining to the function of the A600 gene could not be ascertained on the basis of sequence homology, the amastigote-specific expression profile of this gene made it an exciting target for further study. Only a handful of Leishmania genes were known to be dramatically upregulated in the amastigote stage of the lifecycle and it had been hypothesized that these genes would play a critical role for amastigote differentiation and survival inside the host macrophage. Undoubtedly, the identification and characterization of genes that are essential for amastigote growth and survival will improve greatly our understanding of basic amastigote biology. These discoveries represent a potential as drug targets for the development of more effective and less toxic therapeutics for the treatment of leishmaniasis.  CHAPTER 3 - Characterization of the A600 Locus Multiple copies of the A600 coding sequence had been detected in the L. mexicana genome. A restriction fragment encoding the A600 genes was isolated from a genomic D N A library of Xhol restriction fragments. Restriction mapping experiments identified four A600 coding sequence in the genomic locus. The complete sequence and expression profile was determined for the A600-1 and A600-3.  CHAPTER 4 - Generation and Characterization of A600 Targeted Deletion Mutants Targeted deletion of the A600 locus was accomplished with two round of homologous recombination. The role of the A600 locus for amastigote differentiation, infectivity, and survival in macrophages was examined using the A600 knockout mutants. 42  Chapter 1 - Introduction CHAPTER 5 - Role of the A600-3 3'UTR for Stage-Specific Gene Expression In Leishmania, stage-specific gene expression is regulated by post-transcriptional mechanisms. The A600-3 3'UTR conferred stage-specific expression to the luciferase reporter gene. A putative regulatory element in the A600-3 3'UTR was identified using a PCR-based deletion strategy.  43  Chapter 2 - Methods and Materials 2.  Methods and Materials  2.1  Leishmania Culture Leishmania mexicana (WHO designation MNYC/M379) promastigotes were cultured  at 26°C in M199 medium pH 7.4 (9.8 g/L medium 199, 40 m M HEPES, 6 u M Hemin) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, USA) and I X Pen-Strep (100 U/mL Penicillin G, 100 jag/mL Streptomycin sulphate). Promastigote cultures were maintained at densities ranging from 1 x 10 to 3 x 10 parasites per m L of culture. 5  7  Axenic amastigote cultures were started by culturing log phase promastigotes at 32°C for 1 6 - 1 8 hours. Subsequently, one volume of UM54 medium pH 5.5 (9.8 g/L medium 199, 25 m M HEPES, 6 u M Hemin, 14 m M glucose, 5 mg/mL Trypticase Peptone, 5 m M L Glutamine) supplemented with 20% FBS and I X Pen-Strep. Amastigote cultures were diluted with UM54 culture media to maintain culture densities ranging from 3 x 10 to 5 x 7  10 parasites per mL of culture. 7  2.2  Bacterial Strains and Vectors DH5cc strain E. coli was used routinely as a host strain for maintaining and preparing  plasmid D N A for restriction mapping, subcloning, construction of a partial genomic library. E. coli cultures were started by inoculating a single bacterial colony into 3 m L of Lennox L broth base (86 m M NaCl, 10 mg/mL Peptone 140, 5 mg/mL Yeast extract) growth media (Invitrogen, Burlington, Canada). Bacterial cultures were incubated in a 37°C shaking incubator for 16 to 18 hours. The plasmids pBluescript KS(-) (Stratagene, L a Jolla, U S A ) , pLitmus28 (New England BioLabs (NEB), Beverly, USA), and pGem9z (Promega, Madison, USA) were used 44  Chapter 2 - Methods and Materials routinely for cloning and restriction mapping experiments. The luciferase coding sequence was purified from the pGL3 plasmid to construct luciferase reporter gene constructs (Promega). The pDrive cloning vector (Qiagen, Hilden, Germany) was used to clone P C R amplicons with overhanging 3' adenosine bases. The pGem-Hyg and pGem-Sat plasmids, which contained the Hygromycin B Phosphotransferase (hph) and S-Acetyl Transferase (sat) coding sequences, were constructed previously in the lab by Dr. Ben Kelly. The pLex-Neo plasmid was a Leishmania expression vector constructed in the lab (Joshi et al., 1995).  2.3  Nucleic Acid Isolation  2.3.1  Isolation of Leishmania Genomic DNA L. mexicana genomic D N A was isolated from promastigotes, as described previously  (Medina-Acosta et al, 1993). Briefly, l x l O to l x l O log phase promastigotes were washed 7  with phosphate-buffered  8  saline (PBS) pH 7.4 (137 m M NaCl, 2.7 m M KC1, 10 m M  N a H P 0 , 1.75 m M K H P 0 , ) to remove the FBS protein in complete M l 9 9 medium. Cells 2  4  2  4  were resuspended in 250 uL of lysis buffer (4% Triton X-100, 2.5 M L i C l , 50 m M Tris-HCl pH 8.0, 62.5 m M EDTA) and incubated at room temperature for 5 minutes. A n equal volume of 1:1 phenol/chloroform was added to the lysate and the solution was mixed on a rotating plate for 10 minutes. The phases were separated by centrifugation at 12,000x_r for 10 minutes at room temperature and the upper, aqueous phase was removed to an autoclaved 1.7 mL microfuge tube. Genomic D N A was precipitated by addition of 0.1 volumes of 3 M Sodium Acetate pH 5.2 and 2.5 volumes of 95 % ethanol. The D N A was sedimented by centrifugation at 12,000x_- for 10 minutes. The D N A pellet was washed once with 1 mL of 70% ethanol by gently inverting the micro fuge tube 6 to 8 times. D N A resuspended in 100 ul  45  Chapter 2 - Methods and Materials I X T E pH 8.0 (10 m M Tris-HCl, 1 m M EDTA) containing 0.1 ug/mL RNaseA, and incubated at 37°C for 30 minutes. The concentration and purity of genomic D N A was determined with an UltraSpec 300 spectrophotometer (Amersham, Uppsala, Sweden).  2.3.2  Isolation of Plasmid DNA Small scale isolation of plasmid D N A was performed routinely by the alkaline lysis  method with the QIAprep Spin Miniprep Kit (Qiagen). This method routinely yielded 20 to 30 |o.g of plasmid D N A from 3 mL overnight bacterial cultures. Larger scale plasmid isolations were performed with the High Speed Plasmid Maxi K i t (Qiagen), which was based on the alkaline lysis-based maxiprep method. Greater than 500 |ag of plasmid D N A was isolated routinely from 100 mL overnight cultures. The quality and concentration of plasmid D N A was measured as described for genomic D N A isolation.  2.3.3  Isolation of Leishmania RNA L. mexicana promastigote and amastigote total R N A was isolated using TRIzol  reagent (Invitrogen). Cells were washed with 1 mL PBS and resuspended with 750 uL of TRIzol reagent. Cell lysis was accomplished by incubating cells in TRIzol reagent for 10 minute at room temperature. R N A isolation was performed immediately or the R N A was stored stably in TRIzol reagent at -20°C. Phenol-chloroform extraction was performed by addition of 200 uL of chloroform, briefly vortexing the tube, and incubating the solution at room temperature for 10 minutes. The phases were separated by centrifugation at 12,000x5" for 10 minutes at room temperature. 300 uL of the upper, aqueous phase was transferred to an RNase-free, 1.7 m L microfuge tube. R N A was precipitated at room temperature for 20  46  Chapter 2 - Methods and Materials minutes, following addition of 187.5 uL of high salt solution (1.2 M NaCl, 0.8 M Tri-Sodium Citrate) and 187.5 uL isopropanol. R N A was sedimented by centrifugation at 12,000xg for 10 minutes. R N A was washed with 1 mL of 70% ethanol by gently inverting the tube 6 to 8 times and centrifuged at 7,500xg. The R N A pellet was air dried for 10 minutes and resuspended in 25 \LL of DEPC-treated H2O. DNasel-treatment of isolated R N A was performed routinely in a 20 uL reaction that contained I X DNasel buffer (20 m M Tris-HCl (pH 8.4), 50 m M KC1, 2 m M MgCl ), 2 units of DNasel enzyme, and 10 ug of total R N A . 2  The reaction proceeded at room temperature for 15 minutes and was terminated by addition E D T A to a 2.5 m M final concentration. The DNasel enzyme was heat inactivated at 65°C for 10 minutes.  2.4  Protein Isolation  2.4.1  Protein Isolation from Leishmania  Protein lysates were prepared from Leishmania log phase promastigotes or day 7 axenic amastigotes for Western blot analysis. 1x10 to l x l O cells were washed once with cold PBS s  9  and resuspended in 300 uL cold lysis buffer (1% (v/v) TritonX-100, 20 m M Tris-HCl pH 7.4, 150 m M NaCl, 20 u M 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 10 u M /rans-Epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64),  1.3  u,M N-[2S,3R]-3-  Amino-2-hydroxy-4-phenylbutyryl]-L-leucine hydrochloride (Bestatin), 10 n M Leupeptin, 3 n M aprotinin, and 50 m M E D T A . Cells were incubated, on ice, in lysis buffer for 20 minutes with periodic vortexing. Insoluble protein and cell debris was sedimented by centrifugation at 20,000 x g for 15 minutes. Soluble protein lysate was transferred to a pre-chilled 1.7 mL microfuge tube and the insoluble proteins were resuspended in I X sample buffer (50 m M  47  Chapter 2 - Methods and Materials Tris-HCl (pH 6.8), 2% SDS, 0.2% (w/v) bromophenol blue, 10% (v/v) glycerol, 1% (v/v) fresh P-mercaptoethanol).  2.5  Gel Electrophoresis  2.5.1  Non-Denaturing Agarose Gel Electrophoresis D N A fragments were separated by electrophoresis in non-denaturing agarose gels  containing 0.2 p-g/mL ethidium bromide. Agarose gels were prepared with 0.7 to 2.0% (w/v) agarose (Invitrogen) dissolved in 0.5X T B E buffer (45 m M Tris-HCl (pH 8.0), 45 m M Boric Acid, 1 m M EDTA). D N A fragments were visualized on a U V light apparatus and an image was captured using the Insta-Doc gel documentation system (BioRad, Hercules, USA). A digital image of the ethidium bromide-stained gel was printed using the P90W video copy processor (Mitsubishi Electronics, Singapore). D N A fragments were isolated by excision from an agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen).  2.5.2  Southern Blot Analysis D N A to be analyzed by Southern blot hybridization was restriction digested overnight  and separated on a non-denaturing 0.8% agarose gel containing 0.2 p.g/mL ethidium bromide. After electrophoresis, the gel was incubated in a denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 45 minutes, followed by a 30 minute incubation in a neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.0). Subsequently, the D N A was transferred overnight to Hybond-N " membrane (Amersham) by capillary transfer with 10X SSC transfer solution (1.5 1  M NaCl, 0.15 M Tri-Sodium Citrate). D N A was crosslinked to the membrane at 120,000 ujoules/cm with a U V Stratalinker apparatus (Stratagene). The membrane was incubated for 2  at least 1 hour in a 5 mL prehybridization solution (6X SSC, 5X Denhardt's solution (0.1% 48  Chapter 2 - Methods and Materials (w/v) Ficoll 400, 0.1% (w/v) Polyvinylpyrrolidone (PVP), 0.1% (w/v) Bovine Serum Albumin (BSA)), 0.5% (w/v) SDS, and 10 \xglvnL fragmented, denatured salmon sperm D N A ) at 65°C in a rotating hybridization tube. P -labelled probes were denatured by heating 32  to 100°C for 5 minutes, cooled on ice for 1 minute, and added directly to pre-hybridization solution. Southern blots were incubated with probe for at 16 to 18 hours at 65°C in a rotating hybridization tube. Subsequently, blots were subjected a series of washes at 65°C, which consisted of two 10 minute incubations in wash solution I (2X SSC, 0.1% SDS), one 15 minute incubation in wash solution II ( I X SSC, 0.1% SDS), and one 10 minute incubation in wash solution III (0.1X SSC, 0.1% SDS). Blots were exposed to a storage phosphor screen (Kodak, New Haven, USA), which was scanned using a Molecular Imager F X imaging device (BioRad). Relative band intensities were quantitated using Quantity One software (BioRad).  2.5.3  Northern Blot Analysis Denaturing agarose gels for Northern blots were prepared with 1% (w/v) agarose, I X  MOPS buffer (20 m M MOPS, 5 m M Sodium Acetate, 1 m M EDTA) and 6% (v/v) formaldehyde. 5 jj.g of R N A was prepared for gel electrophoresis with 50% (v/v) formamide, 6% (v/v) formaldehyde, 5 |ag/mL ethidium bromide solution. R N A was separated by gel electrophoresis in a denaturing agarose gel until desired separation was achieved. Postelectrophoresis, the gel was washed for 1 hour in DEPC-treated ddH20 before overnight transfer of R N A to Hybond-N* membrane (Amersham, Canada) by capillary transfer with DEPC-treated 20X SSC transfer solution (3 M NaCl, 0.3 M Tri-Sodium Citrate). R N A was cross-linked to the membrane with a U V Stratalinker apparatus (Stratagene). Northern blot hybridization with radiolabeled probe was performed overnight at 42°C in UltraHyb 49  Chapter 2 - Methods and Materials hybridization (Ambion, Austin, USA). Washing of the blot and detection were identical to the protocol described for Southern blot analysis.  2.5.4  Western Blot Analysis Proteins were separated on 10% SDS-PAGE or 16.5% SDS-PAGE gels at 150V  using the Protean minigel system (BioRad). Proteins were transferred to P V D F membrane at 150 m A for 2 hours using the wet transfer method (48 m M Tris-HCl, 39 m M Glycine, 0.0375% SDS, 20% Methanol). Following blotting, the membrane was stained with 0.5% ponceau red to confirm equal loading. The membrane was washed two times with TBS-T (0.05% Tween20 in TBS) for 5 minutes. The membrane was blocked in 5% skim milk/TBST at 4°C overnight. The next day the membrane was incubated with 1 |a,g/mL primary antibody in 2.5% skim milk/TBS-T for at least 2 hours. Subsequently, the membrane was washed three times with TBS-T. The membrane was incubated for 45 minutes with HRPconjugated secondary antibody at a 1:2000 dilution in 2.5% skim milk/TBS-T. The blot was washed three times with TBS-T. Protein bands were detected with E C L chemiluminescent substrate according to manufacturer's instructions (Amersham).  2.6  Generation and Purification of Polyclonal Antibody  2.6.1  Conjugation of anA600 Peptide to KLH Carrier Protein Antibody was raised against a synthetic A600 peptide (Research Genetics). The A600  peptide was conjugated to the K L H carrier protein via a C-terminal cysteine residue. 10 mg of A600 peptide was resuspended in 0.3 mL of 4 M Guanidine HC1. The peptide was reduced by addition of 7 mg of DTT and incubation at room temperature for 10 minutes. Reduced A600 peptide was purified from DTT using a column packed with Sephadex G10 resin. 50  Chapter 2 - Methods and Materials Briefly, the reduced peptide was passed through equilibriated Sephadex G10 resin by centrifugation at 1000 rpm for 3 minutes at room temperature. Subsequently, the A600 peptide was added to a 1 mL solution of 5 mg of K L H and 0.8 mg S M C C and incubated overnight. The A600 peptide is conjugated to the K L H carrier protein via S M C C .  2.6.2  Preparation of A600-peptide Conjugated Affinity Column The A600 peptide was conjugated to Sulfolink Coupling Gel (Pierce, Rockford,  USA).  Briefly, 0.5 mL of Sulfolink Coupling Gel was equilibriated with 20 mL of a  coupling buffer (50 m M Tris-HCl, 5 m M E D T A pH 8.5). The A600 peptide was resuspended at 10 mg/mL in coupling buffer and incubated with the Sulfolink Coupling Gel at room temperature for 45 minutes. The A600-conjugated column was washed with 3 column volumes of coupling buffer to remove excess A600 peptide. The column was blocked by incubation with 50mM cysteine in coupling solution for 45 minutes. The column was washed with 50 mL 1 M NaCl, followed by 50 mL of 0.05% sodium azide (NaN ) in TBS. 3  2.6.3  Affinity Purification of a-A600 Antibody Immune rabbit serum was collected in 15 mL Falcon tubes and allowed to clot at  room temperature for 30 minutes. The tube was centrifuged at 2,500xg for 10 minutes and the serum was removed to a new Falcon tube. 5 mL rabbit serum was diluted with an equal volume of TBS (0.15 M NaCl, 10 m M Tris-HCl, pH 7.4), followed by addition of 0.5 mL of 1 M Tris-HCl pH 8.0. The A600 peptide-conjugated column was washed with 25 mL of 100 m M Tris-HCl pH 8.0, then 25 mL of 10 m M Tris-HCl pH 8.0, and finally 10 mL of TBS. The diluted a-A600 rabbit serum was passed over the column with a flow rate of one drop per second. The column was washed successively with 50 mL of TBS, 25 mL of 100 m M 51  Chapter 2 - Methods and Materials Tris-HCl pH 8.0, and 50 m L of 10 m M Tris-HCl pH 8.0. ct-A600 antibody was eluted in 10 mL of elution buffer (0.05 M diethylamine, 0.15 M NaCl pH 11.5) and collected as ten 1 mL fractions. Eluate fractions were neutralized immediately with 0.5 mL of 1 M Tris-HCl pH 8.0. The column was neutralized with 5 mL of 1 M Tris-HCl pH 8.0 and washed with 50 mL of TBS. The column was stored at 4°C with 3 mL of 0.05% sodium azide in TBS. Fractions containing protein were identified by measuring the  A280  absorbance,  pooled, and dialyzed against PBS overnight at 4°C. The affinity purified a-A600 antibody was concentrated using a Centricon column with a 30 kDa molecular weight cut off (MWCO) membrane (Millipore, Bedford, USA). The concentration of the ct-A600 antibody was determined by measuring the A280  A280  absorbance, where IgG antibody at 1 mg/mL has an  absorbance value of 1.35.  2.7  Molecular Biology Techniques  2.7.1  Restriction Enzyme Digestion Restriction enzyme digestions were performed according to standard protocols  (Maniatis et al, 1989). A l l reactions were set up in the appropriate restriction enzyme buffer and contained 0.1 mg/mL Bovine Serum Albumin (BSA) and incubated at 37°C for the appropriate amount of time. Restriction digests for small amounts of D N A (less than 5 ug) were performed for at least 1 hour in 20 to 100 uL volumes, using between 5 and 10 units of enzyme. Restriction enzyme digestion of genomic D N A was conducted in volumes ranging from 50 to 500 oL and used at least 3 units of restriction endonuclease per jag of genomic D N A . These reactions proceeded at 37°C for 16 to 18 hours to ensure complete digestion of genomic D N A . Digestion of D N A with more than one restriction enzyme was performed concurrently, in the same tube, when a compatible restriction enzyme buffer was available. 52  Chapter 2 - Methods and Materials 2.7.2  DNA Ligation and Transformation ofBacteria D N A fragments used for subcloning were routinely gel purified. Ligation reactions  contained I X ligation buffer (50 m M Tris-HCl pH7.5, 10 m M M g C l , 1 m M ATP, 10 m M 2  DTT, 25 pg/mL BSA), 50 ng of linear vector, three-fold molar excess of insert D N A fragments, and 40 units/mL of T4 D N A Ligase (NEB).Efficiency of blunt end ligations was improved with 5% (w/v) PEG-8000. Ligations were performed routinely overnight at 16°C. The "heat shock" method was used to transform ligations or circular plasmid into CaCb-competent DH5oc strain E. coli. Briefly, 50 pL of competent DH5ct E. coli and 2 pL of ligation reaction were mixed in a microfuge tube, and incubated on ice for 30 minutes. The tube was heated at 42°C for 45 seconds, and immediately placed on ice for 2 minutes. Transformed bacteria were incubated at 37°C for 1 hour and streaked onto LB-Amp plates (LB media, 1.5% (w/v) Agar) containing the appropriate antibiotic selection. Electroporation was used to improve bacterial transformation efficiency of large plasmids and during construction of genomic D N A libraries. Electro-competent DH5a strain E. coli were mixed with circular plasmid or D N A from a ligation and transferred to a prechilled 0.1 cm electroporation cuvette (BioRad). Electroporation was conducted using a Gene Pulser apparatus with setting of 1.65 k V , 25 pF, and 200C2 (BioRad). Transformed bacteria were collected in 1 mL of L B growth medium and transferred to a 12 mL culture tube. The cells were incubated shaking at 37°C for 1 hour and streaked onto LB-Agar plates containing the appropriate antibiotic selection. Whenever possible, LB-Amp plates were pre-streaked with IPTG and Blue-Gal reagents to differentiate colonies containing recombinant products from those with nonrecombinant products on the basis of blue-white colour screening. Cells from blue colonies  53  Chapter 2 - Methods and Materials were transformed with non-recombinant plasmid, while the cells from white colonies contained recombinant plasmid.  2.7.3  Polymerase chain reaction PCR  reactions were performed using Applied Biosystems Gene Amp 2700  Thermocycler apparatus, with the following reaction conditions: I X Taq buffer, 0.2 m M of dNTP mix (dATP, dCTP, dGTP, and dTTP), 0.4 u M primer, and 2.5 units Taq D N A polymerase (Qiagen). Reactions were performed routinely with 30 cycles of denaturing for 30 s at 94°C, annealing for 30s, and extension at 72 °C. The optimal annealing temperature was determined for each primer pair. The extension time corresponded to 1 minute per 1 kbp length of the PCR product.  2.7.4  Labeling DNA Probes PCR products were radiolabeled routinely with [ P]-dCTP for 30 minutes at 37°C in 32  a reaction containing I X N E B Buffer 2 (10 m M Tris-HCl pH 7.9, 50 m M NaCl, 10 m M M g C l , 1 m M DTT), 5.4 O.D. N6 primer (NAPS Unit, UBC), 1 m M dATP, 1 m M dGTP, 1 2  m M dTTP, 50 uCi [a- P]-dCTP (Perkin Elmer, Wellesley, USA), and 5 U of the Klenow 32  fragment (3' - 5' exo') of D N A Polymerase I (NEB). P-labelled probes were column 32  purified from unincorporated nucleotides using the QIAquick P C R purification kit (Qiagen). The specific activity for 1 uL of probe, in counts per minute (cpm), was measured with a Beckman LS 5000 C E liquid scintillation counter (Beckman, Fullerton, USA). 2.7.5  Exonuclease(III) Deletions Approximately 5 ug of D N A template was mixed with I X N E B restriction enzyme  Buffer 1 (10 m M Bis Tris Propane-HCl, 10 m M M g C l , 1 m M DTT) in final volume of 25 2  54  Chapter 2 - Methods and Materials u.L and prewarmed to 37°C for 2 minutes prior to the addition of 2 p.L Exonuclease III (NEB). Deletion proceeded at 37°C with 3 uL aliquots removed every 50 s, and placed on dry ice for 5 minutes to stop the reaction. With addition of 3 uL dE^O to the aliquots, heat inactivation of Exonuclease(III) was carried out at 70°C for 10 minutes. To eliminate the overhanging single strand of D N A , I X Mung Bean nuclease buffer (50 m M sodium acetate pH 5.0, 30 m M NaCl, 1 m M ZnS04) and 1 unit Mung Bean nuclease (NEB) were mixed with each aliquot and incubated at 30°C for 30 minutes. Each sample was subjected to phenol extraction followed by ethanol precipitation. The ends of the double-stranded D N A were filled by using T4 D N A polymerase (NEB). Briefly, the D N A was diluted in a solution with I X T4 polymerase buffer (50 m M NaCl, 10 m M Tris-HCl, 10 m M M g C l , 1 m M DTT), 50 2  pg/mL B S A , 100 u M dNTP and incubated for 20 minutes at 12°C. E D T A stop buffer was added until a final concentration of 10 m M was reached. T4 D N A polymerase was heat inactivated at 75°C for 20 minutes.  2.7.6  DNA Sequencing D N A sequencing reactions were performed with Big Dye Terminator version 2.0 (PE  Biosystem). Reactions were prepared with 200 ng of D N A template, 5 pmole M13R20 primer, and 2 u.1 B i g Dye ready mix in 20 pX final volume. The cycling condition was as follows: 96°C for 30 s, 52°C for 15 s, and 60°C for 4 minutes. D N A was ethanol precipitated and resuspended in 20 pX HiDi Formamide. Samples were heat denatured at 94 °C for 2 minutes. The D N A sequence was determined by capillary electrophoresis using the A B I 310 Genetic Analyser. Sequence analysis and contig alignments were performed with Sequencher software (Gene Codes Corp., Ann Arbor, USA).  55  Chapter 2 - Methods and Materials 2.7.7  Leishmania Transfection and Cloning Logarithmic phase L. mexicana promastigotes were centrifuged at 500xg for 10  minutes, washed twice in 10 mL electroporation buffer (137 m M NaCl, 20 m M HEPES pH 7.4, 6 m M Glucose, 5 m M KC1, 0.7 m M NaH2P04,) and resuspended in electroporation buffer at 1x10 cells/mL. 4 x l 0 cells were transfected with 10 }4.g of D N A by electroporation s  7  with the Genepulser apparatus using 0.45 k V and 500 uF settings (BioRad). Electroporated Leishmania cells were collected in 10 mL of drug-free M l 9 9 culture media and incubated at 26°C. After 24 hours, the transfected Leishmania promastigotes were centrifuged at 500xg for 10 minutes. The cells were resuspended in a 300 uL volume and streaked onto M l 99Agar plates (Ml99 culture media, 1% (w/v) Agar) containing appropriate drug selection. Promastigotes transfected with constructs for chromosomal integration were streaked onto two selection plates with 150 uL of cell suspension. Due to the high efficiency of plasmid transfection, promastigotes transfected with plasmid D N A were streaked onto two selection plates using 30 ul and 270 ul volumes, respectively. The plates were incubated at 26°C and independent, drug-resistant clones were visible after 7 to 10 days. Clones were transferred to independent wells of a 24 well plate, which contained 1 m L of drug-free M l 9 9 media supplemented with 20% FBS and I X Pen-Strep, and cultured at 26°C. After 3 to 4 days, the 1 mL cultures were expanded to a 5 mL volume of M l 9 9 culture media with drug selection. Cell stocks of drug-resistant clones were made in M l 9 9 culture media containing 15% (v/v) glycerol.  56  Chapter 2 - Methods and Materials 2.8  Generation of Constructs for Transfection  2.8.1  Targeted Deletion Constructs Two targeting constructs that contained different selection markers were generated in  the pGem9z plasmid cloning vector. The positive selection markers used for the targeting constructs were the hygromycin B phosphotransferase (hph) and the S-acetyl transferase (sat) genes. The hph and sat genes had been cloned previously into the pGem9z plasmid as Spel/Hindlll restriction fragments. The intergenic region of an L. major ct-tubulin gene was subcloned downstream of the hph and sat genes as a Hindlll/Xbal restriction fragment. The resulting plasmids were pGem/Hyg-Tub and pGem/Sat-Tub. The D N A sequences immediately upstream of the A600-1 ORF and downstream of the A600-3 ORF were introduced into the targeting vectors. The upstream sequence, referred to as 5'A600, was isolated as a 0.45 kbp P C R product using oligonucleotide primers with artificial 5' Spel (forward primer) and Xbal (reverse primer) restriction sites. A 1 kbp sequence from the A600-3 3'UTR, designated 3'A600, was amplified using primers with artificial 5' Xbal (forward primer) and Kpnl (reverse primer) sites. The 5'A600 and 3'A600 P C R amplicons were initially cloned into the pDrive T/A cloning vector (Qiagen). The 5'A600 fragment was subcloned, as a Spel/Xbal fragment, into the Spel site at the 5' end of the hph and sat markers in each targeting vector. The resulting plasmids were named p5'Hyg-Tub and p5'Sat-Tub. Spel- and .Y&ai-digested D N A have compatible ends for ligation, which prevented directional cloning of the 5'A600 fragment. However, ligation of Spel and Xbal ends produced a new D N A sequence that is not recognized by either restriction enzyme. Therefore, correct orientation of the 5'A600 sequence in the p5'Hyg-Tub and p5'Sat-Tub plasmids was determined with a Spel/Hindlll restriction digest. The targeting constructs were completed by  57  Chapter 2 - Methods and Materials directionally subcloning the 3'A600 sequence into the Xbal and Kpnl sites downstream of the a-tubulin intergenic sequence.  2.8.2  Luciferase Reporter Constructs The pLitmus28 vector initially was digested with BgUI and BamHI restriction  enzymes to remove a 60 bp sequence between Spel and Ncol sites in the multiple cloning region. The pLit28' plasmid was created by self-ligation of this plasmid as BgUI and BamHI digestion produced compatible ends for ligation. The luciferase open reading frame (ORF) was subcloned from the pGL2 luciferase plasmid (Promega) into the pLit28' vector, as an NcoIIXbal fragment. Subsequently, the pHyg/Luc plasmid was created by cloning a 2.3 kbp Spel/Xbal restriction fragment of 5'A600-/j/?/j-atubLR into a Spel site immediately upstream of the luciferase ORF. This 5'A600-/z/?/z-cctub insert contained a 450 bp sequence from upstream of the A600-1 gene, the hygromycin phosphotransferase (hph) gene for positive selection, and the a-tubulin intergenic region for constitutive expression of the hph selection marker. This vector was designed to allow for cloning various A600-3 3'UTR and intergenic region (3'UTR+LR) sequences, full length or truncated versions, into the Xbal and Kpnl sites located downstream of the luciferase reporter gene.  2.9  Macrophage Techniques  2.9.1  Peritoneal Macrophage Isolation Macrophages were isolated from 6 to 8 week old B A L B / c mice by peritoneal lavage.  A small incision was made in the skin above the diaphragm and extended in the posterior direction along the ventral midline. This procedure left the peritoneal membrane intact and exposed. 3 mL of HBSS supplemented with I X Pen-Strep was injected into the peritoneal 58  Chapter 2 - Methods and Materials cavity with a 26 guage needle. The cavity was gently massaged to mix the solution. A 1 to 2 mm incision was made in the peritoneal membrane approximately 1 cm posterior to the diaphragm. Approximately 2.5 mL of solution was withdrawn with a 1 m L serological pipette and transferred to a sterile 15 mL Falcon tube. The peritoneal cavity was washed two additional times and the lavages were pooled. The cells from the lavage were washed once with 10 m L HBSS containing I X Pen-Strep and resuspended at 2 x 10 cells/mL in complete 5  RPMI medium.  2.9.2  Infection of Peritoneal Macrophages with Leishmania Peritoneal macrophage infections were conducted in flat bottomed 24 well plates.  Sterile 12 mm circular coverslips were placed into each well. 2 x 10 peritoneal macrophages 5  were seeded into each well in a 1 mL volume of complete R P M I medium and allowed to adhere to the coverslips overnight at 34°C in 5% CO2. The next day non-adherent cells were removed by washing the wells with HBSS. Day 4 stationary phase Leishmania parasites were washed once with HBSS and resuspended at 6 x 10 cells/mL in pre-warmed, complete R P M I media. 2 x 10 6  6  promastigotes were seeded into each well, providing a multiplicity of infection (MOI) of 10 parasites per macrophage. After a 4 hour infection period, coverslips were withdrawn from the well and dipped 4 to 5 times in HBSS to removed Leishmania that had not adhered to macrophages. Coverslips with Leishmania-infected macrophages were transferred to fresh wells with 1 mL of complete RPMI media. Infected macrophages were cultured in an incubator at 34°C in 5% CO2 and the media was replaced after 72 hours. Macrophages were fixed by incubation in 4% formaldehyde/PBS at room temperature for 10 minutes and subsequently washed three times with PBS. Intracellular amastigotes were stained with 59  Chapter 2 - Methods and Materials 66uM Hoescht 33342 stain for one minute. The coverslips were washed three times with PBS and mounted onto glass microscope slides with Vectashield mounting medium (Vector Laboratories, Burlingame, USA). The slides were viewed under U V light with an upright fluorescent microscope (Zeiss, Oberkochen, Germany). Images were collected from the microscope with Digital Focus software.  2.9.3  BALB/c Mouse Infections with Leishmania 6 to 8 week old B A L B / c mice were housed in the animal facility at the Jack Bell  Research Centre and maintained according to the standard University of British Columbia (UBC) Animal Care practices. Mouse infections using Leishmania were conducted according to U B C Animal Care protocol number A02-0207. Briefly, day 4 stationary phase Leishmania promastigotes were washed twice with PBS and resuspended at 1 x 10 cells/mL in PBS. 7  Groups of five mice (n=5) were injected subcutaneously in the right flank with a 0.1 mL cell suspension (1 x 10 stationary phase promastigotes) using a tuberculin syringe with a 27 G 6  needle. Lesion progression was measured weekly with calipers. Mice were euthanized in a CO2 chamber when lesions were greater than 10 mm in diameter.  2.9.4  Isolation of Lesion Amastigotes Mice were euthanized with CO2 and sterilized with 70% ethanol. The fibrotic lesions  that formed on the flank (site of infection) were excised and transferred to a sterile 10 cm culture dish (Falcon) with 3 mL HBSS containing I X Pen-Strep. Excess hair was trimmed from the lesion. Parasites were extracted from the lesion by sheering the tissue into a single cell suspension. Briefly, the lesion was cut into smaller pieces with scissors and subsequently into a slurry using a razor blade. The tissue was forced through a fine mesh sieve using the 60  Chapter 2 — Methods and Materials plunger of a 3 mL syringe. The mesh was washed with 5 mL of HBSS to collect residual cells. The cell suspension was passed through a 22 G needle ten times to further solubilize the lesion tissue into a single cell suspension. Macrophages were lysed, releasing intact lesion amastigotes, by passaging the cell suspension through a 26 G needle ten times. The resulting solution was centrifuged at 100x_r for 2 minutes to collect tissue debris and intact mouse cells. This step was repeated and the supernatant, which contained amastigotes released from macrophages, was centrifuged at lOOOxg for 10 minutes to pellet amastigote cells. Leishmania parasites purified from mouse lesions were used to prepare total R N A or differentiated to promastigotes by culturing the cells in M l 9 9 medium at 26°C.  61  Chapter 3 - Results 3.  Characterization of the A600 locus The A600-3 gene had been cloned previously using a suppressive-substraction PCR  (SS-PCR) approach to identify genes preferentially expressed in the amastigote stage of the L. mexicana lifecycle. This chapter describes cloning an Xhol restriction fragment of genomic D N A that contained the tandemly repeated A600 coding sequences. The remainder of this chapter describes restriction mapping, sequencing, and characterization of the individual A600 genes contained within the genomic locus. A comparative analysis was performed for two divergent genes, A600-1 and A600-3, within the A600 locus.  3.1  Results  3.1.1  Genomic Arrangement of Related A600 Genes The arrangement of A600 genes in the L. mexicana genome was examined by  Southern blot analysis of genomic D N A digested with a panel of restriction enzymes. A random labelled probe for the A600-3 coding sequence hybridized to a single band in Xhol digested D N A , while multiple bands were detected when the D N A had been digested with other restriction enzymes (Figure 6). Additional experiments had shown that a single band in Xmal digested D N A also hybridized to a probe for the A600-3 coding sequence. The Xhol and Xmal restriction fragments that hybridized to the A600-3 probe were larger than the 12 kbp marker, which indicated that these restriction fragments contained all of the A600 coding sequences. These results provided evidence for additional A600 genes in the L. mexicana genome and that these genes were tandemly arranged at a single genomic locus. Southern blot experiments were performed by double restriction digestion of L. mexicana genomic D N A with Xhol and a panel of additional restriction enzymes (Figure 7). The aim of this experiment was to identify a smaller restriction fragment that contained the 62  Chapter 3 - Results  1  Figure 6.  2  3  4  5  6  7  Southern blot analysis of the L. mexicana A600 genes.  Southern blot analysis of L. mexicana genomic D N A digested overnight with (1) HindHI, (2) PvuII, (3) Xhol, (4) Sail, (5) BamHI, (6) Sad, or (7) PstI restriction enzyme. The blot was hybridized with a probe for the A600-3 coding sequence. 1 kb molecular weight marker (1 kb) sizes are displayed on the left side of the blot.  63  Chapter 3 - Results  entire A600 locus. As previously observed, a probe for the A600-3 coding sequence hybridized to a single, high molecular weight band in D N A digested with either Xhol or Xmal restriction enzyme; however, this probe clearly hybridized to smaller restriction fragment in Xhol digested D N A (Figure 7, lanes 1 and 2). When D N A was double digested with Xhol and Xmal, Alfl, BspDl, Dral, Ndel, or Xbal enzyme, the A600-3 coding sequence probe hybridized to a band that was equivalent in size to the Xhol band (Figure 7, lanes 3, 4, 5, 6, 10, and 11). It was concluded that these restriction sites probably do not exist between the Xhol sites that flank the A600 locus. Multiple bands hybridized to the A600-3 coding sequence probe when genomic D N A was double digested with Xhol and EcoRI, Fspl, or Ncol (Figure 7, lanes 7 - 9). This result indicated that these restriction sites exist more than once within the A600 locus.  3.1.2  Cloning the A600 Locus It was necessary to clone a restriction fragment that contained the entire A600 locus,  so that detailed restriction mapping and characterization of the locus could be performed. The pBluescript K S plasmid was digested to completion with Xhol restriction enzyme to generate a linear cloning vector. The ATioZ-digested pBluescript plasmid was dephosphorylated with Calf Intestinal Phosphatase (CIP) to prevent self-ligation of vector. Test ligations with this vector showed that there was minimal self-ligation and re-phosphorylation of the vector with polynucleotide kinase (PNK) restored the ability of the vector to self-ligate. This pBluescript vector was used for the construction of an L. mexicana genomic D N A library of high molecular weight Xhol restriction fragments. Successful cloning of an Xhol restriction fragment containing the A600 genomic  64  Chapter 3 - Results  1  Figure 7.  2  3  4  5  6 Std  7  8  9  10  11  12  Southern blot analysis to restriction map the L. mexicana A600 locus.  Southern blot analysis of L. mexicana genomic D N A digested overnight with (I) Xhol alone, (2) Xmal alone, or Xhol in combination with (3) Xmal, (4) Alfl, (5) BspDI, (6) Dral, (7) EcoRI, (8) F ^ 7 , (9) Tvco/, (10) Ndel, (11) ^7ja7, (12) Xmnl. The blot was hybridized with a probe for the A600-3 coding sequence. 1 kb molecular weight marker (Std) sizes are displayed on the left side of the blot.  65  Chapter 3 - Results locus proved to be a challenge. L. mexicana genomic D N A was digested with Xhol restriction enzyme and various methods were used to isolate restriction fragments larger than 12 kbp. Initial attempts to recover high concentrations of high molecular weight Xhol restriction fragments were unsuccessful. This was largely attributed to the requirement of extracting D N A from an agarose gel by dissolving the agarose gel slice or by electroelution of D N A from the gel slice. To overcome this limitation, 600 pg of .YfcoT-digested genomic D N A was separated by sucrose gradient centrifugation. A hole was bored in the bottom of each tube and the gradient was recovered in 0.25 mL fractions. A small volume of each fraction was assayed on an analytical agarose gel to confirm that the restriction fragments had been separated adequately on the basis of molecular weight. A Southern blot of the D N A from fractions 7 - 1 4 , which contained high molecular weight restriction fragments, was hybridized with a probe for the A600-3 coding sequence. D N A from fractions 11 - 13 of the sucrose gradient hybridized strongly to the probe, which indicated that the A600 locus was enriched in these fractions (Figure 8). D N A from fractions 1 1 - 1 3 was pooled together, ethanol precipitated, and resuspended in a small volume of TE. A library of the A600enriched Xhol restriction fragments was ligated into the dephosphorylated, pBluescript vector mentioned above. This partial genomic library was transformed into electro-competent D H 5 a strain E. coli and streaked at high density onto LB-Amp plates (LB agar plates with 100 ug/mL Ampicillin selection). A colony screen was employed to identify bacterial colonies that contained the A600 locus as an insert in the pBluescript plasmid. The bacterial colonies were transferred onto Hybond membrane by laying the membrane on top of the colonies. The orientation of the membrane on the plate was recorded such that ^o'OO-positive colonies could be isolated from the plate after screening. Bacteria were lysed on the membrane, which facilitated binding of 66  Chapter 3 - Results bacterial D N A to the membrane. Twenty-one ^dOO-positive colonies were identified by autoradiography after hybridizing the membranes with a radiolabeled probe for the A600 coding sequence. These colonies were transferred from the LB/Amp plates into 100 pi L B Amp in a 96 well plate and subjected to a PCR-based colony screen. The 282 bp A600-3 coding sequence was amplified from eleven of the twenty-one colonies identified by the primary colony screen. The /4<500-positive clones were re-streaked at a lower density onto LB-Amp plates for a second colony screen to ensure that individual colonies could be selected. Four of the eleven ^AO-positive colonies produced a strong signal after the colony blot membrane was hybridized with the probe for the A600-3 coding sequence. These A600positive clones were from the E6, E8, G2, and G6 wells of a 96 well plate. Plasmid could be isolated from five independent colonies of the E8, G2, and G6 clones Plasmid isolated from bacterial colonies on the E8, G2, and G6 plates were named pBST-E8.1-5, pBST-G2.1-5, and pBST-G6.1-5. A n Xhol restriction digest of the pBST-E8, G2, and -G6 plasmids produced the expected 2.9 kbp plasmid band and an approximately 14 kbp genomic D N A insert in each sample (Figure 9). Southern blot analysis was performed on L. mexicana genomic D N A and pBST-E8 plasmid D N A to confirm that the entire genomic A600 locus had been cloned (Figure 10). D N A was digested with Xhol restriction enzyme alone and Xhol in combination with PvuII or PstI enzyme. A direct comparison was performed for the number and molecular weight of bands that hybridized to a probe for the A600-3 coding sequence. The results from this experiment demonstrated that the A600-3 probe hybridized to D N A bands of identical molecular weight in the plasmid and L. mexicana genomic D N A . Furthermore, the relative intensities of the bands that hybridized to the A600-3 probe in XhoI/PvuII and XhoI/PstI digested samples were consistent in both the  67  Chapter 3 - Results  a  b Sucrose fraction 7  Figure 8.  8 9 10 1 1 1 2 13 14  Sucrose fraction 7  8  9  10 11 12 13 14  Enrichment for Xhol restriction fragments that encode the A 600 genes.  Two identical sucrose gradients (tubes 1 and 2) were used to separate 600 pg of Xholdigested L. mexicana genomic DNA. A Southern blot of D N A from fractions 7-14 from the gradient, which contained high molecular weight Xhol restriction fragments, was analyzed by hybridization with a probe for the A600-3 coding sequence. Enrichment of the A600 genes in fractions 1 1 - 1 3 was clearly shown by comparison of the result from (a) the Southern blot and (b) the agarose gel prior to Southern blot transfer.  68  Chapter 3 - Results  Figure 9.  The pBluescript-E8 and -G2 plasmids contained large genomic D N A inserts.  (a) Plasmid map of the pBST-E8 plasmid. The A600 locus was cloned into the pBluescript K S plasmid as an Xhol restriction fragment. The A600 coding sequences are represented by red arrows, (b) Plasmid isolated from five colonies of pBST-E8 (lanes 1-5) and pBST-G2 (lanes 7-11) was digested for 2 hours with Xhol enzyme prior to agarose gel electrophoresis. The pBluescript K S vector was 2.9 kb and the genomic D N A insert was larger than the 12 kb marker. Undigested pBluescript-E8 and - G 2 plasmid was loaded as a control (lane 6,12). 1 kb molecular weight marker (Std) sizes are displayed on the left side of the agarose gel.  69  Chapter 3 - Results  Figure 10.  Comparison of L. mexicana genomic D N A and the pBST-E8 plasmid D N A .  Genomic D N A (lanes 1,3,5) and pBST-E8 plasmid D N A (lanes 2,4,6) digested overnight with Xhol alone (lanes 1,2), Xhol + PstI (lanes 3,4), or Xhol + PvuII (lanes 5,6) was analyzed by Southern blot hybridization with a probe for the A600-3 coding sequence. 1 kb molecular weight marker (Std) sizes are displayed on the left side of the blot  70  Chapter 3 - Results plasmid and genomic D N A samples. Taken together, these results provided further evidence that the all of the coding sequences from L. mexicana A600 locus had been cloned into the pBluescript plasmid.  3.1.3  Restriction Map of the A600 Locus The pBST-E8, -G2, and -G6 plasmids were digested with different combinations of  the Xhol, EcoRI, and Ncol restriction enzymes. XhoI/EcoRl digestion of these plasmids produced three 4.3 kbp bands and single 1.7 kbp and 2.9 kbp bands. A n Xhol/Ncol restrictiondigest produced two 4.3 kbp bands and single bands at 3.7 kbp, 2.9 kbp, and 2.3 kbp. Identical restriction product profiles were produced by each of these plasmids. The 2.9 kbp band observed in both digests was pBluescript K S plasmid that had been digested from the insert at the Xhol cloning sites. The EcoRI/Ncol restriction product profile of the pBSTE8 plasmid (three 3.7 kbp bands and single 4.3 kbp and 0.6 kbp bands) differed from the pBST-G2 and -G6 plasmids (two 3.7 kbp bands and single bands at 6.8 kbp, 1.8 kbp, and 0.6 kbp) (Figure 11). A more extensive list of restriction sites within the A600 locus was determined by digestion of the pBST-E8 plasmid with the Xhol enzyme alone or in combination with a second restriction enzyme. This experiment demonstrated that the A600 locus contained multiple sites for the following restriction enzymes: Hindi, Hindlll, BamHI, PstI, EagI, Apal, and Sail. The different  restriction profiles produced by EcoRI/Ncol digestion of the  pBluescript-E8, -G2, and - G 6 plasmids was not surprising, given that it was possible for the A600 locus to ligate into the pBluescript vector in two different orientations. The A T G start codon for the A600-3 ORF was only 1.3 kbp upstream from the Xhol restriction site at position 1425 bp of the A600-3 cDNA. Using this knowledge, a PCR-based assay was 71  Chapter 3 - Results  Xhol + EcoR1 E8 Size  (kb)  12  Xhol + Nco E8  G6  G2  3 4 12  3 4 12  3 4  Size 1 2  (kb)  3  G2 4  G6  5 6 7 8 9101112  12-1  5H  «m* » * M i .<*» <%» «£ « a (pp  **  4•V  4*  «t* »»» * « " • » ' *# M  ..• <''-/"»«' * * «*•• * -  W  * * *«• fc*.  2— 1.6—  1 —  EcoRI  Ncol  G2 G6 1 2 1 2 12  E8 G2 G6 12 1 2 1 2  EcoRI + Ncol E8 Size  (kb)  12  3 4 12  G2  E8  G6  3 4 12  3 4  Size  12 —  Figure 11.  Restriction Digests of the pJ3ST-E8, -G2, and - G 6 Plasmids.  pBST-E8, -G2, and -G6 independent plasmids (1-4) were digested with (a) Xhol + EcoRI, (b) Xhol + Ncol, (c) EcoRI + Ncol and (d) EcoRI or Ncol alone. Restriction fragments were separated on a 0.8% agarose gel. 1 kb molecular weight marker (Std) sizes are displayed on the left side of the agarose gels. 72  Chapter 3 - Results employed to determine the orientation of the A600 locus in the pBST-E8 and -G2 plasmids. PCR reactions were performed with a vector-specific primer, M13F27 or M13R20, and the A600-Fwd primer, which annealed at the 5' end of the A600-3 ORF. The A600Fwd/M13R20 primer pair specifically amplified the expected 1.3 kbp P C R product from the pBST-E8 plasmid, while only the A600-Fwd/M13F27 primer pair amplified this P C R product from pBST-G2 plasmid. This result clearly demonstrated that identical genomic D N A fragments in the pBST-E8 and-G2 plasmids had ligated into the pBluescript vector in opposing orientations. The restriction mapping experiments indicated that the cloned Xhol restriction fragment, which contained the A600 genes, was 1 4 - 1 5 kbp in length. A restriction map of EcoRI and Ncol sites was constructed from these data and, based on this map, the A600 locus appeared to have a repetitive arrangement of EcoRI and Ncol restriction sites (Figure 12a). The A600 coding sequences were named according to their relative position in the locus. The A600-1 gene is positioned at the 5' end of the locus, upstream of the other A600 genes. Two A600 genes were predicted in the middle of the locus, which are referred to A600-2.1 and A600-2.2. The A600-3 gene corresponds to the A600 cDNA, which contained an Xhol site in the 3'UTR sequence. Therefore, the A600-3 coding sequence is the generic "A600 coding sequence" that was used to probe the Southern blots in Figures 1 and 2. Subsequent sections in this chapter will describe efforts to determine the complete sequence for each of the A600 genes. In order to accomplish targeted deletion of the A600 locus via homologous recombination, it was important to obtain the D N A sequence immediately upstream of the A600-1 coding sequence. A 3.7 kbp Xhol/Ncol restriction fragment from the 5'end of the pBST-E8  plasmid  was  subcloned  into 73  a  pLitmus28  vector  to  generate  the  Chapter 3 - Results  ^ A^O-1  A600-2.1  m  I  A6^-2.2 H  \  Distance (kbp)  Figure 12.  10  A600-3  15  Restriction Map of the A600 Locus.  (a) Restriction map of Ncol and EcoRI sites in the cloned A600 locus. The A600 ORFs are shown as red boxes. Distances (kb) are shown below the map. (b) The pLit-5'E8 plasmid was constructed from an Xhol/Ncol restriction fragment that contained the proximal 3.7 kb of the A600 locus. The A600-1 ORF is shown as a red arrow, (c) The pLit-midE8 plasmid was constructed from a mixture 4.3 kb Ncol restriction fragments, which map to the middle of the A600 locus. These Ncol restriction fragments contain the A600-2.1 and A600-2.2 ORFs shown as red arrows.  74  Chapter 3 - Results pLit-5'E8 plasmid (Figure 12b). A series of restriction enzyme digests were performed to create a restriction map for this region of the A600 locus and a PstI restriction site mapped to 1.5 kbp downstream of the Xhol cloning site. A Southern blot of pLit-5'E8 plasmid D N A digested with Xhol and PstI restriction enzymes was hybridized with a probe for the A600-3 coding sequence to determine the position of the A600-1 gene relative to the Xhol cloning site. The A600-3 ORF probe hybridized to the 1.5 kbp XhoI/PstI restriction fragment from the 5' end of the A600 locus. This XhoI/PstI restriction fragment was subcloned into a pBluescript vector and the complete D N A sequence was determined by end sequencing from the M13F27 and M13R20 primer sites. Sequence analysis showed that the A T G start codon for the A600-1 ORF was only 450 bp downstream from the Xhol site used to clone the A600 locus from L. mexicana genomic D N A . A restriction map for the A600 locus also indicated that three Ncol sites were arranged at 4.3 kbp intervals in the middle of the locus (Figure 12a). The pBST-E8 plasmid was digested with Ncol enzyme and produced bands at 7 kbp, 4.3 kbp, and 2.3 kbp. The 4.3 kbp band, which corresponded to two Ncol restriction products from the central 8.6 kbp of the A600 locus, was excised from an agarose gel and the D N A extracted from the gel slice. Subsequently, these 4.3 kbp Ncol restriction fragments were cloned into the pLitmus28 plasmid to generate pLit-midE8 (Figure 12c). As the 4.3 kbp Ncol band isolated from pBSTE8 contained a mixture of two restriction fragments, a PstI D N A fingerprint was performed on the pLit-midE8 plasmid isolated from twenty white colonies. Interestingly, all twenty plasmids produced identical PstI restriction product profiles, which suggested that the two 4.3 kbp Ncol restriction fragments had very similar sequences. This result provided evidence that the tandem arrangement of A600 genes may have resulted from recent gene duplication of the A600 locus. 75  Chapter 3 - Results One of the primary objectives of this thesis was to characterize the mechanism(s) that confer preferential expression to the A600-3 gene in amastigotes. It had been hypothesized that differential gene expression in Leishmania was mediated by cis-regulatory elements in the 3'UTR sequence of m R N A transcripts. However, sequence was available only for the proximal 2.3 kb of the 3.3 kb A600-3 cDNA, so it was important that complete sequence was obtained for the A600-3 3'UTR and downstream intergenic region (A600-3 3'UTR+IRj. The sequence downstream of the A600-3 gene was examined by Southern blot analysis of L. mexicana genomic D N A digested with the restriction enzymes PvuII, PstI, and Ncol alone, or in combination with Xhol; a single copy of the restriction site for these enzymes was identified in the distal 2.3 kb of the A600 locus (Figure 13a). The blot was hybridized with a probe for the A800 sequence, which was isolated as an amastigote c D N A fragment and mapped downstream of the Xhol site in the A600-3 3'UTR (Figure 13b; filled black box). The probe hybridized to a single band in each lane and PstI, PvuII, and Xhol sites were mapped to positions 4.2 kb, 6.0 kb, and 10 kb downstream of the A600-3 coding sequence. A high molecular weight band was observed in Ncol digested D N A , so it was difficult to map the downstream Ncol site accurately. A restriction map of Xhol, PstI, and PvuII sites was generated from the results of this experiment (Figure 13b). The 3.7 kb PstI restriction fragment, which contained the A600-3 3'UTR+ER. sequence, was cloned from an L. mexicana genomic D N A library of 3 r 5 kb PstI restriction fragments. L. mexicana genomic D N A was digested with PstI enzyme prior to separation by agarose gel electrophoresis. The 3 - 5 kb restriction fragments were ligated into the pLitmus28 vector. The ligation was transformed into DH5a strain E. coli and colony blots of the resulting bacterial colonies were produced on nitrocellulose membranes. In the primary colony screen, eleven colonies were selected by hybridization of the colony blots 76  Chapter 3 - Results  End of known A600-3 sequence  PstI  575  PvuII 800  Xhol 1425  PstI 4200  / 1 kb  Figure 13.  2kb  3kb  PvuII 6000  Xhol & Ncol  /  4kb  10 kb  Southern Blot Analysis of the A600-3 Downstream Sequence.  (a) L. mexicana genomic D N A digested overnight with (1) Xhol, (2) PvuII, (3) PvuII + Xhol, (4) PstI, (5) PstI + Xhol, (6) Ncol, and (7) Ncol + Xhol was analyzed by Southern blot hybridization using a probe for the A800 3'UTR sequence. 1 kb molecular weight marker (Std) sizes are displayed on the left side of the blot, (b) Schematic restriction map for the positions of PstI, PvuII, Xhol, and Ncol sites relative to the A600-3 ORF (red box). The position of the A800 3'UTR sequence is shown as a black box. 77  Chapter 3 - Results with a probe for the A800 sequence sequence in the A600-3 3'UTR. Two of these eleven colonies were validated by a secondary colony screen with the probe for the A800 sequence; the plasmids isolated from the A800-positive colonies were named pLit-A800/6 and pLitA800/10. Only the pLit-A800/6 plasmid produced the expected 2.9 kb and 0.85 kb restriction products after digestion with the Xhol and PstI restriction enzymes, which confirmed that the desired A600-3 UTR+IR sequence had been cloned. A contiguous D N A sequence for the A600-3 coding sequence and 3'UTR+IR was constructed with a two-step cloning strategy. First, the pLit-A600yA plasmid was constructed by cloning a 0.7 kb EcoRI/PstI restriction fragment from pBST-E8 into the pLitmus28 plasmid. This sequence included the A600-3 ORF and the 3'UTR sequence up to the PstI site at position 575 bp of the A600-3 cDNA. pLit-A600yC, which contained the contiguous A6003 3'UTR+IR sequence, was constructed by subcloning the 3.7 kb PstI restriction fragment from pLit-A800/6 into pLit-A600yA. D N A sequencing across the PstI cloning site in the pLit-A600yC plasmid confirmed that a contiguous sequence had been constructed accurately for the A600-3 3 'UTR+IR.  3.1.4  Sequencing the Complete A600-1 and A600-3 Genes The complete sequence of the A600-1 and A600-3 genes, including the 3'UTR and  downstream intergenic region, was determined using sequencing templates that were generated by Exonucleaselll (ExoIII) deletion. D N A corresponding to the A600-1 and A6003 genes had been cloned previously into the pLitmus28 vector to generate pLit-5'E8 and pLit-A600yC, respectively. The A600 insert D N A from these plasmids was subcloned into the pBluescript vector to create pBST-5'E8 and pBST-A600yC (Figure 12b and Figure 14a).  78  Chapter 3 - Results  Std  Figure 14.  1  2  3  4  5  6  7  8 Std  Exonuclease (III) Deletion Panel of the pBST-A600yC Plasmid.  pBST-A600yC plasmid was linearized by restriction digestion with Ncol (Exo(III)susceptible) and Kpnl (Exo(III)-resistant) enzymes. Exonuclease digestion of the linear plasmid proceeded from the Ncol end for 50, 100, 150, 200, 250, 300, 350, and 400 seconds (lanes 1 - 8), followed by Mung Bean nuclease digestion to produce blunt end products to enable plasmid re-ligation. 1 kb molecular weight marker (kb) sizes are displayed on the left side of the gel. 79  Chapter 3 - Results These plasmids were digested at the Ncol and Kpn sites, which produced linear D N A molecules with Exo(III)-susceptible (5' overhang) and -resistant (3' overhang) ends. A panel of deletions was created by a time course of Exo(III) digestion from the Ncol end, followed by Mung Bean nuclease treatment to produce blunt ends (Figure 14b). The digested D N A was self-ligated, transformed into D H 5 a strain E. coli, and the isolated plasmids were used as sequencing templates. The panel of Exo(III) deletion clones constructed from pBST-5'E8 and pBST-A600yC were sequenced from the M13F20 primer site by the BigDye dideoxy sequencing method. Overlapping D N A sequences obtained from the Exo(III) deletion panels were aligned into sequence contigs using Sequencher computer software. The complete sequence of the L. mexicana A600-3 3'UTR and downstream intergenic region was determined from sequencing of the pBST-A600y plasmid. A gene encoding the 60S ribosomal protein was identified downstream of the A600-3 3'UTR, which confirmed that the complete sequence of the A600-3 gene had been determined. In addition, the complete sequence of the A600-1 coding sequence and the proximal 2.7 kb of the A600-1 3'UTR were obtained from the Exo(III) deletion panel of pBST-5'E8. ClustalX sequence alignment software showed that the L. mexicana A600-1 and A600-3 coding sequences shared 78% sequence identity across the A600-3 sequence. The A600-1 coding sequence contained a 150 bp extension at the 3' end (Figure 15). The L. mexicana A600-1 and A600-3 predicted protein sequences shared only 57% amino acid sequence identity (Figure 16). In the annotated L. major genome sequence database, a single A600 gene homolog was identified. The L. major A600 homolog shared 88% D N A sequence identity with the L. mexicana A600-1 coding sequences (Figure 17). A second L. major A600 homolog, which had not been annotated from the genome sequence, was identified in 8 kbp of downstream intergenic sequence. A ClustalX alignment showed that the second L. major A600 gene 80  Chapter 3 - Results  LmxA6001  1 ATGCCCTCTATGCTCAACCTTGTCCCGGCGACCGCGATCGCTGTGGGCGC  50  LmxA6003  I I I I I I I I I I I I I II I I I I I I I I I I I II I I 1 ATGCCCTCTATGCTCAACCTTGTCCCGGCG  33  LmxA6001  III GTG  51  GATAGCCCTCCCTGCGGCTGCGACGACGACGACGACTGCGGCTCCTGTTC  100  LmxA6003  34  I . I I I I I I I I• I I I I • I I • I • I I •• I • • GAGACGACGATGAC—CCGC-ACCCCGATGT  61  LmxA6001  101  LmxA6003 LmxA6001  150  62  CTGTCAACCTCAGGCTGAACATCATCACGGCGGTGCTGATTCTAGGTGTG . I I I I • I • . I • I II • I I I I • • • I • I • • I I • • I • I I • I I • I • • I • I I I I I • ATGTCGAGGTGAGGGTGAATGCCGTGCCGTTGATGATGGTCTTTGGTGTC  151  TCACTTGTGTTGACGCTGGTGTACACCCTGTGGAAGCTTCTCCCGAGGAT  200  LmxA6003  112  I I I I II I I I . I 1 - I I I I I I I I I I I I I - I I I I I I I I I I I I I I I I I I I I I I I TCACTTGTGCTGGCGCTGGTGTACACTCTGTGGAAGCTTCTCCCGAGGAT  161  LmxA6001  201  CCGCAGTGGCGAGCTCTCCTTCTCGAAGTTCGAGTTCGACTGGCGTGCGG  250  LmxA6003  162  I I I I I I I I I I I I I I I I II • I • I II I I I I I I • • I • I II • • I I I I I I I CCGCAGTGGCGAGCTCTCGTCCTCGAATACGGAGGCCAACTTTCGTGCGG  211  LmxA6001  251  AGCTGCTGAACCAGACGCCGAAGAAGGAGAAGGCGCGCCGCGCGACGGA-  299  LmxA6003  212  . I I I I I I I I I I I • I I • I I • I I II I • I I I I I I II . I II I GGCTGCTGAACCGGAAGCTGAAGAGGGAGAAGGTGCGC  .1111 TCGGAG  255  LmxA6001  300  GAAGGCTCGCCGTGAGGAGGAGATGGCGTCCGGGTGCAACCGCGACAACG  34 9  LmxA6003  256  I I. I . . I I . . I I I• I I I GATGATTCATC-TGCGGA  LmxA6001  350  ACGAGGGACGCGTGCAGTACGCCCACACGCAGCCGCGGGTGGAGGTGGGC  LmxA6003  283  LmxA6001  400  LmxA6003  283  LmxA6001  450  LmxA6003  283  Figure 15.  I •• I I I I• II CATGGTGTAA  111  282 399 282  GAGGGCGACGCCGCGGCTGCCAGATCGCAGCGCAAGGGACAGAGGCACGT  44 9 282  CGAGGCCGATGTGAGCGTTGCGGTGACGGTGCCCCGCGAGTAG  4 92 282  Alignment of the L. mexicana A600-1 (LmxA6001) and A600-3 (LmxA6003) Coding Sequences. 81  Chapter 3 - Results  LmxA6001 LmxA6003  1 MPSMLNLVPATAIAVGAIALPAAATTTTTAAPVPVNLRLNIITAVLILGV 11 1 11 1 1 1 1 | . . | | . | . . | : . | . : | : | . : . . : : : . II A V E TTMT RT PM Y V E VRVN AV P LMMV FG V 1 MPSMLNLVP  LmxA6001  51  Lmx6003  38  LmxA6001  101  50 37  SLVLTLVYTLWKLLPRIRSGELSFSKFEFDWRAELLNQTPKKEARRATEK 100 11 1 1 . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . 1 . . 1 . : : M . 1 1 1 : . . 1 : SLVLALVYTLWKLLPRIRSGELSSSNTEANFRAGLLNRKLKR EK 81 ARREEEMASGCNRDNDEGRVQYAHTQPRVEVGEGDAAAARSQRKGQRHVE l l • • • 1 • 1  150  LmxA6003  82  VRSEDDSSADMV*  94  LmxA6001  151  ADVSVAVTVPRE*  163  LmxA6003  95  Figure 16.  94  Alignment of the L. mexicana A6001 (LmxA6001) and A6003 (LmxA6003) Protein Sequences. 82  II  Chapter 3 - Results  LmxA6001  1 ATGCCCTCTATGCTCAACCTTGTCCCGGCGACCGCGATCGCTGTGGGCGC  50  LmjA6001  I I I I I I I I I M I .. I I I . I I . I I I I I. I I . I I I I I I I. I I . . . I . I • I I I 1 ATGCCCTCTATGAGCAAGCTCGTCCCAGCCACCGCGAGCGAAATTGCCGC  50  LmxA6001  51  GATAGCCCTCCCTGCGGCTGCGACGACGACGACGACTGCGGCTCCTGTTC  100  LmjA6001  51  I I I • I I I I I I I•• I I I I • I I I I I I I I I I I I I I I I I I I I I I I •I GATTGTTTCCCCTGCTTCTGCAACGACGACGAC—C-GCGGCTCCTGTCC  97  LmxA6001  101  LmjA6001 LmxA6001  98  CTGTCAACCTCAGGCTGAACATCATCACGGCGGTGCTGATTCTAGGTGTG • I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I • I I • I I • I I I TTGTCAACCTCAGGCTGAACATCATCACGGCGGTGCTGATCCTTGGCGTG  147  151  TCACTTGTGTTGACGCTGGTGTACACCCTGTGGAAGCTTCTCCCGAGGAT  200  LmjA6001  148  I I • I I I I I I I I I • I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I • I I I I I TCGCTTGTGTTGGCGCTGGTGTACACCCTGTGGAAGCTTCTCCCAAGGAT  197  LmxA6001  201  CCGCAGTGGCGAGCTCTCCTTCTCGAAGTTCGAGTTCGACTGGCGTGCGG  250  LmjA6001  198  I I I I I I I I I I I I I II I I I I I I I I I I I II • I • I I I I I I I I I I I I I I I I I I I CCGCAGTGGCGAGCTCTCCTTCTCGAAGCTTGAGTTCGACTGGCGTGCGG  247  LmxA6001  251  AGCTGCTGAACCAGACGCCGAAGAAGGAGAAGGCGCGCCGCGCGACGGAG  300  LmjA6001  248  I I I I I II I I I I I I I I • I I I I II I I I I I I I I I I I • I I I I I I • I I I • I • I I I AGCTGCTGAACCAGATGCCGAAGAAGGAGAAGGTGCGCCGGGCGGCTGAG  297  LmxA6001  301  AAGGCTCGCCGTGAGGAGGAGATGGCGTCCGGGTGCAACCGCGACAACGA  350  LmjA6001  298  I I I I I I I I I I I . I II I I I I I I I I I I I I I I . I II I II • I II I • II • I I • • I AAGGCTCGCCGCGAGGAGGAGATGGCGTCGGGGTGCGACCGGGATAAGAA  34 7  LmxA6001  351  CGAGGGACGCGTGCAGTACGCCCACACGCAGCCGCGGGTGGAGGTGGGCG  400  LmjA6001  348  I I I I I I I I . I I I I I II I I I I I I . . I I • I I I I II I I I I I I I I I I I II I I II CGAGGGACACGTGCAGTACGCCGCCATGCAGCCGCGGGTGGAGGTGGGCG  397  LmxA6001  401  AGGGCGACGCCGCGGCTGCCAGATCGCAGCGCAAGGGACAGAGGCACGTC  450  LmjA6001  398  I I I I I I I I I I • I I I I I II I • I I • I I I I I I I I I I • • I I • I I I I I I I I II • I AGGGCGACGCGGCGGCTGCTAGGTCGCAGCGCAGTGGGCAGAGGCACGGC  447  LmxA6001  4 51  GAGGCCGATGTGAGCGTTGCGGTGACGGTGCCCCGCGAGTAG  4 92  LmjA6001  I I I I I I I I • I • I I II I I I I I I I I I I II • I I I II I I • I I I I I I 44 8 GAGGCCGACGAGAGCGTTGCGGTGACGATGCCCCGGGAGTAG  Figure 17.  150  489  Alignment of L. mexicana A600-1 (LmxA6001) and L. major A600-1 (LmjA6001) Coding Sequences. 83  Chapter 3 - Results  LmxA6003  GC  29  I I I I I I I I I I I i I . I I I . I I . I II I I • II ATGCCCTCTATGCGCAAGCTCGTCCCAGCCACCGCGAGCGAAATTGCCGC  50  1 ATGCCCTCTATGCTCAACCTTGTCCCG  LmjA6003  1  LmxA6003  30  GGTGG  AGACGACGATGACCCGCA-CCCCGATG  60  LmjA6003  51  I.I.I . I I I I I I I I I I I I I I I I I . I I I I . I GATTGTTTCCCCTGCTTCTGCAACGACGACGATGACCCGCAGTCCCGC-G  99  LmxA6003  61  TATGTCGA-GGTGAGGGTGAATGCCGTGCCGTTGATGATGGTCTTTGGTG  109  LmjA6003  100  I I I I I I I I I I I I I. I I I I ... I I. . I I I I I I I I I I I • I • I I . . TATGTCGACGGTG-GCGTGCGGATCACTCCTGTGATGATGGTCGTCGGCA  148  LmxA6003  110  LmjA6003  14 9  LmxA6003  TCTCACTTGTGCTGGCGCTGGTGTACACTCTGTGGAAGCTTCTCCCGAGG I • I I • I I II I I • I I I I I • I I I II I I I I I • I I I I I I I I I I I I I I I I I • I I I TGTCGCTTGTGTTGGCGTTGGTGTACACCCTGTGGAAGCTTCTCCCAAGG  159  160  ATCCGCAGTGGCGAGCTCTCGTCCTCGAATACGGAGGCCAACTTTCGTGC  209  LmjA6003  199  I I I I I I I I I I I I I I I I I• I I I I I• I I I I I I • I •• I I I I I I II ATCCGCAGTGGCGAGCTTTCGTCTTCGAAGCTTGGGTTCAAGGGGCGTGC  248  LmxA6003  210  GGGGCTGCTGAACCGGAAGCTGAAGAGGGAGAAGGTGCGCTCGGAGGATG  259  LmjA6003  I I. I I . • I I I I• I I I • I I I I• I I 24 9 GGAGCCACTGAGCCGAAAGCCGA  LmxA6003  260  AT  TCATCTGCGGACATGGTGTAA  282  LmjA6003  293  II II • I I I I I I I • I I I I I I I I I • ATTGCTCGTCTGCGGGCATGGTGTAG  318  Figure 18.  I I II I I I I I • I I I I I I • II • I AGAAGGTGCACTCGGAAGACG  198  292  Alignment of the L. mexicana A600-3 (LmxA6003) and L. major A600-3 (LmjA6003) Coding Sequences. 84  Chapter 3 - Results shared 67% D N A sequence identity and 60% amino acid sequence identity with the L. mexicana A600-3 gene; this observation suggest that this L. major gene was a homolog to the L. mexicana A600-3 gene (Figure 18). The L. mexicana A600-1 and A600-3 3'UTR sequences did not contain any significant sequence identity. However, the L. major genes corresponding to the L. mexicana A600-1 and A600-3 genes showed a high degree of identity across both the coding and 3'UTR sequences. A 60S ribosomal subunit gene was also annotated downstream of the A600 locus in the L. major genome.  3.1.5  Expression Profiles of the A600-1 and A600-3 Genes The relative level of A600 gene expression in promastigotes and amastigotes was  determined by Northern blot analysis. Previously, it was shown that the abundance of the A600-3 m R N A transcript was 8-fold greater in L. mexicana axenic amastigotes, compared to log phase promastigotes (Bellatin et al, 2002). A600-3 gene expression was examined in lesion amastigotes, which had been isolated from L. mexicana-infected B A L B / c mice, to validate the L. mexicana axenic amastigote model. A Northern blot of total R N A isolated from promastigotes and lesion amastigotes was hybridized with a probe for the A600-3 coding sequence (Figure 19). The probe hybridized to a 3.3 kb m R N A transcript, which was significantly upregulated in lesion amastigotes. The blot was stripped and re-probed for the 16S ribosomal rRNA to provide a control for equal loading. Quantitative analysis determined that the A600-3 R N A transcript was 7-fold more abundant in axenic amastigotes than promastigotes. Upregulation of A600-3 gene expression was followed during differentiation of promastigotes into axenic amastigotes. Briefly, amastigote differentiation was induced by culturing log phase promastigotes at 32 °C for 16 hours. UM54 amastigote media (pH 5.0) 85  Chapter 3 - Results  Figure 19.  Northern Blot Analysis for A600-3 Gene Expression.  Northern blot was prepared with L. mexicana R N A isolated from log phase promastigotes (LP), stationary phase promastigotes (SP), and lesion amastigotes (LA), (a) The blot was hybridized with a probe for the A600-3 coding sequence. The membrane was stripped and reprobed for the 16S r R N A sequence (lower panel). Molecular weight standard sizes (kb) are shown on the left of the blot, (b) Quantification of changes in A600-3 mRNA transcript abundance. The relative changes in R N A abundance were expressed as an amastigote/promastigote (A/P) ratio, normalized to rRNA abundance. 86  Chapter 3 - Results was added to the cultures after 16 and 96 hours at 32 °C, and the culture was maintained for 7 days. A600-3 gene expression was assayed at various time intervals during amastigote differentiation by Northern blot analysis. The temperature shift or early addition of UM54 amastigote media did not induce a noticable change in A600-3 gene expression until the 48 hour time point (Figure 20, panel A). During a 7 day time course, impressive upregulation of A600-3 expression only occurred after the second addition of UM54 media (Figure 20, Panel B). Expression of the A600-1 and A600-3 genes was examined by Northern blot analysis with gene-specific probes. Northern blots were prepared with total R N A isolated from L. mexicana log phase promastigotes and axenic amastigotes and hybridized with random labelled probes for the A600-1 and A600-3 coding sequences (Figure 21, Panels A and C). The A600-1 ORF probe hybridized to 4.4 kb and 3.3 kb m R N A transcripts denoted as A6001top and A6001-bottom, respectively. The 4.4 kb A6001-top transcript was slightly induced in axenic amastigotes, while the 3.3 kb A6001-bottom transcript was expressed almost exclusively in amastigotes. The A600-3 ORF probe hybridized only to a 3.3 kb transcript, which was significantly more abundant in amastigote R N A . It was not possible to determine whether the 3.3 kb A600-3 transcript had crosshybridized to the A600-1 ORF probe, so gene-specific probes were generated from the unique 3'UTR sequences of the A600-1 and A600-3 genes (Figure 21, Panels B and D). The A600-1 U T R probe was a random labelled 500 bp sequence from the A600-1 3'UTR that amplified from the pBST-5'E8 and pBST-midE8 plasmids, but not from pBST-A600yC plasmid. This probe hybridized only to the 4.4 kb transcript. A probe for the A800 sequence in the A600-3 3'UTR hybridized only to a 3.3 kb transcript. B y quantitative analysis, the A6001-top transcript was 1.5-fold more abundant in amastigotes, while the A600-bottom 87  Chapter 3 - Results transcripts was almost 6-fold increased in amastigotes. B y comparison, probes for the A600-3 coding and 3'UTR sequence hybridized to a 3.3 kb transcript that had 7-fold higher abundance in amastigotes. The expression profile of the L. mexicana A600-2.1 and A600-2.2 genes during the Leishmania lifecycle was not examined as the complete sequence of these genes has not been determined.  88  Chapter 3 - Results  c  d  Time (hours)  Figure 20.  Time (days)  A600-3 Expression During Axenic Amastigote Differentiation.  Northern blots were prepared with 5 pg of total R N A isolated from cells after (a) 0, 16, 18, 20, 27, 40, and 48 hours and (b) after 0, 1, 3, 3, 5, and 7 days of culture at 32 °C. A600-3 m R N A transcript abundance was analyzed by Northern blot hybridization with a random labelled probe for the A600-3 coding sequence. The blot was stripped and re-probed for the 16S r R N A sequence (lower panels). R N A molecular weight marker (Std) sizes are shown on the left side of each blot. (c,d) Relative changes in A600-3 R N A abundance was expressed as an amastigote/ promastigote (A/P) ratio, normalized to rRNA abundance. 89  Chapter 3 - Results  C  a Probe:  Sizes (Kb)  A600-1 coding  PP AA  Probe:  A600-1 UTR  Sizes (Kb)  PP AA  Probe:  A600-3 coding  Sizes (Kb)  PP AA  9.49 — 7.46 —  9.497.46"  4.40 —  4.40-  2.37-  2.37 —  2.37-  1.35—I  1.35 —  1.35-  9.497.46" 4.40-  mtm -mm  d Probe:  Sizes (Kb) 9.49—] 7.46 —| 4.402.37-  A600-3 UTR  PP AA  10.00  c o '</)  8.00  $>  CL  X  7.00  I  6.00  m  5.00  <D  4.00  LU  Od CL  1.35—I  9.00  3.00 2.00 1.00 0.00  A6001 Top Figure 21.  A6001 Bottom  A6003  Comparison of L. mexicana A600-1 and A600-3 Gene Expression.  Northern blots were prepared with 5 \xg of total R N A isolated from log phase promastigotes (LP) and day 7 axenic amastigotes (AA). The blots were hybridized with random labelled probes for the (a) A600-1 coding sequence, (b) A600-1 3'UTR sequence, (c) A600-3 coding sequence, and (d) the A800 sequence from the A600-3 3'UTR. Molecular weight markers (kb) are displayed on the left side of the blot, (e) Quantification of changes in A600-1 and A600-3 m R N A transcript abundance. The relative changes in m R N A abundance were expressed as an amastigote/ promastigote (A/P) ratio. "A6001 Top" and "A6001 Bottom" refer to the 4.4 kb and 3.3 kb A600-1 transcripts, respectively. "A6003" refers to the 3.3 kb A600-3 transcript. 90  Chapter 3 - Discussion 3.2  Discussion The primary objective of this chapter was to clone and characterize the A600 locus of  the L. mexicana genome. The A600-3 gene was identified previously as an amastigotespecific c D N A and Southern blot analysis showed multiple A600 genes in the genome (Bellatin et al, 2002). The A600-3 D N A and protein sequence did not share homology with any known genes, which made it difficult to directly infer gene function. The lack of identity to annotated genes from other organisms was not surprising, as 64% of Leishmania genes annotated from the L. major genome sequencing project were novel genes that did not share identity with any known genes (Ivens et al, 2005). There was great interest in determining the function of this novel gene, which had dramatically elevated expression in the amastigote stage of the Leishmania lifecycle. The only amastigote-specific genes that had been identified previously were the L. donovani A2, L. infantum amastin, and the L. mexicana cysteine protease genes (Souza et al, 1992; Charest et al, 1994; Wu et al, 2000). Homologs of the A600 genes also have been identified in genome sequences for L. major and L. infantum, which are Old World Leishmania species. However, the A600 genes were not identified in genome sequence for the related T. bruci or T. cruzi protozoan parasites. These observations indicate that the A600 genes constitute a Leishmania-spQcific gene family, which is well conserved in Old and New World Leishmania species that cause both cutaneous and visceral leishmaniasis in the mammalian host. Southern blot analysis of L. mexicana genomic D N A , performed after a variety of single and double restriction enzyme digestion, showed that the A600-3 gene belonged to a multi-gene family. A probe for the A600-3 coding sequence hybridized to a high molecular weight band in Xhol- or Xmaldigested genomic D N A . These results indicated that the multiple A600 genes were tandemly arranged at a single genomic locus. In Leishmania, tandemly arranged gene families are 91  Chapter 3 - Discussion common as this confers higher expression to these genes. The leishmanolysin (gp63) genes are tandemly repeated at a single genomic loci in the L. major and L. chagasi genomes (Button et al., 1989; Roberts et al., 1993; Voth et al., 1998). Other tandemly repeated genes include the L. mexicana glucose transporter genes, paraflagellar rod (PFR2), and cysteine protease b (cpb) genes (Moore et al., 1996; Bart et al., 1997; Burchmore et al., 1998). The A2 and amastin genes from the L. donovani genome exist in a more complex arrangement, as these genes are tandemly repeated at more than one genomic locus (Charest et al., 1994; Zhang et al, 2001; Rochette et al, 2005). Additionally, the gp46, a-tubulin, and P-tubulin genes also are tandemly repeated in Leishmania genomes (Spithill et al, 1987; Lohman et al, 1990). Initial attempts to clone Xhol and Xmal restriction fragments that encode the entire A600 locus into the pBluescript K S cloning vector proved unsuccessful. These failures were attributed primarily to two factors. First, the A600 locus was approximately five times larger than the plasmid vector, so a high concentration of large Xhol restriction fragments was required to achieve a 3-fold molar excess of genomic D N A fragments for the ligation reaction. The sucrose gradient method was a practical approach to isolate and purify a large amount of high molecular weight Xhol restriction fragments. Second, the A600-3 ORF probe hybridized to a significantly larger band in X/noT-digested genomic D N A , relative to the 14 kb Xhol restriction fragment. It could be inferred from this data that the Xmal restriction fragment would have been much more difficult to isolate by cloning into a plasmid vector. A strategy of cloning these genomic D N A fragments into a cosmid vector may have been more appropriate, as D N A fragments larger than 10 kb are notoriously difficult to ligate into plasmid vectors.  92  Chapter 3 - Discussion A detailed restriction map of the A600 locus was generated using the pBST-E8 plasmid. It was interesting to observe that the EcoRI and Ncol restriction sites were repeated at regular intervals across the A600 locus, as this suggested that recent A600 gene duplication may have occurred. The position of the A600-1 gene was determined by mapping the 5' end pBST-E8 plasmid. The A600-1 coding sequence shared 78% sequence identity across the A600-3 coding sequence. The extension at the 3' end of the A600-1 coding sequence may encode a C-terminal functional protein domain that is absent in the A600-3 protein. The full length, predicted amino acid sequences of the A600-1 and A600-3 proteins only shared 57% identity across the A600-3 amino acid sequence, although both proteins contained hydrophobic N-termini and a highly conserved, twenty-five amino acid sequences in the central region. The hydrophobic N-termini contained predicted transmembrane regions that would tether these proteins to the membrane. It is hypothesized that the highly conserved amino acid sequence represents a functional domain of these proteins. Based on their relatively small size, the A600 proteins were not predicted to function as enzymes. Rather, it is possible that these proteins form the subunit of a larger protein complex and the conserved amino acid sequence is critical for proper interaction with other factors. The pBST-midE8 plasmid was used to obtain partial sequence for the A600-2 gene, which shared a high degree of sequence identity with the A600-1 coding and proximal 3'UTR sequences. The pBST-midE8 plasmids were constructed from two 4.3 kb Ncol restriction fragments, which mapped to the middle of the A600 locus (Figure 12). A PstI fingerprinting experiment indicated that the independent pBST-midE8 plasmids consisted of highly conserved D N A sequences. In combination, these results suggest that four A600 coding sequences exist in the L. mexicana genome, whereby the A600-1, A600-2.1, and A600-2.2 coding sequences share a high degree of sequence identity and the A600-3 coding 93  Chapter 3 - Discussion and 3'UTR sequence have diverged. Interestingly, the last gene in the hsp70 gene cluster also had a highly divergent 3'UTR sequence compared to the upstream gene copies (Quijada et al., 1997). It was also interesting to note that although the L. mexicana A600 genes share sequence identity, there was a noticeable difference in the size of the A600-1 and A600-3 genes. The coding sequences were 492 bp and 282 bp, respectively, and produced 4.4 kb and 3.3 kb R N A transcripts. B y inference, the A600-1 3'UTR should be approximately 3.8 kb, while the A600-3 3'UTR would be 2.9 kb. On average, Leishmania genes preferentially expressed in the amastigote stage have slightly longer 3'UTR sequences than promastigotespecific transcripts. The amastigote-specific A2, Imcpb, and amastin R N A transcripts each had 2.0 kb 3'UTR sequences, while the promastigote-specific gp63, gp46, PFR2-B, and LmGT2 transcripts had 3'UTR sequences ranging from 1.0 to 1.5 kb. Only the constitutively expressed L. mexicana LmGTl and LmGT3 genes, which encode of 1.8 kb coding sequences, had longer 3'UTR sequences than the A600 genes. These glucose transporter transcripts were approximately 7.5 kb in length, and contained greater than 5.5 kb of 3'UTR sequence. In summary, the structure for both A600 transcripts was unusual, as the relatively small coding sequences were followed by unexpectedly large 3'UTR sequences. It is tempting to hypothesize that the relatively large of A600 3'UTR sequences may contribute to R N A transcript stability in amastigotes; however, the A600-1 and A600-3 3'UTR sequences are almost completely diverged, which suggests that discrete regulatory elements within these sequences, rather than the overall size, confers stage-specific gene expression. Southern blot analysis with a probe for the A600-3 coding sequence detected multiple bands when genomic D N A was digested with restriction enzymes that cut one or more times within the A600 locus. However, Northern blots hybridized with a probe for the A600-3 94  Chapter 3 - Discussion coding sequence only detected a 3.3 kb transcript, while an A600-1 coding sequence probe detected both 4.4 kb and 3.3 kb transcripts. Based on the Southern blot results, it was expected that the A600-3 probe also should detect the 4.4 kb A600-1 transcript. This discrepancy was addressed by probing Northern blots with probes specific for the A600-1 or A600-3 3'UTR sequences. The A600-1 3'UTR probe hybridized only to a 4.4 kb transcript, while the probe for the A600-3 3'UTR detected only a 3.3 kb transcript. These results did not elucidate the source of the 3.3 kb transcript that hybridized to the A600-1 coding sequence probe; however, it was observed that the A600-3 transcript was present at a much higher abundance than the A600-1 transcript. It is proposed that the A600 coding sequence probes hybridize to related A600 R N A transcripts, although the hybridization efficiency would be lower for divergent sequences. Therefore, the 3.3 kb transcript detected by the A600-1 ORF probe may be the highly abundant A600-3 transcript. If the blot hybridized with the A600-3 ORF probe were exposed for a longer period of time, one would probably detect a low intensity 4.4 kb band corresponding to the A600-1 transcript. The results from the Northern blot experiments indicated that the abundance of the A600-1 m R N A transcript was approximately equivalent in promastigotes and amastigotes. However, the accuracy of these results was compromised with the high degree of sequence identity shared by the A600-1 and A600-2 genes. The random labelled probes for the A600-1 coding and 3'UTR sequences would have hybridize to the A600-2.1 and A600-2.2 mRNA transcripts, which may have different expression profiles than the A600-1 gene. It will be necessary to determine the complete sequence of the A600-2 genes to design probes that will differentiate expression of these genes. From these experiments, it was not possible to conclude definitively the expression profile of the L. mexicana A600-1 gene.  95  Chapter 3 - Discussion In 2004, the annotated L. major genome sequence was released in the GeneDB database at http://www.Renedb.org. A B L A S T search with the L. mexicana A600-3 coding sequence identified a single L. major A600 gene, which had significant identity to the L. mexicana A600-1 coding and 3'UTR sequences. The 8 kb sequence downstream of the L. major A600 coding sequence suggested that additional A600 homologs may be encoded at this locus. The L. mexicana A600-1 and A600-3 R N A transcripts were 4.4 kb and 3.3 kb, respectively. In combination with knowledge that the average intergenic distance in the Leishmania genome was shown to be 500 bp, these results indicated that a second A600 gene was encoded downstream of the L. major A600-1 homolog. Indeed, a second L. major A600 homolog was identified, which shared sequence identity with both the L. mexicana A600-3 coding and 3'UTR sequences. The L. mexicana A600-1 and A600-3 genes had completely divergent 3'UTR sequences, so it was concluded that the L. major A600 locus consisted of conserved A600 gene homologs. Although only two A600 genes were annotated from the complete L. major genome sequence, there exists the strong possibility that additional copies of the A600-1 gene exist. It is difficult for alignment software to predict accurately the copy number for tandemly repeated D N A sequences, so this may explain copy number differences for A600 genes in the L. major and L. mexicana genomes. Considering that the Old World L. major and New World L. mexicana species are evolutionary divergent, it should be concluded that sequence divergence of the A600-1 and A600-3 genes occurred in an ancestral Leishmania species. Furthermore, it would appear that higher selective pressure has been placed on conservation of the A600-1 gene sequence. The A600-1 D N A sequences in L. major and L. mexicana showed 88% sequence identity, while the A600-3 gene showed only 67% sequence identity.  96  Chapter 4 - Results 4.  Generation and Characterization of A 600 Knockout Mutants This chapter describes the generation of A600 knockout mutant parasites by  homologous recombination. Initially, targeting constructs were constructed and knockout mutants were produced by two rounds of homologous recombination. The A600-1 mdA6003 genes were re-introduced into the knockout parasites to complement the knockout phenotype. The role of the A600 genes for intracellular survival and proliferation of amastigotes was examined with in vitro macrophage infections and in vivo mouse infections using the B A L B / c model for leishmaniasis.  4.1  Results  4.1.1  Generation of A600 Deletion Mutant Parasites by Homologous Recombination One of the primary objectives of this thesis was to functionally characterize the A600  genes by targeted deletion of the entire L. mexicana A600 locus. Two targeting constructs, pHygKO and pSatKO, were generated in the pGem9z plasmid cloning vector (Figure 22a,b). Briefly, these constructs contained different positive selection markers (the hygromycin B phosphotransferase (hph) or S-acetyl transferase (sat) genes), which allowed recombinant parasites to be selected on the basis of resistance to Hygromycin B or Nourseothricin drug, respectively. The selection markers were flanked by D N A sequence from immediately upstream A600-1 ORF (5'A600) and downstream of the A600-3 ORF (3'A600). As Leishmania parasites replicate asexually, it was necessary to perform two rounds of homologous recombination to generate A600 knockout mutant parasites (Figure 22c). Log phase wildtype promastigotes were transfected with either HygKO or SatKO linear targeting constructs that had been purified from the pGem9z plasmid backbone. Briefly, 50 jxg of the pHygKO or pSatKO plasmid were digested overnight with restriction enzymes to isolate the 97  Chapter 4 - Results  Figure 22.  Schematic for Knockout Constructs and Targeted Deletion of the A600 Locus.  Plasmid maps of the (a) pHygKO and (b) pSatKO plasmid vectors. The Hygromycin phosphotransferase (hph) and S-acetyl transferase (sat) positive selection markers are shown as blue and red arrows, respectively. The a-tubulin intergenic region, cloned downstream of the hph and sat genes, is represented as a light blue box. The flanking sequences used for homologous recombination, 5'A600 and 3'A600, are indicated with grey boxes, (c) A schematic model for targeted deletion of the A600 locus by homologous recombination. The HygKO and SatKO targeting constructs were purified from the plasmid constructs as Spel and Kpnl restriction fragments. A600 knockout mutants were generated by two rounds of homologous recombination. 98  Chapter 4 - Results targeting construct from the plasmid vector. Spel and Kpnl enzymes excised the targeting constructs from the plasmid vector, while Seal cut the vector within the Ampicillin-resistance (Amp ) gene. The resulting D N A restriction fragments were separated by agarose gel R  electrophoresis and the linear HygKO and SatKO targeting constructs were purified for transfection. Mid-log phase L. mexicana wildtype promastigotes were transfected with the purified HygKO or SatKO construct. Transfected cells were cultured in drug-free media for 24 hours prior to being selected on Ml99-Agar plates containing 35 pg/mL Hygromycin B (Hyg) or 100 pg/mL Nourseothricin (Nour) drug, respectively. Eleven Hyg-resistant and twelve Nourresistant colonies were selected and cultured in liquid M l 9 9 media containing 35 pg/mL Hyg or 100 pg/mL Nour drug selection, respectively; these clones were denoted as 9 5 H y g l - l l and 95Satl-12. A PstI site in the a-tubulin sequence of the targeting constructs was introduced into the chromosomal D N A during the targeted replacement of one allele at the A600 locus (Error! Reference source not found.a). Southern blot analysis was performed with P_£/-digested genomic D N A isolated from drug-resistant clones (Error! Reference source not found.b,c). A probe for the A800 sequence of the A600-3 3'UTR hybridized to a 3.5 kbp band in wildtype genomic D N A , whereas a 3.5 kbp and a 4.5 kbp band were detected in each of the 22 drug-resistant, A600"' clones. The 95Hygl and 95Satl A600t'- clones were transfected with the SatKO and HygKO targeting constructs, respectively, to replace the remaining wildtype allele at the A600 locus. As described for the first round of transfection, linear targeting constructs were isolated by restriction digestion of pSatKO or pHygKO plasmid. Mid-log phase L. mexicana A600* ' 1  promastigotes were transfected with purified HygKO or SatKO constructs and double drugresistant clones were selected on M199-Agar plates containing both Hyg (35 and 99  Chapter 4 - Results  Figure 23.  Targeted Replacement of the A600 Locus in L. mexicana Wildtype Cells.  (a) Illustration for integration of a targeting construct at the A600 locus. A PstI restriction site, from the a-tubulin intergenic sequence, was introduced into chromosomal D N A by integration of the targeting constructs. A probe for the A800 sequence from the A600-3 3'UTR (black box) hybridized to a 3.5 kb PstI fragment from the wildtype allele and a 4.5 kb PstI fragment after integration of the targeting constructs at the A600 locus. Southern blot analysis was performed with Pstf-digested genomic D N A from wildtype transfected with the HygKO or SatKO constructs, (b) Southern blot of genomic D N A from wildtype (lane 1) and the A600+/- 95Hygl-10 clones (lanes 2 - 11). (c) Southern blot of genomic D N A from wildtype (lane 1) and the A600+/- 95Satl-12 clones (lanes 2 - 13). The blots were hybridized with a probe for the A800 sequence of the A600-3 3'UTR, which hybridized to a 3.5 kb band from the wildtype A600 locus and a 4.5 kb band after integration of the HygKO or SatKO constructs at the A600 locus. 100  Chapter 4 - Results  A600 l o c u s  o  in  A800 Probe  ft.  o. 3.5 kb  I_  ft.  4.5 kb  A600 " 95Hyg Clones +/  8  4.5 3.5  9  10  11  kb _ | kb •  A600 - 95Sat C l o n e s +/  10 4.5 3.5  kb kb  101  11  12  13  Chapter 4 - Results  Figure 24.  Targeted Replacement of the Remaining at the A600 Locus.  (a) Illustration for targeted integration of both the SatKO and HygKO constructs at the A600 locus. A PstI restriction site, from the oc-tubulin intergenic sequence, was introduced at the A600 locus by integration of the targeting constructs. A probe for the A800 sequence from the A600-3 3'UTR (black box) hybridized to a 4.5 kb PstI restriction fragments after integration of the targeting constructs at the A600 locus. (B) Southern blot analysis was performed with /W-digested genomic D N A from wildtype (lane 1), A600+/- (lane 2), and seven A600-/- clones (lanes 3 - 9). The blot was hybridized with a probe for the A800 sequence of the A600-3 3'UTR. The probe hybridized to a 3.5 kb band from the wildtype A600 locus and a 4.5 kb band after integration of the HygKO and SatKO constructs at the A600 locus. 102  Chapter 4 - Results 70 pg/mL) and Nour (120 pg/mL) drugs. Thirty-five double drug-resistant colonies were transferred from the agar plates to liquid M l 9 9 culture media supplemented with the Hyg and Nour drugs at 70 pg/mL and 120 pg/mL, respectively. Both targeting constructs introduced the a-tubulin PstI site into the chromosomal D N A at the remaining wildtype allele at the A600 locus (Figure 24a). Southern blot analysis of .ftoT-digested genomic D N A showed that only seven of thirty-five, or 20%, of the drug-resistant clones had a targeted deletion at the second wildtype allele of the A600 locus. The Southern blot experiment was repeated with the wildtype, 95Hygl, and A600* (KOHyg70.2, KOSat35.2, KOSat35.3, KOSat35.4 KOSat35.6, KOSat35.7 KOSat70.5 and KOSat70.11) clones. A probe for the A800 sequence of the A600-3 3'UTR hybridized only to a 4.5 kbp band in the seven selected A600~'~ clones (Figure 24b). The probe hybridized to a 3.5 kb band in wildtype D N A , while 3.5 kbp and 4.5 kbp bands were detected in the A60Cf'~ clone.  4.1.2  A600 Knockout Confirmation The result from Figure 24 demonstrated that the HygKO and SatKO constructs had  integrated at the 3' end of the A600 locus. It was imperative to confirm that the A600-1 and A600-2 genes had been deleted by integration of the targeting constructs. Southern blot analysis was performed with ^7jo/-digested genomic D N A isolated from wildtype, A60O"', and A600~'~ clones. The blot was probed for the A600-3 coding sequence, which had crosshybridized with related on A600 genes in previous Southern blot experiments (Figure 6 and Figure 7). In this experiment, the probe hybridized to a 14 kbp band in wildtype and A6QQf'~ D N A . Importantly, this band was not detected in D N A from each of the A600~'~ clones, which indicated that the A600-1 and A600-2 genes had been deleted from the genome (Figure 25a). It was also necessary to show that A600 gene expression could not be detected in the A600~'~ 103  Chapter 4 - Results clones. Northern blot analysis was performed with total R N A isolated from log phase promastigote, stationary phase promastigote, and axenic amastigote cells. The blot was hybridized with a probe for the A600-1 coding sequence, which detected 3.3 kb and 4.4 kb transcripts that were preferentially expressed in amastigotes (Figure 26a). When the blot was hybridized with a probe for the A600-3 coding sequence, a 3.3 kb transcript was detected only in amastigotes (Figure 26b). The probes for the A600-1 and A600-3 coding sequences did not hybridize to A600 R N A transcripts in the KOSat35.2 A600~'~ clone, which further confirmed that targeted deletion of the A600 locus had been accomplished. The membrane was stripped and re-probed with a 16S rRNA probe to control for loading.  4.1.3  A600 Protein was Absent in L. mexicana A600~ Amastigotes A  Antibody was raised against a synthetic peptide, which was conserved in the predicted A600-1 and A600-3 amino acid sequences. New Zealand white rabbits were immunized with A600 peptide that had been conjugated to the carrier protein, Keyhole Limpet Hemocyanin (KLH). Polyclonal aA600 antibody was affinity purified from immune rabbit serum using a column conjugated to the A600 peptide. Soluble and insoluble cellular fractions were prepared from Triton X-100 lysates of L. mexicana wildtype and A600~'~ axenic amastigotes. Western blot analysis was performed with protein lysates, which had been separated on a 16.5% Tris-Tricine SDS-PAGE gel to ensure that low molecular weight proteins were separated efficiently (Figure 27). The blot was hybridized with affinity purified a-A600 antibody, which detected A600 protein in the Triton X-100 insoluble fraction of L. mexicana wildtype amastigotes. The A600 protein was not detected in the Triton X-100 insoluble fraction of A6Q0~'~ amastigotes, or the Triton X-100 soluble fraction of wildtype or A60Q~ ~ amastigotes. I  104  Chapter 4 - Results  Figure 25.  Confirmation for Targeted Deletion of the L. mexicana A600 Locus.  (a) Southern blot analysis was performed on ^7jo/-digested genomic D N A from L. mexicana wildtype (lanel), A600 " (lane 2), and seven A600 knockout clones (lanes 3 - 9 ) cells. The blot was hybridized with a probe for the A600-3 coding sequence, (b) A n agarose gel showing the J(7jo/-digested genomic D N A that was used for the Southern blot analysis. The molecular weight marker (kb) sizes are shown on the left of the Southern blot. +/  105  Chapter 4 - Results  Figure 26.  Absence of A600-1 and A600-3 Expression in L. mexicana A600 (KO) Parasites.  Northern blot analysis with total R N A isolated from log phase promastigotes (LP), stationary phase promastigotes (SP), and axenic amastigotes (AA). (a) The blot was hybridized with a probe for the A600-1 coding sequence, (b) The membrane was stripped and probed for the A600-3 coding sequence. The membrane was stripped and re-probed for the 16S rRNA (lower panels) to control for loading. Molecular weight marker (kb) sizes are shown of the left of the blot. 106  Chapter 4 - Results  Soluble protein  Figure 27.  Insoluble protein  Western Blot Analysis for A600 Protein Expression.  1% Triton X-100 protein lysates were prepared from L. mexicana wildtype (WT) and A600 " (KO) axenic amastigotes. Triton X-100 soluble proteins (lanes 1,2) and Triton X-100 insoluble proteins (lanes 3,4) were separated on a 16.5% Tris-Tricine SDS-PAGE gel and probed with a-A600 polyclonal antibody. Molecular weight marker (kDa) sizes are shown on the left. 7  107  Chapter 4 - Results 4.1.4  Knockout Reconstitution with the A600-1 and A600-3 Genes The A600 genes were re-introduced into the KOSat35.2 A60fr'~ clone to complement  any observed knockout phenotype. The A600-1 and A600-3 coding sequences were amplified with a proofreading D N A Taq polymerase, using primer pairs that contained 5' artificial Xmal (forward primer) and Xbal (reverse primer) restriction sites. The A600-1 and A600-3 amplicons were cloned into the pDrive T/A cloning vector (Qiagen) and the D N A sequences were confirmed by direct D N A sequencing to ensure that errors had not been introduced. The A600-1 and A600-3 coding sequences were subcloned into the Leishmania expression vector, pLexNeo, at Xmal and Xbal sites upstream of the hexbp 3'UTR and intergenic region (Joshi et al., 1995) (Figure 28a,b). The pLexNeo-A6001 and pLexNeo-A6003 plasmids expressed chimeric R N A transcripts with the A600 coding sequences fused to the hexbp 3'UTR. Cells from the KOSat35.2, A600" " clone were transfected with 5 jag of circular /  pLexNeo-A6001 or pLexNeo-A6003 plasmid, and selected on Ml99-Agar plates with 10 l^g/mL Geneticin (G418) drug selection. G418-resistant clones were transferred to liquid M199 media containing 20 ug/mL G418. The KO+A6001.1-3 and KO+A6003.1-3 clones were isolated after transfection of A600" cells with pLexNeo-A6001 and pLexNeo-A6003 /_  plasmids, respectively. Re-introduction of the A600-1 or A600-3 gene was demonstrated by P C R amplification of 500 bp and 300 bp products, respectively. The KO+A6001 clones amplified a 500 bp P C R product, but failed to produce the 300 bp P C R product (Figure 28c); conversely, the 300 bp P C R product was specifically amplified from the KO+A6003 clones (Figure 28d). P C R products corresponding to both the A600-1 and A600-3 genes were amplified from D N A of the L. mexicana wildtype strain, but could not be amplified using D N A from the A60a clone. A  Northern blot analysis was performed with promastigote and amastigote total R N A 108  Chapter 4 - Results isolated from wildtype, A 6 0 0 ' , KO+A6001.1, and KO+A6003.1 cells. The blot was hybridized with a probe for the A600-1 coding sequence, which detected 3.3 kb and 4.4 kb R N A transcripts in wildtype cells. The A600-1 transcripts were absent in knockout cells, but 1.8 kb, 4.0 kb, and a high molecular weight band were detected in the KO+A6001.1; unexpectedly, the 1.8 and 4.0 kb A600-1 transcripts were preferentially expressed in the amastigote stage (Figure 29a). The membrane was stripped and hybridized with a probe for the A600-3 coding sequence, which preferentially hybridized to a 3.3 kb transcript from wildtype amastigotes. A600-3 R N A transcripts were absent in knockout cells, and a high molecular weight band was detected in the KO+A6003.1 clone (Figure 29b). The A600-3 R N A transcript was expressed preferentially in amastigotes of the KO+A6003.1 clone.  4.1.5  The A600-1 Gene Is Essential for Proliferation ofAxenic L. mexicana Amastigotes The role of the A600 genes for L. mexicana amastigote survival and proliferation was  assayed with promastigote and axenic amastigote cultures of L. mexicana wildtype, A600 ', 7  KO+Genel.l, and KO+Gene3.1 strains. Promastigote growth was not affected by targeted deletion of the A600 locus, as shown with a 5 day growth curve (Figure 30a). L. mexicana promastigotes were differentiated to axenic amastigotes by culturing the cells in UM54 media (pH 5.0) at 32°C. Briefly, 5 m L log phase promastigote cultures were transferred from 26°C to 32°C; after 24 and 96 hours, one volume of UM54 media was added to these flasks. Growth curves were determined every 24 hours during amastigote differentiation (Figure 30b). Interestingly, the growth curves were identical for cell line during the initial 96 hours of amastigote differentiation, after which wildtype parasites continued to proliferate until day 7. A600" cells failed to proliferate and cell densities had decreased by day 7. Amastigote A  growth was restored partially by re-introduction of the A600-1 gene in KO+A6001 cells. 109  Chapter 4 - Results  c  d 1 2 3 4 5 6 7 8  Figure 28.  1 2 3 4 5 6 7 8  Reconstitution of the A600~'~ Strain with the A600-1 or A600-3 Genes.  The (a) pLexNeo-A6001 and (b) pLexNeo-A6003 plasmids were created by cloning PCR amplicons for the A600-1 and A600-3 coding sequences into the Leishmania expression plasmid, pLexNeo. Re-introduction of the A600-1 or A600-3 coding sequences was confirmed for the KO+A6001.1-3 and KO+A6003.1-3 clones. Genomic D N A isolated from the wildtype (lane 1), A600' (lane 2), KO+A6001.1-3 (lanes 3-5), and KO+A6003.1-1 (lanes 6-8) clones was used as a template for PCR. (c) A 500 bp P C R product from A600-1 gene was amplified selectively in the KO+A6001 clones, (d) A 300 bp P C R productfromA600-3 was amplified selectively in the KO+A6003 clones. The A600-1 and A600-3 coding sequences were amplified from wildtype D N A (lane 1), and were not detected in D N A from the A600' clone (lane 2). A  A  110  Chapter 4 - Results  Figure 29.  Expression of the A600-1 and A600-3 Genes in the L. mexicana Wildtype, A600''-, KO+A6001.1, and KO+A6003.1 Clones.  Northern blot analysis was performed with R N A isolated from (P) log phase promastigotes and (A) axenic amastigotes. (a) The blot was hybridized with a probe for the A600-1 coding sequence, (b) The membrane had been stripped and hybridized with a probe for the A600-3 coding sequence. The membrane was stripped and re-probed for the 16S r R N A to control for loading (lower panels). The molecular weight standard (kb) sizes are indicated on the left of each blot. Ill  Chapter 4 - Results However, reconstitution with the A600-3 gene (KO+A6003) failed to complement the knockout phenotype. These results demonstrated clearly that targeted deletion of the A600 locus abolished the ability of L. mexicana to replicate as axenic amastigotes. Cultures of wildtype and KO+A6001.1 amastigotes were able to acidify the culture medium. The A600~ and KO+A6003.1 cultures did not acidify the culture medium to the A  same degree as the wildtype amastigotes. A s the acidification of the culture medium correlated with abrogated cell proliferation in A600' cells, it was hypothesized that the A600 A  protein may function to maintain a neutral intracellular pH. The intracellular pH of day 7 amastigotes was measured with a pH-sensitive fluorescent dye, 5-CFDA, which is activated by cytosolic esterases. A n intracellular pH standard curve was constructed by incubating cells in solutions of known pH that contained the N a / H transporter inhibitor, Nigericin. +  +  These samples produce broad histogram curves and the fluorescence intensity decreased as the intracellular pH decreased. Sharp histogram peaks of equal fluorescent intensity were observed when day 7 axenic amastigotes were stained with 5-CFDA. The results from this experiment had two implications. First, a neutral intracellular pH was maintained in the L. mexicana wildtype, A600"'", KO+Genel.l, and KO+Gene3.1 clones. Second, targeted deletion of the A600 locus did not affect amastigote viability as the 5-CFDA dye is only activated in live cells. The importance of the A600-1 gene for amastigote survival and proliferation in host macrophages was examined using in vitro and in vivo models of Leishmania infection.  4.1.6 The A600-1 Gene is requiredfor Intra-Macrophage Amastigote Proliferation Peritoneal macrophages isolated from 6-8 week old B A L B / c mice were infected with the L. mexicana wildtype and A600 knockout strains to determine whether the A600 genes 112  Chapter 4 - Results  1x10 1 8  Days cultured at 26°C  b =F  7.00  __  6.00  0  1  2  3  4  5  6  7  8  Days cultured at 32°C  WT—KO  Figure 30.  KO+A6001 - * - K O + A 6 0 0 3  Leishmania Growth Curves.  The cell density of L. mexicana wildtype (WT, dark blue), A600-/- (KO, pink), KO+A6001.1 (yellow), and KO+A6003.1 (light blue) cultures was determined at various time intervals, (a) Promastigote cultures were started at 1x10 cells/mL and a growth curve was constructed during a 6 day time course, (b) A n axenic amastigote growth curve was constructed from promastigotes ( l x l O cells/mL) that were transferred to 32°C and expanded in UM54 amastigote culture media. A l l culture densities were determined by counting cells using a hemocytometer. s  7  113  Chapter 4 - Results were required for intra-macrophage amastigote proliferation. Briefly, peritoneal macrophages were incubated with day 4 stationary phase promastigotes for 4 hours and the infection proceeded for 72 or 120 hours. The nuclei of both macrophages and intracellular Leishmania amastigotes were stained with Hoescht 33342 D N A dye and visualized by fluorescence microscopy. The number of amastigotes per macrophages, determined as an average for 100 macrophages, was quantified after 72 or 120 hour infections. After 72 hours, a parasite burden of 4.5 parasites per macrophage was observed for macrophages infected with L. mexicana wildtype, A6Q0~'\ KO+A6001.1, or KO+A6003.1 strains (Figure 31 and Figure 33). After 120 hours, significant differences in parasite burden were observed between macrophages infected wildtype or A600''' parasites. Peritoneal macrophages infected with L. mexicana wildtype parasites contained, on average, 100 intracellular amastigotes per cell. Conversely, macrophages infected with L mexicana A600' ' parasites contained only 4 amastigotes per macrophage (Figure 32). Re/  introduction of the A600-1 gene, but not the A600-3 gene, partially restored the ability of amastigotes to replicate within primary macrophage. Macrophages infected with KO+A6001.1 parasites contained 50 amastigotes per macrophage, while KO+A6003.1-infected macrophages had 5 amastigotes per macrophage (Figure 33). These experiments clearly demonstrated that the A600-1 gene was required for in vitro intra-macrophage amastigote replication.  4.1.7 A600 genes are required for the development of murine leishmaniasis The in vivo relevance for the A600 genes was tested using the B A L B / c mouse model for leishmaniasis. Groups of five B A L B / c mice were subcutaneously inoculated, in the flank, with cells from day 4 stationary phase cultures of the L. mexicana wildtype or A600' ' strains (Figure /  34). In mice infected with L. mexicana wildtype parasites, lesions developed between 6 and 8 weeks post-infection. Lesions increased in diameter as the disease progressed during the  114  Chapter 4 - Results subsequent six months. Mice infected with the A600' ' parasites did not develop lesions in the 6 1  months following infection.  115  Chapter 4 - Results  Wildtype  %  A600-/-  • • amastigotes  /  •  •  m W  macrophage nucleus  K O + A600-1  K O + A600-3  # *  *  *  " 'A*  i  •  • m  Figure 31.  72 hour Infection of B A L B / c Peritoneal Macrophages.  Macrophages were infected for 4 hours with the L. mexicana wildtype, A600-/-, KO+A6001.1, and KO+A6003.1 clones and cultured for 72 hours. Macrophages were fixed and stained with Hoescht 33342 D N A stain. Intracellular amastigotes are the small white dots in the cytoplasm; macrophage nuclei are the large, bright dots. 116  Chapter 4 - Results  Wildtype  A600-'-  •  •  •  •  K O + A600-1  m  K O + A600-3  amastigotes  mm"  •  macrophage nucleus  Figure 32.  •  +  120 Hour Infection of B A L B / c Peritoneal Macrophages.  Macrophages were infected for 4 hours with the KO+A6001.1, and KO+A6003.1 clones and cultured for and stained with Hoescht 33342 D N A stain. Intracellular in the cytoplasm; macrophage nuclei are the large, bright 117  L. mexicana wildtype, A600-/-, 120 hours. Macrophages were fixed amastigotes are the small white dots dots.  Chapter 4 - Results  100 o  U)  c  (0 Q.  iI 1  8  E  (0 o  2 .2>  2 IS  E < KO Leishmania  KO+1 Strain  172 hour • 120 hour  Figure 33.  Quantitation of Parasite Burden in Pertional Macrophages.  Peritoneal macrophages isolated from B A L B / c mice were infected for 72 or 120 hours with L. mexicana wildtype (WT), A600 (KO), KO+A6001.1 (KO+1), or KO+A6003.1 (KO+3) parasites. Infected macrophages were fixed and stained with Hoescht 33342 D N A stain to detect intracellular amastigotes. The average number of intracellular amastigotes was determined for 100 macrohages. Blue bars illustrate parasite burden after infection for 72 hours. Red bars illustrate parasite burden after infection for 120 hours. A  118  Chapter 4 - Results  2  3  4  5  Infection T i m e (months)  W T , n=5  Figure 34.  KO, n=5  Lesion Development in Leishmania-infected B A L B / c Mice.  Groups of five mice (n=5) were infected in the flank with 1 x 10 L. mexicana wildtype (WT) or A6QQ~ ~ (KO) parasites from day 4 stationary phase cultures. Lesion formation and progression was measured at weekly intervals during 6 month infection. 6  /  119  Chapter 4 - Discussion 4.2  Discussion This chapter described the generation of a L. mexicana A600' " strain. The unique /  expression profile of the A600 genes made them attractive targets to investigate as potential factors for amastigote survival or virulence factors. It had been hypothesized that large changes in global gene expression would be associated with the morphological and biochemical changes  associated with parasite  differentiation from promastigotes  to  amastigotes; however, only a small number of genes with amastigote-specific expression profiles have been characterized to date. More recently, large scale R N A and protein expression profiling experiments have indicated that almost all Leishmania genes have less than two-fold changes in expression throughout the parasite lifecycle (Saxena et al., 2003; Akopyants et al., 2004; Nugent et al., 2004). These observations provide additional importance to elucidating the function of genes that are preferentially expressed in amastigotes. Presumably this expression profile reflects a unique, and perhaps essential, function for the encoded protein in amastigotes. There have been intensive efforts to identify genes involved in Leishmania survival and virulence within the sandfly vector and mammalian host macrophages. Investigators have identified putative Leishmania virulence factors on the basis of homology to well characterized genes from other organisms, or novel genes preferentially expressed in a particular stage of the parasite lifecycle. For example, Leishmania enzymes required for the complex glycosylation of membrane lipids were cloned on the basis of homology to known enzymes (Spath et al, 2000; Spath et al, 2003; Zhang et al, 2004). Other Leishmania genes have been identified on the basis of developmentally regulated gene expression. To this end, the A2, amastin, and A600-3 genes were cloned as c D N A fragments with amastigote-specific expression (Charest et al, 1994; Wu et al, 2000; Bellatin et al, 2002). The Imcpb gene was 120  Chapter 4 - Discussion cloned on the basis of sequence homology to cysteine proteases from other organisms (Souza et al., 1992). The functional role of these and other Leishmania genes has been examined by targeted gene deletion. A  system for targeted deletion of genes by homologous recombination for  Leishmania has proved to be a powerful tool to study gene function. (Cruz et al, 1990; Cruz et al, 1991). As Leishmania parasites replicate asexually, two rounds of gene replacement are required to generate a homozygous knockout mutant. Fortunately, there are a number of positive selection markers, which confer resistance to the following drugs: Geneticin (G418), Hygromycin B , Puromycin, Phleomycin, Nourseothricin, and Blasticidin (Cruz et al, 1990; Laban et al, 1990; Freedman et al, 1993; Joshi et al, 1995; Goyard et al, 2000). Targeted deletion has been accomplished for several single copy Leishmania genes, including the DHFR-TS, HEXBP, cysteine protease a (cpa), LPG1 (galacto-furanosyl transferase), LPG2 (guanosine mono- phosphate transporter), and LPG3 genes (Cruz et al, 1991; Souza et al, 1994; Webb et al,  1994; Bart et al,  1997; Spath et al, 2000; Spath et al, 2003).  Furthermore, this approach has been used successfully for targeted deletion of large multigene loci, including the L. major gp63, L. mexicana cysteine protease b (cpb), L. mexicana glucose transporters (LmGT), and the L. mexicana paraflagellar rod (PFR2) loci (Mottram et al, 1996; Santrich et al, 1997; Joshi et al, 2002; Burchmore et al, 2003). The ability to knockout genes tandemly arranged at a single genomic locus provides a tremendous advantage to elucidate gene function. However other multi-gene families, which include the amastigote-specific amastin and A2 genes, are encoded at more than one genomic locus and are not amenable to targeted gene deletion. Exceptions to this rule are the cpa/cpb double mutant strain and the triple deletion mutant for three LPG1 homologs (Alexander et al, 1998; Zhang et al, 2004). R N A interference (RNAi) has been used as a strategy to 121  Chapter 4 - Discussion "knockdown" gene expression in T. brucei, although R N A i biochemical pathways do not appear to exist in Leishmania. The tandem arrangement of the A600 genes made it possible for the entire genomic locus to be replaced by targeted gene deletion. The HygKO and SatKO targeting constructs were generated with flanking sequence from upstream and downstream of the A600 locus. It was relatively easy to generate A600 " clones, as we achieved 100% success for homologous +/  recombination at the A600 locus during the first round of transfection. However, attempts to generate A600 " clones with a second round of transfection proved to be a challenge. Initial _/  attempts produced false positive colonies, and in transfections that produced true A600'" clones, only 25% of the colonies had targeted deletion of the remaining wildtype allele at the A600 locus. The relative difficulty of generating a true "knockout" has been described previously for essential genes in Leishmania, including the a-tubulin, DHFR-TS and A2 gene clusters (Curotto de Lafaille et al, 1992; Cruz et al, 1993; Zhang et al, 2001). One could interpret the difficulty of replacing the remaining A600 allele as being consistent with an important gene, although with the amastigote-specific expression of the A600 genes one would not expect to encounter problems generating A600~ mutants in promastigotes. A  Targeted deletion of the A600 locus was confirmed by Northern and Western blot analysis. Northern blots of L. mexicana total R N A were hybridized with probes for the A6001 and A600-3 coding sequences. The results from these experiments convincingly demonstrated that the A600 genes were not expressed in the KOSat35.2 A6QQ~'~ clone. Western blot analysis of protein isolated from L. mexicana wildtype and A600~ axenic A  amastigotes showed that A600 protein was not expressed in the A600' clone. The oc-A600 A  antibody was raised against a synthetic peptide corresponding to sequence conserved in the A600-1 and A600-3 predicted proteins. Western blot analysis performed with soluble and 122  Chapter 4 - Discussion insoluble TritonX-100 protein lysates showed that the A600 protein was detected specifically in the insoluble fraction of wildtype cells. It is hypothesized that the A600 proteins are membrane proteins that localize to detergent-resistant membrane domains, which sediment with cell debris and cytoskeletal structures. A similar result was observed during characterization of an 11 kDa T. brucei protein, k m p l l , which was a highly abundant, immunogenic membrane protein (Jardim et al,  1995). TritonX-100 detergent-resistant  membrane domains could be disrupted with stronger detergents to isolate the A600 proteins in the soluble protein fraction. The presence of A600 protein in the insoluble fraction from wildtype amastigotes, and not in the A600' ' amastigotes, validated our predicted open reading 1  from the A600-3 c D N A sequence. In assigning a more specific function to the A600 proteins, it would be  helpful  to  obtain  information  regarding  subcellular  localization by  immunostaining fixed amastigote cells. The A600~'~ mutant parasites demonstrated an interesting growth phenotype as axenic amastigotes. Proliferation of wildtype and A600~'~ axenic amastigotes was equivalent during the initial four days of culture at 32°C in UM54 culture medium. Thereafter, wildtype cells continued to replicate, while the A600~ ~ cells remained viable but stopped dividing. The /  growth inhibition phenotype was also observed during in vitro infection of B A L B / c peritoneal macrophages. The wildtype and A6QQ~'~ parasite burden was equivalent during the initial 72 hours of infection, which indicated the wildtype and A600~'~ clones have an equivalent capacity for attachment, uptake, and to establish an infection inside macrophages. At 120 hours post-infection, there was a clear distinction between macrophages infected with wildtype and A6QQ~'~ parasites. The wildtype cells continued to replicate inside macrophages with approximately 100 parasites per macrophage, while macrophages infected with A600 " v  parasites maintained a persistent low level of infection. Interestingly, A600' ' cells 1  123  Chapter 4 - Discussion reconstituted with the A600-1 gene, KO+A6001, regained the capacity to proliferate as both axenic and intramacrophage amastigotes. However, re-introduction of the A600-3 gene into A600" cells did not complement the growth defect for these axenic amastigotes. It is A  important to note that A600-3 expression from the pLexNeo-A6003 plasmid produced a high molecular weight m R N A transcript, which may not be translated efficiently. It is possible that the inability of the A600-3 gene to complement the knockout phenotype resulted from faulty R N A processing. Fortuitously, the A600-1 and A600-3 R N A transcripts were preferentially expressed in the amastigote stage. Although it may appear that the macrophages were able to control an infection by A6Q0~'~ cells, one could argue that abrogation of A600~'~ amastigote proliferation was due to an inherent defect in the ability of those amastigotes to replicate at a higher temperature or more acidic environment. To our knowledge, the failure of A600 " mutants to replicate as axenic amastigotes is v  only the second such phenotype to be described in Leishmania. The Imgf ' mutant strain, 1  which was generated by targeted deletion of the three gene L. mexicana glucose transporter locus, had major defects of promastigote growth and showed a complete inability to replicate as axenic amastigotes. Furthermore, Imgf ' parasites were unable to establish infections of 1  peritoneal macrophages or to generate lesions in B A L B / c mice. These experiments convincingly showed that promastigote and amastigote growth was dependent on glucose uptake by Leishmania (Burchmore et al, 2003). The Imcpb' ' parasites, which were generated 1  by targeted deletion of the L. mexicana cysteine protease b locus, showed a slight growth defect as axenic amastigotes. However, re-introduction of the CPB2.8 gene did not restore normal amastigote growth, which suggested that the growth phenotype may be related to that particular clone (Bart et al,  1997). Antisense R N A mediated knockdown of A2 gene  expression did not affect the viability or growth of L. donovani axenic amastigotes. 124  Chapter 4 - Discussion Interestingly, the A2 "knockdown" cells established successful infections in peritoneal macrophages, while infected B A L B / c mice had a dramatically lower hepatic parasite burden than mice infected with L. donovani wildtype parasites (Zhang et al., 1997). The discrepancy in parasite burden from in vitro and in vivo infections, in combination with normal growth of axenic amastigotes, suggested that the A2 genes were not required for intra-macrophage amastigote survival and replication; rather, the A2 genes may act as an L. donovani virulence factor to modulate the host immune response to infection. Expression of the A2 gene in L. major abrogated the ability of L. major promastigotes to establish progressive lesions in infected B A L B / c mice, which provided evidence that the A 2 protein may be involved in L. donovani tissue tropism (Zhang et al., 2003; Garin et al., 2005). There has been a great deal of effort to characterize the importance of Leishmania surface glycolipids and sphingolipids for parasite virulence. The Leishmania enzymes involved in biosynthesis of complex glycoconjugates has been studied in L. major, which is not amenable to culture as axenic amastigotes. Targeted deletion of the Ipgl gene caused a defect in the ability of amastigotes to establish an infection in macrophages, and these parasites showed a delay in lesion formation (Spath et al., 2003). A n identical phenotype was observed for a sphingolipid-free L. major mutant generated by targeted deletion of an essential subunit of the serine palmitoyltransferase (spt) enzyme, which is required for de novo sphingolipid biosynthesis (Zhang et al., 2005). A n elegant approach was used to determine whether reduced parasite virulence occurred as a result of direct effects on amastigote virulence or defects in the ability of metacyclic promastigotes to establish macrophages infections. Lesion amastigotes of wildtype, Ipgl' ', and spt' ' were equally 1  1  capable of establishing and maintaining infections of peritoneal macrophages, which showed that amastigotes virulence was not dependent on L P G or sphingolipids (Spath et al., 2000; 125  Chapter 4 - Discussion Zhang et al, 2005). Targeted deletion of the L. major gp63 locus also resulted in delayed lesion formation in B A L B / c mice, which was attributed to increased sensitivity to complement-mediated killing (Joshi et al, 2002). It would be interesting to examine the ability of gp63~'~ parasites to infect peritoneal macrophages, and determine whether these parasites are more susceptible to the microbicidal activities of macrophages. It was hypothesized that the growth inhibition of A600' ' cells may be related to 1  acidification of the local environment. The small size of the A600 proteins suggested that they may be cofactors or subunits of a larger enzyme complex. As amastigotes have a neutral intracellular pH, they must maintain a two-log proton gradient across the membrane in a pH 5.0 macrophage phagolysosome or UM54 amastigote culture media. The intracellular pH of wildtype, A600' ', KO+A6001, and KO+A6003 amastigotes was determined with a pH 1  indicator fluorescent dye, 5-CFDA. The results from these experiments showed that the intracellular pH of the wildtype and A600' ' parasites was equivalent. Furthermore, these 1  experiments demonstrated that the A600' ' parasites were viable as the 5-CFDA dye must be 1  cleaved by cytosolic esterases to yield a fluorescent molecule. Our understanding of amastigote biology and virulence is limited by the fact that this stage of the lifecycle does not appear to actively modify the surrounding acidic and hydrolytic environment of the macrophage phagolysosome. The process of Leishmania uptake by host macrophages has been described as "silent" as phagocytosis failed to stimulate the inflammatory and microbicidal pathways of activated macrophages. Indeed, parasite virulence has been characterized as the ability of promastigotes to infect macrophages without eliciting the production of nitric oxide or inflammatory cytokines (Spath et al, 2003). A slight delay in phagolysosome fusion has been described during  126  Chapter 4 - Discussion macrophage infections with L. donovani promastigotes, although phagosome maturation is complete by 48 hours post-infection (Desjardins et al., 1997). Preliminary experiments showed that macrophages infected with L. mexicana wildtype or A600' ' for 4 hours produced only basal levels of tumor necrosis factor-ct (TNF1  ct) and nitric oxide. This result indicated that A600' ' stationary phase promastigotes retained 1  the ability to infect macrophages without activating an antimicrobial or inflammatory response. Additional in vitro infection experiments could be performed using peritoneal macrophages deficient for oxidative burst. These experiments could determine whether the cessation of intramacrophage proliferation by A600' ' amastigotes was the result of increased 1  sensitivity to reactive oxygen intermediates. The molecular mechanism for Leishmania-mediated suppression of macrophage activity has been attributed to activation of the host SHP-2 phosphatase, which inhibited signalling through the IFN-y receptor. A Leishmania homolog of E F - l a has been directly implicated in SHP-2 activation, as it was secreted into the macrophage cytosol and immunoprecipitated with SHP-2. This mechanism for intracellular amastigote survival provided new insight into the immune evasion strategies of Leishmania, although the strategies for parasite survival within the mature phagolysosome remain to be determined. It would be interesting to infect macrophages with L. mexicana wildtype or A600' ' parasites 1  and subsequently  determine whether infected macrophages  are equally resistant to  stimulation by inflammatory stimuli, such as LPS and/or IFN-y. The in vivo virulence of these parasites was assayed using the B A L B / c mouse model for leishmaniasis. Mice infected with L. mexicana wildtype parasites developed progressive lesions, while A600 " parasites failed to develop lesions. Unfortunately, continued passage of 7  these parasites in culture had attenuated virulence and the wildtype parasites only developed 127  Chapter 4 - Discussion lesions two months post-infection. Recently, lesions failed to develop when B A L B / c mice were infected with WT, A600' ', KO+A6001, and KO+A6003 cells; attenuated parasite 1  virulence was attributed to prolonged in vitro culturing of these cells. Fortunately, virulent parasites can be isolated from mice infected with high infectious dose (5x10 ) of stationary phase promastigotes. Subsequently, in vivo mouse infections will be performed with l x l O  6  virulent, stationary phase promastigotes to determine whether A600' ' parasites are able to 1  develop progressive lesions. It is expected B A L B / c mice will either fail to form lesions or develop small, self-limiting lesions due to the previously observed growth defects for A600' ' 1  amastigotes. If the mouse infections parallel the in vitro peritoneal macrophage infection results, the KO+A6001 clone would be expected to produce smaller lesions than wildtype parasites, and perhaps with a slightly delayed onset. Mice infected with the KO+A6003 clone are expected to show an identical phenotype to an A600' "infection, as observed previously /  for the in vitro infection experiments.  It also will be interesting to quantitate the parasite  burden per mouse by limiting dilution, as amastigotes have been known to persist at the site of infection in asymptomatic mice (Spath et al., 2003).  128  Chapter 5 - Results 5.  Role of the A600-3 3'UTR for Stage-Specific Gene Expression This chapter describes a set of experiments that define the regulatory mechanisms for  stage-specific expression of the A600-3 gene. The current model for Leishmania gene expression is that large, polycistronic transcription units are processed into unique mRNA transcripts for each gene. There is accumulating evidence that regulation of gene expression is mediated by regulatory elements in the 3'UTR sequence of processed m R N A transcripts. Stage-specific expression of the A600-3 gene was examined using reporter gene constructs with the A600-3 3'UTR cloned downstream of the luciferase coding sequence.  5.1  Results  5.1.1  Luciferase Reporter Constructs A luciferase reporter gene construct was used to demonstrate that the A600-3 3'UTR  conferred stage-specific expression to the heterologous luciferase reporter gene and to identify regulatory sequence elements within the 3'UTR sequence. The reporter construct was contained by sequences that flank the A600 locus to facilitate targeted integration of the reporter constructs at the A600 locus. In addition the reporter constructs contained the hph selection marker and a-tubulin intergenic region, which provided stable expression of the selection marker (Figure 35). The rationale for targeted integration of the reporter constructs at the A600 locus was eliminate the confounding effect of differences in plasmid copy number and the possibility that local chromatin arrangement at the A600 locus may contribute to regulation of A600-3 gene expression. The reporter constructs were generated with A600-3 3'UTR and intergenic (3'UTR+IR) sequences, full length or truncated versions, cloned downstream of the luciferase gene.  129  Chapter 5 - Results  130  Chapter 5 - Results 5.1.2  The A600-3 3 'UTR Regulates Amastigote-Specific Gene Expression In proof-of-principle experiments, the full length A600-3 3'UTR+IR was amplified  from the pBST-A600yC plasmid using the UTR1 "forward" primer the M13R20 "reverse" primer from the pBluescript K S vector. The 3.6 kbp 3'UTR+IR amplicon was flanked by Xbal sites, which were introduced from the UTR1 primer and the multiple cloning region of the vector. The pHyg/Luc-Fwd and pHyg/Luc-Rev plasmids were constructed by cloning the 3'UTR+IR P C R product into the pHyg/Luc vector at the Xbal site downstream of the luciferase gene. The pHyg/Luc-Fwd plasmid was generated by cloning the 3'UTR+IR in the sense orientation, while pHyg/Luc-Rev plasmid was formed by cloning the 3'UTR+IR fragment in the antisense orientation (Figure 35a,b). The 0.65 kbp A600-3 intergenic region (IR) was amplified with the A600IR-F and A600IR-R primers that contained 5' artificial Xbal and Kpnl restriction sites, respectively. The pHyg/Luc-IR plasmid was constructed by cloning the A600IR into the pHyg/Luc vector. (Figure 35c). Luciferase reporter constructs also could be purified from the pLitmus28 plasmid vector, as Spel/Kpnl restriction fragments, for targeted integration at the A600 locus (Figure 35d). L. mexicana promastigotes were transfected, by electroporation, with circular plasmids of pHyg/Luc-Fwd, pHyg/Luc-Rev, and pHyg/Luc-IR. Transfected cells were selected on Ml99-Agar plates that contained 35 u,g/mL of Hygromycin B (Hyg) drug. Hygresistant clones were transferred into liquid M l 9 9 media and the cultures were maintained in 50 p-g/mL Hyg drug. Southern blot analysis was performed with KpnI-digested D N A isolated from two pHyg/Luc-Fwd, six pHyg/Luc-Rev, and three pHyg/Luc-IR independent clones. The blots were hybridized with a probe for the luciferase coding sequence to confirm that these clones contained the luciferase reporter constructs (Figure 36a,b). The pHyg/Luc-Fwd  131  Chapter 5 - Results  a Sizes (kb)  pLuc-Rev  3  4  5  pLuc-IR  3  4  5  10« 8' 6" 5"  pLuc-Rev  Figure 36.  pLuc-Fwd  L. mexicana Clones Transfected with the Luciferase Reporter Plasmids.  Southern blot analysis was performed with KpnI-digested D N A isolated from promastigotes. (a) pLuc-Rev clones 3-5 and pLucIR clones 3-5. (b) pLuc-Rev clones 1-2 and pLuc-Fwd clones 3 and 6. The blots were hybridized with a probe for the luciferase coding sequence. Molecular weight marker (kb) sizes are indicated at the left of the blots. 132  Chapter 5 - Results and pHyg/Luc-Rev plasmids were detected as 11 kbp bands, while the pHyg/Luc-LR plasmid was seen as a 7.5 kbp band. These clones were referred pLuc-Fwd, pLuc-Rev, and pLuc-IR. Northern blot analysis was performed to compare the steady state level of luciferase m R N A transcripts in promastigotes and amastigotes of the pLuc-Fwd, pLuc-Rev, and pLucIR clones (Figure 37). In the pLuc-Fwd clone, the sense orientation A600-3 3'UTR+LR conferred preferential amastigote expression to the luciferase m R N A transcript, which was three-fold more abundant in amastigotes. In the pLuc-Rev clone, the luciferase probe hybridized to a broad smear that was detected predominantly in promastigotes. The luciferase probe hybridized very weakly to pLuc-IR promastigote and amastigote R N A , which indicated that the downstream intergenic region of the A600-3 gene was not sufficient for stable gene expression. This experiment clearly demonstrated that expression of the heterologous luciferase reporter gene was regulated by the downstream 3'UTR sequence. Western blot analysis was used to assay for luciferase protein expression from the luciferase reporter plasmids. Luciferase protein expression from the pLuc-Fwd construct was approximately three-fold higher in amastigotes, relative to promastigotes (Figure 38). Large differences in relative luciferase protein expression were observed in pLuc-Fwd and pLucRev amastigotes,  while luciferase expression was equivalent in the  corresponding  promastigotes. A quantitative comparison of luciferase band intensity determined that there was a nine-fold difference in expression between the pLuc-Fwd and pLuc-Rev amastigotes. Luciferase protein expression from the pLuc-IR construct was undetectable in the promastigote and amastigote stages.  133  Chapter 5 - Results  pLuc -IR  Figure 37.  pLucRev  pLuc Fwd  Luciferase Gene Expression as a Chimeric Transcript with the A600-3 3'UTR.  Northern blot analysis was performed with total R N A isolated from promastigotes (P) and amastigotes (A) of the pLuc-IR, pLuc-Rev, and pLuc-Fwd clones. The blot was hybridized with a probe for the luciferase coding sequence. The membrane was stripped and re-probed for the 16S rRNA to control for loading (bottom panel). Molecular weight marker (kb) sizes are shown at the left of the blot. 134  Chapter 5 - Results  pLuc- pLucFwd IR Sizes (KDa)  P A  P A  pLuc -Rev P A  < 62  •-  1 a  2  UJ  1  Fw d  Rev  IR  Luciferase Construct  | Promastigote | Amastigote  Figure 38.  Effects of the A600-3 3 ' U T R for Luciferase Protein Expression.  Western blot analysis for luciferase expression was compared in the promastigote (P) and amastigote (A) stages of the lifecycle. 25 u,g of Triton X-100 protein lysate from of the wildtype (WT), pLuc-Fwd, pLuc-IR, and pLuc-Rev clones was separated on a 10% SDSP A G E gel. Protein was transferred to P V D F membrane and hybridized with polyclonal ctluciferase antibody. The 62 kDa luciferase protein was detected with HRP-conjugated antigoat secondary antibody. Molecular weight markers (kDa) sizes are indicated at the left of the blot, (b) Quantitation of relative luciferase protein abundance in each sample 135  Chapter 5 - Results 5.1.3  A Regulatory Element Exists at Position 1500 - 2500 of the A600-3 3 'UTR A deletion strategy was used to identity regulatory regions within the 2.9 kb of  3'UTR sequence. A600-3 3'UTR deletions were created by a PCR-based deletion strategy, which used the pBST-A600yC plasmid as a template. Progressively larger deletions were introduced at the 5' end of the A600-3 3'UTR, while maintaining a trans-splicing signal in the downstream intergenic sequence. Six "forward" P C R primers (UTR1 - UTR6), with artificial 5' Xbal sites, were designed at 500 bp intervals along the sense strand of the 2.9 kbp A600-3 3'UTR (Figure 39a). The six 3'UTR deletion fragments (UTR1 - AUTR6) were produced with P C R reactions using these forward primers and the common A600LR-R (intergenic region) reverse primer (Figure 39b). P C R amplification was performed with a proof reading Taq polymerase to ensure that sequence errors were not introduced into the 3'UTR deletion fragments. The P C R products were amplified with these deletion primers and blunt-end cloned into the pBluescript K S vector; the UTR1 - AUTR6 deletion products of these P C R reactions were 3.6 kbp, 3.1 kbp, 2.6 kbp, 2.1 kbp, 1.6 kbp, and 1.1 kbp, respectively (Figure 39c). These deletion products were subcloned into the pHyg/Luc vector at the Xbal and Kpnl sites downstream of the luciferase gene. Linear luciferase reporter constructs were purified from 50 pg of each plasmid by digestion with the Spel and Kpnl restriction enzymes. Recombinant L. mexicana parasites were generated by targeted integration of the L u c - U T R l , -AUTR3, -AUTR4, and -AUTR6 constructs at the A600 locus. Log phase, wildtype parasites were transfected with 10 pg of purified reporter construct and Hyg-resistant clones were isolated from Ml99-Agar plates containing 35 pg/mL Hygromycin B selection. There were nine independent clones of LucU T R l (Luc-UTRl. 1-9), five clones with Luc-AUTR3 (Luc-AUTR.3.1-5), seven clones of  136  Chapter 5 - Results Luc-AUTR4 (Luc-AUTR4.1-7), and eight clones of Luc-AUTR6 (Luc-AUTR6.2-8). An Xhol site from the ct-tubulinlR sequence was introduced at the A600 locus by targeted integration of the Luc-AUTR reporter constructs. A restriction map for the integrated reporter constructs indicated that a probe for the hph sequence would hybridize to a 1.5 kbp Xhol restriction fragment after targeted integration at the A600 locus (Figure 40a). Southern blot analysis was performed with .Yftoi-digested genomic D N A from the L u c - U T R l , Luc-AUTR3, LucAUTR4, and Luc-AUTR6 clones. The blot was probed for the hph coding sequence to identity clones which successfully had integrated these constructs at the A600 locus. Integration of the L u c - U T R l , -AUTR3, and -AUTR4 constructs at the A600 locus was less efficient than expected, as chromosomal integration was confirmed only for the L u c - U T R l . 1, -AUTR1.5, -AUTR3.3, -AUTR3.4, and -AUTR4.7 clones. However, all eight Luc-AUTR6 clones were shown to have integration of the reporter construct at the A600 locus (Figure 40b,c). These clones were generated with luciferase reporter constructs that were created with progressively larger deletions from the proximal end of the A600-3 3'UTR. Northern and Western blot analysis was performed with the L u c - U T R l . 1, Luc-AUTR4.7, and LucAUTR6.2 clones to examine the effect of these 3'UTR deletions for expression of the heterologous luciferase gene  5.1.4  Analysis of Luciferase Expression in Transfected Leishmania It was shown previously that the A600-3 downstream intergenic region was unable to  confer expression to luciferase R N A or protein expression (Figure 37 and 38). As expected, Northern and Western blot analysis of luciferase expression in the L u c - U T R l . 1 clone was similar to the data obtained from the pHyg/Luc-Fwd clone. Preferential expression of the  137  Chapter 5 - Results  UTR1  UTR2  A600-3  UTR3  UTR4  UTR5  UTR6  A600-3 3'UTR + IR A600IR-R  PCR amplification of 3'UTR deletion fragment  A600-3  3'UTR + IR  UTR 1 AUTR2 AUTR3 AUTR4 AUTR5 AUTR6  Figure 39.  Sizes (kb)  1 2 3 4 5 6  4 3 2 1.5 1  0.5  Schematic of PCR-based A600-3 3'UTR Deletion Strategy.  (a) Diagram of the A600-3 3'UTR+LR with the UTR1 - UTR6 forward primers designed at 500 bp intervals along the sense strand, and the A600IR reverse primer on the antisense strand, (b) Schematic of the PCR amplicons (UTR1 - AUTR6), which contain artificial Xbal and Kpnl restriction sites for cloning into the pHyg/Luc vector, (c) Agarose gel with the gel purified P C R amplicons for the A600-3 3'UTR+IR deletions, UTR1 - AUTR6 (lanes 1-6). Molecular weight marker (kb) sizes are indicated at the right of the figure. 138  Chapter 5 - Results  a  —  o  X  1  A600 l o c u s  2.U  5'A600  o X  /ip/7 i |TublR  (0  2.2.  luciferase  o  X  4600-3 3'UTR + IR AUTR 1 AUTR 2 AUTR 3  o  X i  1.5 kb  X  AUTR 4  |  AUTR 5  hph probe  AUTR 6  Luc-UTRl Sizes (kb) 106 "  1  WT  Luc-AUTR6  2 3 4 5 6 7 8 9  21.5 " 1"  Luc-AUTR3 2 3 4 5  Luc-AUTR4 1 2 3 4 5 4 7  10 6 2 1.5 1  Figure 40.  Targeted Integration of Luciferase Reporter Constructs at the A600 Locus.  (a) Illustration for integration of the reporter construct at the A600 locus. The wildtype A600 locus is shown with the A600 coding sequences (red arrows). The integrated reporter construct introduced an Xhol site, from the cctubulin intergenic region (TublR), into chromosomal D N A . A probe for the hph coding sequence (black box) hybridized to a 1.5 kb Xhol fragment i f the construct integrated at the A600 locus. The a-tubulin intergenic region of the Luc-AUTR reporter constructs introduced an Xhol site into chromosomal D N A at the A600 locus. (b,c) Southern blot analysis was performed with ^7/o/-digested genomic D N A isolated from L. mexicana wildtype (WT), nine L u c - U T R l clones, four Luc-AUTR3 clones, seven Luc-AUTR4, and eight Luc-AUTR6 clones. The blots were hybridized with a probe for the hph coding sequence, which detected a 1.5 kb band when the reporter constructs had integrated at the A600 locus. Molecular weight marker (kb) sizes are indicated at the left of the blots. Red arrowheads show the 1.5 kb band that demonstated integration of the reporter construct at the A600 locus. 139  Chapter 5 - Results luciferase gene also was observed in amastigotes of the Luc-AUTR3.3 and Luc-AUTR4.7 clones which consisted of 1 kb and 1.5 kb deletions from the proximal end of the A600-3, 3'UTR (data not shown). Northern blot analysis was performed to compare luciferase expression in promastigotes and amastigotes of the L u c - U T R l . l , Luc-AUTR4.7, and LucAUTR6.2 clones. The blot was hybridized with a probe for the luciferase coding sequence, which detected 4.8 kb L u c - U T R l , 3.3 kb Luc-AUTR4, and 2.3 kb Luc-AUTR6 mRNA transcripts (Figure 41a). The Northern blot was stripped and re-probed for the A600-3 coding sequence, which hybridized to a 3.3 kb R N A transcript in the amastigote samples (Figure 41b). The relative abundance of the luciferase and A600-3 transcripts was approximately equal in L u c - U T R l . l and Luc-AUTR4.7 amastigotes. However, the luciferase transcript almost was undetectable in Luc-AUTR6.2 amastigotes, which had similar levels of the A6003 transcript to the L u c - U T R l . l and Luc-AUTR4.7 clones. This result indicated that deletion of a regulatory sequence within the sequence at position 1500 - 2500 bp A600-3 3'UTR abrogated luciferase gene expression. This region, referred to as the "regulatory domain" (RD), clearly contained a sequence that conferred stage-specific expression to the heterologous luciferase gene. Relative levels of luciferase protein expression were assayed by Western blot analysis. The L u c - U T R l . l clone showed almost identical luciferase protein expression to the pLuc-Fwd3 clone. The luciferase protein was preferentially expressed in the L u c - U T R l . l and Luc-AUTR4.7 amastigotes, although lower expression was seen in both Luc-AUTR4.7 promastigotes and amastigotes. In the Luc-AUTR6.2 clone, the luciferase protein was expressed preferentially in the promastigote stage (Figure 42a). A Coomassie stained SDSP A G E gel, which was run in parallel, indicated that equal amounts of protein were  140  Chapter 5 - Results  c  o "</)  A600-3 3'UTR + IR UTR 1  a x  AUTR4  0) CD  AUTR6  2 o Q.  5 WT  Luc1.1  Luc4.7  Luc6.2  Clone  Figure 41.  Effects of A600-3 3'UTR Deletions on Luciferase Expression.  Northern blot analysis was performed with total R N A isolated from promastigotes (P) and amastigotes (A) of wildtype (WT), L u c - U T R l . 1, -AUTR4.7, and -AUTR6.2 clones, (a) The blot was hybridized with a probe for the luciferase coding sequence, (b) The blot was stripped and re-probed for the A600-3 coding sequence. The membrane was hybridized with a probe for the 16S r R N A to control for loading (bottom panels). Molecular weight marker (kb) sizes are shown at the left of the blot, (c) Quantitation of amastigote:promastigote (A/P) relative luciferase R N A abundance after correction for differences in r R N A band intensity, (d) Schematic of the A600-3 3'UTR deletion constructs. 141  Chapter 5 - Results  b  c WT  LucLucLucAUTR1 AUTR4 AUTR6  P A P  Figure 42.  A  P  A  P  A  A600-3 3'UTR + IR  Effects of the A600-3 3'UTR Deletions On Luciferase Protein Expression.  (a) 25 pg of Triton X-100 protein lysate from promastigotes (P) and amastigotes (A) of the wildtype (WT), L u c - U T R l . 1, Luc-AUTR4.7, and Luc-AUTR6.2 clones was separated on a 10% SDS-PAGE gel. (a) Western blot analysis for luciferase expression. Protein was transferred to P V D F membrane and hybridized with polyclonal ct-luciferase antibody. The 62 kDa luciferase protein was detected with HRP-conjugated anti-goat secondary antibody (red arrow), (b) Coomassie stained 10% SDS-PAGE gel shows equal loading of promastigote and amastigote protein lysate. (c) Schematic of A600-3 3'UTR deletion constructs. Molecular weight markers (kDa) sizes are indicated at the left of the blot. 142  Chapter 5 - Results loaded in each well (Figure 42b). The results from these experiments also indicated that a regulatory sequence existed between positions 1500 - 2500 bp of the A600-3 3'UTR. Additional 3'UTR deletion mutants were generated to more precisely map the regulatory sequence element.  5.1.5  Fine Mapping the Regulatory Sequence in the RD Region A series of 250 bp deletions were created from the proximal end of the R D region of  the A600-3 3'UTR, using the P C R strategy described previously. The ARD1, ARD2, and ARD3 PCR products were generated using the RD1, RD2, or RD3 "forward" primers and the A600IR-R "reverse" primer (Figure 43a). The resulting PCR products were blunt-end cloned into the pBluescript K S vector as 1900 bp, 1650 bp, and 1400 bp fragments, respectively. The ARD1, ARD2, and ARD3 fragments were subcloned into the pHyg/Luc vector at the Xbal and Kpnl sites downstream of the luciferase gene. The resulting L u c - A R D l , LucARD2, and Luc-ARD3 constructs were transfected into wildtype L. mexicana and integrated at the A600 locus. Northern blot analysis was performed with total R N A isolated from promastigotes and amastigotes of the L u c - U T R l , -AUTR3, -AUTR4, -AUTR6, -ARD1, -ARD2, and -ARD3 clones (Figure 44). The blot was hybridized with a probe for the luciferase coding sequence, which indicated that the luciferase R N A transcript was preferentially expressed in the LucU T R l , -AUTR3, and -AUTR4 amastigotes. As expected, the luciferase transcript almost was undetectable in Luc-AUTR6 amastigotes. The luciferase transcript abundance had decreased significantly in amastigotes, relative to expression from the Luc-AUTR4 clone. In Luc-ARDl and Luc-ARD2 promastigotes, however, relative luciferase R N A expression was comparable  143  Chapter 5 - Results to the Luc-AUTR3 and Luc-AUTR4 promastigotes. The Luc-ARD3 and Luc-AUTR6 clone luciferase R N A transcript was almost undetectable in both promastigotes and amastigotes. The levels of luciferase protein expression in amastigotes was highest in the LucU T R l clone, although equivalent levels of luciferase protein were detected in amastigotes of the Luc-AUTR3, -AUTR4, -AUTR6, -ARD1, -ARD2, and -ARD3 clones. Luciferase protein expression in the promastigote stage clearly was affected by the A600-3 3'UTR deletions. The L u c - U T R l , -AUTR3, -AUTR4 promastigotes had low levels of luciferase protein, while Luc-AUTR6 promastigotes had upregulated expression of the luciferase protein (Figure 45). Low levels of luciferase protein were detected in Luc-ARDl promastigotes, while expression was elevated significantly in Luc-ARD2 and -RD3 promastigotes. These results suggest that translation of the luciferase protein was regulated by a sequence element in the RD2 region (2000 - 2250 bp) of the A600-3 3'UTR. Data from these R D deletion mutants indicated that R N A stability and protein expression were regulated by distinct elements within the R D region. These experiments provided loss-of-function evidence for a regulatory element in the RD2 region. It was hypothesized that regulatory elements may have been conserved in the A600-1 and A600-3 3'UTR sequences, which only share 36% identity. D N A Block Aligner software,  available at http://www.ebi.ac.uk/Tools/sequence.htmL. identified a 15 bp,  pyrimidine-rich D N A sequence (CTCACCTCCGTTTT)  in the RD2 region that was  conserved in the A600-1 3'UTR (Figure 46). It is proposed that this 15 bp sequence may influence expression of A600-1 and A600-3 genes by negative regulation of A600 protein translation in L. mexicana promastigotes.  144  Chapter 5 - Results  Figure 43. 3'UTR.  Targeted Deletions Across the Regulatory Domain (RD) in the A600-3  The RD1, RD2, and RD3 "forward" primers (red arrows) were designed at 250 bp intervals across the 1000 bp R D sequence. The ARD1, ARD2, and ARD3 P C R products were cloned downstream of the luciferase gene. Grey bars represent the A600-3 3'UTR. Maroon bars represent the A600-3 IR. 145  Chapter 5 - Results  UTR1  Sizes (kb)  AUTR3  A  AUTR4  P  A  ARD1  P  A  ARD2  ARD3  P A  P A  AUTR6  P  A  9.5. 7.4-  4.4-1 2.3 1.3  Figure 44.  Effects of A600-3 3'UTR Deletions for Expression of the Luciferase Gene.  Northern blot analysis was performed with total R N A isolated from L. mexicana promastigotes (P) and amastigotes (A) of L u c - U T R l , -AUTR3, -AUTR4, -AUTR6, -ARD1, ARD2, and -ARD3 clones. The blot was hybridized with a probe for the luciferase coding sequence. The membrane was hybridized with a probe for the 16S r R N A to control for loading (bottom panels). Molecular weight marker (kb) sizes are shown at the left of the blot. 146  Chapter 5 - Results  UTR1  Figure 45.  AUTR3  AUTR4 AUTR6  ARD1  ARD2  ARD3  Effects ofA600-3 3'UTR Deletions for Expression of the Luciferase Protein.  Western blot analysis of L. mexicana promastigotes (P) and axenic amastigotes (A) for luciferase protein expression. 25 pg of Triton X-100 protein lysate from the L u c - U T R l , AUTR3, -AUTR4, -AUTR6, -ARD1, -ARD2, and -ARD3 clones was separated on a 10% SDS-PAGE gel. Protein was transferred to P V D F membrane and hybridized with polyclonal a-luciferase antibody. The 62 kDa luciferase protein was detected with HRP-conjugated antigoat secondary antibody (red arrow). Molecular weight markers (kDa) sizes are indicated at the left of the blot. 147  Chapter 5 - Results  A  6  0  ,,  0  n n  1  TGACGGCCAGCTCGGCTTGCTTCTGTTCGATTG  ,  A6 00 3 A6001  I - . I. I I . . I I. I. I . . . . I |.  CGTGCCCCTAC  I I I. I I. I I  TTATTTATTTTTATGTTTATTTATATATATTTTTTTCTCCGTCCCTCT— TGCGCGTGCGCTCACCTCCGTTTTTCCTCTTC  C-GCCTCG  a f i n n  ,  H  0  U  U  J  —CAC  A  6  0  0  1  CCACTATGCGCACT—TGC-GCAGCGCACG  TCGTCGTTGC  I •• N I I . . . . I I I  II. II.. I.I  A6003  Figure 46.  Illlllllll.ll.il I CTCACCTCCGTTTTTTCTGTTCTGTTTTTACACGGCCTTT  Ml  . II I. |. | | |  CTTCTATTATTACTTTGTGCTACAGGGAACGGGGGCTGCCTCCTCCCTCC  Conserved 15 nt Sequence in the RD2 Region of the A600-3 3 'UTR.  The L. mexicana A600-1 and A600-3 3'UTR sequence were analyzed for conserved sequence blocks using D N A Block Aligner software. A 15 nt sequence (shown in red) from the RD2 region of the A600-3 3'UTR was conserved in the A600-1 3'UTR. 148  Chapter 5 - Discussion 5.2  Discussion  This chapter described the mechanisms that confer preferential expression to the A600-3 gene in L. mexicana amastigotes. Stage-specific gene expression in Leishmania has been examined for other Leishmania genes. It is widely believed that gene expression is not transcriptionally regulated, as R N A Pol II promoters have not been identified upstream of protein-coding genes (Martinez-Calvillo et al, 2004). Furthermore, nuclear run-on assays have demonstrated that several other differentially expressed Leishmania genes were transcribed at equivalent rates (Aly et al, 1994; Ramamoorthy et al, 1995; Charest et al, 1996; Beetham et al, 1997; Wu et al, 2000; Mishra et al, 2003; Martinez-Calvillo et al, 2004). The current model for gene expression describes large, polycistronic precursor R N A transcripts, which are co-transcriptionally processed to generate monocistronic mRNA transcripts. The process of trans-splicing a capped, 39-nt mini-exon onto the 5' end of each R N A is coupled to polyadenylation at the 3' end of the upstream transcript (LeBowitz et al, 1993; Matthews et al, 1994). There is a plethora of evidence that developmental regulation of gene expression is mediated by regulatory elements in 3'UTR of m R N A transcripts (Aly et al, 1994; Charest et al, 1996; Beetham et al, 1997; Burchmore et al, 1998; Quijada et al, 2000; Wu et al, 2000; Kelly et al, 2001; Boucher et al, 2002; Mishra et al, 2003; Purdy et al, 2005). In L. mexicana, intercistronic regulatory elements have also been described (Brooks et al, 2001). Therefore, it was hypothesized that preferential expression of the A600-3 transcript in amastigotes was controlled by regulatory elements in the 3'UTR sequence. Considering also the polycistronic mechanism of transcription in Leishmania, one can assume that amastigote-specific expression of the A600-3 gene is not regulated at the transcriptional level. Rather, it is more likely that amastigote differentiation triggers 149  Chapter 5 - Discussion induction or degradation of frans-factors operating on cw-acting regulatory element(s) within the A600 3'UTR (Derrigo et al, 2000). At the level of mRNA, steady state accumulation could be influenced by two factors: the efficiency of m R N A transcript processing/maturation or the rate of R N A degradation. Although differential m R N A stability is often implicated as the mechanism of stage-specific regulation, heat shock has been shown to increase the efficiency for processing and maturation of the developmentally regulated hsp83 R N A transcript (Shapira et al, 2001). A pLitmus28-derived luciferase reporter construct was designed to be maintained as a circular plasmid or chromosomally integrated at the A600 locus. Stable expression of exogenous genes in Leishmania has been demonstrated with the pUC19-based, pLex Leishmania expression vectors. As the pLitmus28 plasmid contains the same high copy number pUC19 origin of replication, it was expected to replicate autonomously in Leishmania as extrachromosomal circular concatemers containing 4-8 units of vector monomers (Joshi et al, 1995). Chimeric plasmids were constructed with the luciferase reporter gene cloned upstream of the A600-3 3' U T R and intergenic region (3'UTR+IR). The hygromycin phosphotransferase (hph) gene was included in the construct as a selectable marker during transfection to isolate parasites containing the reporter plasmids. The sequence upstream of the A600-1 gene and the A600-3 3'UTR+IR flanked the reporter construct to facilitate targeted integration at the A600 locus. In Leishmania, previous gene expression studies have been performed using episomal constructs with chloramphenicol acetyltransferase (CAT), B-galactosidase (P-gal), thymidine kinase (TK), or luciferase reporter genes (Aly et al, 1994; Beetham et al, 1997; Wu et al, 2000; Kelly et al, 2001). Other groups have re-introduced the endogenous gene back into deletion mutants. For example, regulation of the promastigote-specific pfr2C gene was 150  Chapter 5 - Discussion studied using episomal constructs transfected into pfr2~'~ cells (Mishra et al., 2003). Only two examples of integration of constructs in the proper chromosomal context previously have been described. The neo selectable marker was integrated at the A2 locus, which demonstrated that non-coding sequences at the A2 locus conferred stage-specific expression to a heterologous gene (Charest et al., 1996). Also, the metacyclic-specific CPB2 and amastigote-specific CPB2.8 genes were re-integrated at the cpb locus of cpb' ' parasites 1  (Brooks et al, 2001). To our knowledge, the experiments described in this chapter are the first to integrate reporter gene constructs with progressive deletions from the 5' end of the 3'UTR at the proper genomic context. The strategy of generating deletions from the 5' end of the A600-3 3'UTR was important for maintaining the trans-splicing site in the downstream intercistronic sequence. Integration of the luciferase reporter construct at the A600 locus provided the opportunity to determine whether the A600-3 3'-UTR mediated differential expression of a heterologous gene in the proper chromosomal context. Only a single copy of the luciferase gene was stably integrated into each Leishmania cell, thus eliminating the confounding effect of plasmid copy number difference on gene expression. Chromosomal integration of the reporter construct made the data interpretation simpler, as this approach eliminated the need to normalize the results to plasmid copy number. However with the chromosomal integration approach, reporter constructs with antisense A600-3 3'-UTR cloned downstream of the luciferase gene could not be integrated at the A600 locus by homologous recombination. The merit of episomal transfection is that plasmids with sense as well as antisense 3'-UTR can give rise to viable transfectants. The luciferase reporter construct served as excellent proofof-principle constructs to demonstrate that the A600-3 3'UTR was able to confer upregulated expression to a heterologous gene in amastigotes. 151  Chapter 5 - Discussion The pHyg/Luc-Fwd, pHyg/Luc-Rev, and pHyg/Luc-IR constructs were transfected stably into L. mexicana promastigotes  as circular plasmids. The pLuc-Fwd clones  demonstrated persuasively that the full length A600-3 3'UTR, in the sense orientation, conferred amastigote-specific expression to luciferase R N A and protein. The pHyg/Luc-Rev plasmid was constructed by cloning the A600-3 3'-UTR in the antisense orientation, so that the orientation specificity of its regulatory role could be studied. The antisense A600-3 3'UTR produced a promastigote-specific smear when Northern blots were hybridized with a probe for the luciferase transcript. The broad range of transcript sizes may have occurred as a result of rolling circle transcription (Lebowitz et al., 1993). The lack of appropriate transi  splicing signal in the antisense 3'-UTR abolishes the distinct hybridizing band observed in pLuc-Fwd clone. O f course, the luciferase protein, which also had promastigote-specific expression, was detected as a distinct band because the stop codon for translation termination was present in each of the R N A transcripts. The pHyg/Luc-IR plasmid was constructed by cloning only the A600-3 intercistronic region (IR) downstream of the luciferase coding sequence. The rationale for this reporter construct was to examine luciferase gene expression in the absence of the putative regulatory function of A600-3 3'-UTR. This would reveal whether the A600-3 intercistronic sequence contained regulatory properties that would complicate the analysis of luciferase expression from 3'UTR deletion constructs. In our experiments, the A600-3 IR was not sufficient for luciferase R N A or protein expression, which indicated that changes in luciferase expression could be attributed directly to deletion of cw-acting regulatory elements in the A600-3 3'UTR. This result differed from other gene expression studies in which the downstream intercistronic sequences of the amastin, CPB2, and CPB2.8 genes were sufficient for, and even regulated, gene expression. The metacyclic-specific CPB2 and amastigote-specific 152  Chapter 5 - Discussion CPB2.8 genes contained identical 3'UTR sequences, which indicated that stage-specific expression of these genes was not regulated by 3'UTR sequences. A 120 bp insertion sequence (InS) in the CPB2 downstream intercistronic sequence was shown to cause destabilization of the this R N A transcript in amastigotes (Brooks et al., 2001). The downstream intercistronic sequence of the L. infantum amastin gene conferred stable expression to the luciferase reporter gene in both promastigotes and amastigotes. In these experiments, luciferase expression mediated by the amastin intercistronic region served as a baseline against which other transfectants could be evaluated; the increase in luciferase activity, relative to the intercistronic construct, was compared across promastigotes and amastigotes (Wu et al., 2000; Boucher et al., 2002). It is possible that the amastigote microenvironment disrupts luciferase activity, and as a result, luciferase protein expression levels cannot be compared directly across promastigotes  and amastigotes.  In our  experiments, Western blot analysis was used to assay for luciferase protein expression in the L. mexicana promastigotes and amastigotes. Deletion of the proximal 1.5 kb of the A600-3 3'UTR did not significantly alter luciferase R N A or protein expression, which indicated that the putative regulatory elements were encoded in the distal 1.4 kb of the 3'UTR. A dramatic reduction of luciferase mRNA abundance occurred in amastigotes when the proximal 2.5 kb of the A600-3 3'UTR was deleted. This discovery suggested that a regulatory element was positioned in the R D region, between position 1500 - 2500 bp of the A600-3 3'UTR. Progressive 250 bp deletions across the R D region resulted in decreased abundance of the corresponding luciferase mRNA transcript in amastigotes, while only a small reduction in luciferase protein expression was observed. These results provided convincing evidence that the RD2 region, position 2000 2250 bp of the full length A600-3 3'UTR, negatively regulated luciferase protein expression 153  Chapter 5 - Discussion in promastigotes. However, deletion of the RD2 region did not alter luciferase protein abundance in amastigotes. It can be concluded from these results that regulation of mRNA abundance does not correlate directly with protein abundance. Taken together these results indicated that positive and negative regulatory elements in the A600-3 3'UTR conferred developmentally regulated expression to the luciferase m R N A and protein, respectively. This existence of negative regulatory elements has been described several times for regulation of gene expression in Leishmania. Promastigote-specific expression of the pfr2C gene was mediated by a 10 nt negative regulatory element, which repressed expression of this gene in L. mexicana amastigotes. This negative regulatory element was shared by other member of the pfr gene family (Mishra et al., 2003). As mentioned previously, the InS region in the CPB2 intercistronic sequence acted as a negative regulatory element to decrease CPB2 mRNA transcript stability in amastigotes (Brooks et al, 2001). L. chagasi gp46A and mspS mRNA transcript abundance increased during the transition from log phase to stationary phase promastigotes. A 92 bp sequence conserved in the 3'UTR of both transcripts was hypothesized to confer preferential expression to these genes in stationary phase promastigotes (Beetham et al, 1997). Targeted deletion of the conserved, 92 bp sequence from the 3'UTR of both genes did not disrupt gene expression; however, the sequences immediately upstream and downstream of the 92-nt conserved sequence acted as positive regulatory  elements  to  increase  mRNA  transcript  abundance  in stationary phase  promastigotes (Myung et al, 2002). The abundance of L. chagasi mspL gene expression was increased by cyclohexamide treatment of cells, which indicated that mspL m R N A transcripts were destabilized by trans-acting protein factors in stationary phase parasites. Furthermore, mapping the mspL 3'UTR identified a 200-nt, negative regulatory sequence that conferred sensitivity to cyclohexamide treatment. A pyrimidine-rich sequence was repeated within this 154  Chapter 5 - Discussion region of the 3'UTR (Purdy et al, 2005). It has been proposed that the pyrimidine-rich sequences are interaction sites for RNA-binding proteins that mediated  transcript  degradation. A 450 bp positive regulatory element has been described in the amastin 3'UTR, which specifically improved translation efficiency without affecting m R N A transcript abundance (Boucher et al, 2002). This regulatory sequence was identified in more than sixty Leishmania genes and Northern blot analysis showed that many of these genes were developmentally regulated in the amastigote stage of the parasite lifecycle. Our analysis of the A600-3 3'UTR may have identified a negative regulatory element for A600-3 protein translation or stability in promastigotes. A 15 bp pyrimidine-rich sequence in the 250 bp RD2 region also was conserved in the highly divergent A600-1 3'UTR sequence. It is hypothesized that this conserved 3'UTR sequence may function as cw-acting translation repressor element in promastigotes. One model of translation regulation has described the 5' and 3' ends of a mRNA transcript interacting, via trans-acting RNA-binding proteins, to stabilize or destabilize the complex proteins that mediate translation. Therefore, the RD2 exacting element may interact with an RNA-binding protein that represses translation by preventing assembly of translation machinery on the A600-3 m R N A transcript in promastigotes. Future experiments should selectively delete the conserved 15 bp conserved sequence using the Spliced Overlap Extension (SOE) P C R strategy to determine whether this sequence has critical role for stage-specific expression of the A600-3 gene. Also, it would be interesting to determine whether cyclohexamide treatment increases luciferase R N A and protein abundance in promastigotes. This experiment would provide evidence for transacting protein factors suppressing luciferase expression by a 3'UTR dependent mechanism.  155  Chapter 5 - Discussion In addition to the positive and negative regulatory elements that have been identified in the A600-3 3'UTR, the temporal pattern of A600-3 gene expression was also curious. Significant induction of A600-3 gene expression was observed only after five days of culture in pH 5.0 media at 34°C (see Chapter 3, Figure 20). For comparison, expression of the A2 and amastin genes was upregulated dramatically after 3 hours and 16 hours, respectively, under similar culture conditions (Charest et al., 1996; Wu et al., 2000). The unique expression kinetics for the A600-3 gene indicate that the function of this novel gene is critical for maintaining a persistent infection in the mammalian host, rather than establishing the infection. Delayed induction of the A600-3 expression in amastigotes may be explained by the slow degradation of a trans-acting factors that target the A600-3 mRNA for rapid degradation in promastigotes. Conversely, expression of the A600-3 gene may be dependent on the accumulation of an amastigote trans-acting factor that protects the A600-3 m R N A transcript from degradation and enhances translation.  156  Chapter 6 - Summary 6.  Summary The experimental results from this study provide novel insight into the biological  function of the A600 locus in L. mexicana amastigotes. The A600 locus was cloned into a plasmid vector, which facilitated restriction mapping experiments. Four genes were mapped within the A600 locus and the complete sequence of the A600-1 and A600-3 gene was determined. Sequence analysis provided evidence that the A600-1 and A600-3 genes had diverged in an ancestral Leishmania species as sequence identity was conserved in both Old World and New World Leishmania species. Northern blot analysis showed that the A600-1 gene was upregulated only 1.5-fold in amastigotes. However, this result was confounded by cross-hybridization with A600-2 m R N A transcripts, which contain strong sequence identity to the A600-1 coding and 3'UTR sequences. Expression of the A600-3 gene was 7-fold higher in amastigotes and a dramatic increase in expression was observed only at the later stages of axenic amastigote differentiation. The A600 deletion mutants demonstrated clearly that the A600 locus was essential for replication of axenic and intramacrophage amastigotes. Promastigote growth was unaffected by targeted deletion of the A600 locus. The A600' parasites differentiated to axenic A  amastigotes and demonstrated similar morphology and growth kinetics to wildtype parasites during the initial four days of culture. Thereafter, A6QQ~'~ amastigotes failed to proliferate, while wildtype amastigotes continued to divide. The infectivity and survival of L. mexicana parasites was assayed using peritoneal macrophages isolated from B A L B / c mice. A similar parasite burden was observed 72 hour post-infection with wildtype and A6QQ~'~ parasites. However, a dramatic reduction in the number of intracellular amastigotes was observed 120 hours post-infection with A600~'~ parasites relative to wildtype parasites. These observations indicated that A600' ' parasites were phagocytosed f  157  efficiently by macrophages and  Chapter 6 - Summary established early infections, but failed to proliferate as intracellular amastigotes. As growth inhibition was observed also with axenic amastigotes, it is proposed that the failure of the A600' parasites to replicate as intramacrophage amastigotes was caused by an inherent A  defect in amastigote replication and was not the result of macrophage-dependent growth inhibition. The knockout phenotype was complemented by the A600-1 gene, and not the A600-3 gene, which indicated that only the A600-1 gene was essential for sustained amastigote replication. The predicted A600-1 protein contains a 70 amino acid C-terminal extension, which may be an essential functional domain of the protein. It was interesting to note that increased A600-3 m R N A abundance correlated strongly with the observed growth deficiency phenotype of both axenic and intramacrophage A600'  A  amastigotes. The A600-1 and A600-3 genes may have similar expression profiles, although it has not been possible to differentiate the expression profiles of the A600-1 and A600-2 m R N A transcripts. The complete sequence of the A600-2.1 and A600-2.2 genes must be determined, so that A600-1 and A600-2 gene-specific sequences can be identified. In light of the knockout complementation studies, elucidation of the expression profile of the A600-1 gene will be of great value to interpreting these results. 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