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Salmonella Typhimurium genes induced upon bacterial invasion into mammalian cells Pfeifer, Cheryl Gurine 1999

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SALMONELLA  GENES INDUCED UPON  TYPHIMURIUM  BACTERIAL INVASION INTO MAMMALIAN CELLS by  CHERYL GURINE PFEIFER B . S c , U n i v e r s i t y o f S a s k a t c h e w a n , 1989 M . S c , U n i v e r s i t y o f S a s k a t c h e w a n , 1992  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES Department o f M i c r o b i o l o g y and I m m u n o l o g y and the B i o t e c h n o l o g y  Laboratory  W e accept this thesis as c o n f o r m i n g to the r e q u i r e d standard  T H E UNIVERSITY OF BRITISH C O L U M B I A August  1999  © C h e r y l G u r i n e Pfeifer,  1999  In presenting this thesis in partial fulfilment  of  the  requirements  for  an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  MKEOfclDiafrK / W  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  T MtAUtiO  LO 6rV  ii  Abstract In causing gastroenteritis, Salmonella typhimurium are able to invade and grow within non-phagocytic intestinal cells. They are also able to survive within phagocytic cells, and have the potential to cause systemic disease.  The ability of these bacteria to be intracellular is  important in the disease process. Salmonella express an unique set of genes inside macrophages (not expressed with other stresses, e.g. heat, low p H , starvation), however, only a few have been identified. Therefore, the goal of this study was to search for Salmonella genes only expressed inside cells, thereby identifying genes essential for intracellular survival and perhaps virulence. A light-based reporter system was developed to specifically detect gene expression from intra-cellular bacteria. Vibrio harveyi luciferase genes, luxAB, were fused to Salmonella plasmid virulence genes, spvRAB,  and the construct was shown to be upregulated  after  Salmonella invasion into epithelial cells. Upregulation was also demonstrated using a similar B-galactosidase (lacZ) reporter gene fusion.  The results indicated luciferase was a sensitive  reporter, able to monitor bacterial gene expression from within host cells, and able to differentiate live from dead bacteria. Subsequently, a library of S. typhimurium mutants was made by randomly inserting promoterless luxAB genes into the bacterial chromosome. Individual mutants were screened for luciferase expression (i.e. light production) within phagocytic cells. Extracellular bacterial mutants expressing light during growth in rich media or in the presence of cell-secreted factors were eliminated. From this screen, four S. typhimurium  genes were identified as upregulated within  both phagocytic and non-phagocytic cells. Three genes were found located within "Salmonella pathogenicity islands" (SPI): sigD/sopB and pipBwithin SPI-5 and ssaR within SPI-2. sigD/sopB  The  gene encodes an inositol phosphate phosphatase affecting host cell signalling  pathways and chloride secretion.  The ssaR gene potentially encodes a type III secretion  apparatus component. Bacterial type III secretion systems are highly regulated and specialized  iii for  secretion  o f bacterial proteins directly into host cells.  mtracellularly) was completely novel.  work  confirms  intracellular environment. has  led  to  an  enhanced  (iicA  for  T h e f o u r b a c t e r i a l m u t a n t s r e t a i n e d t h e i r a b i l i t y to  and g r o w w i t h i n c u l t u r e d cells, a n d all but This  T h e fourth gene  Salmonella  iicA  induced invade  were required for virulence in a m o u s e m o d e l .  pathogenicity  includes  genes  expressed  in  the  T h e a b i l i t y to i d e n t i f y s u c h g e n e s , a n d t h e i r f u r t h e r c h a r a c t e r i z a t i o n understanding  about  how  Salmonella  p a t h o g e n , i n a d d i t i o n to i d e n t i f y i n g s p e c i f i c v i r u l e n c e factors.  functions  as  an  intracellular  iv Table of Contents  Abstract  ii  Table of Contents  iv  List of Tables  viii  List of Figures  i  List of Abbreviations  xi  x  List of Bacterial Genetic Abbreviations  xiii  Acknowledgements  xvi  Dedication  xvii  Chapter 1: Introduction 1.1. Salmonella  1 and salmonellosis  1.2. Model Systems for the Study of Bacterial Pathogens  1  2  1.2.1. Bacterial culture  3  1.2.2. Cell culture  4  1.2.3. Animal models  7  1.3. Bacterial physiology  9  1.3.1. Secretion systems (Gram-negative bacteria only) 1.3.2. Virulence genes  9 13  a. Pathogenicity islands  15  b. Other virulence genes  17  1.3.3. Regulation of virulence genes  21  a. Environmental regulation  22  b. Genetic regulation  22  1.4. Reporter systems for bacterial gene expression  26  1.4.1. Reporter genes  28  a. B-galactosidase  28  b. Bacterial luciferase  28  V  c. Other reporter genes  29  1.4.2. Other detection methods  30  1.5. Summary of thesis  32  Chapter 2: Materials and Methods  35  2.1. Media and chemicals  35  2.1.1. Chemicals and Assay Reagents  35  2.1.2. Antibiotics  36  2.1.3. Molecular Biology Reagents  36  a. Reagents  36  b. Primers  37  2.1.4. Media  38  2.1.5. Buffers  38  2.2. Equipment  39  2.3. Strains and Plasmids  40  2.3.1. Cells lines  40  2.3.2. Bacteria  40  2.3.3. Bacteriophage  41  a.  Preparation of phage P22 stock  b. Transduction of Salmonella  with phage P22  2.3.4. Plasmid Preparation 2.4. Molecular Biology 2.4.1. D N A Isolation  42 42 43 44 44  a. Plasmid preparation  44  b. Chromosome preparation  44  2.4.2. Basic Method of D N A Precipitation with Ethanol  45  2.4.3. Isolation of D N A from Agarose  45  a. Sephaglas Bandprep Kit  45  vi b. Freeze Squeeze Method for Isolating D N A Fragments  45  c. Spin-column Method for Isolating D N A Fragments  46  2.4.4. Electroporation  46  a. Preparation of electrocompetent bacteria  46  b. Electroporation  47  2.4.5. T w o plasmid competition system  47  2.4.6. Inverse P C R  48  2.4.7. Sequencing  48  2.5. Invasion and Survival Assays  49  2.6. Reporter gene assays  50  2.6.1. B-galactosidase assays  50  a. B-galactosidase assay using fluorescent substrate  50  b. B-galactosidase assay using chemiluminescent substrate  51  2.6.2. Luciferase assay  51  2.7. Screen for Transformed Salmonella Exhibiting L o w Luciferase Activity Outside Host Cells  52  2.7.1. Extracellular Bacterial Screen  52  2.7.2. Intracellular versus Extracellular Bacterial Screen  52  2.11. Mouse Studies  54  2.11.1. Typhoid Mouse M o d e l  54  Chapter 3: Development of an Intracellular Reporter System  55  3.1. Results  55  3.1.1. Invasion assay to determine intracellular bacterial numbers  55  3.1.2. B-galactosidase as a reporter of intracellular bacterial expression  58  3.1.3. Luciferase as a reporter of intracellular bacterial gene expression  62  a. Measurement of light production  62  b. Effects of aldehyde concentration on bacteria and luciferase activity  65  vii 3 . 1 . 4 . C o m p a r i s o n o f 6-galactosidase and luciferase as reporters o f intracellular bacterial gene expression 3.2. D i s c u s s i o n  71 72  Chapter 4: D e v e l o p m e n t o f Screen for B a c t e r i a l Genes 4.1. Results  76 76  4.1.1. Transformation o f Salmonella and Screen for U p r e g u l a t e d B a c t e r i a l Genes 76 4.1.2. S c r e e n i n g for B a c t e r i a l G e n e Induction Inside C u l t u r e d M a c r o p h a g e s . . 8 1 4.1.3.  Transfer o f G e n e s to E n s u r e Induction Phenotype is L i n k e d to G e n e  Insertions  85  4.2. D i s c u s s i o n  85  Chapter 5: Characterization o f Genes Upregulated by Intracellular Salmonella 5.1. R e s u l t s  90 90  5.1.1. Inverse P C R and S e q u e n c i n g  90  5.1.2. E x t e n t o f G e n e E x p r e s s i o n b y the M u t a n t s  95  5.1.3. C o m p a r i s o n o f G r o w t h Rate o f the M u t a n t s  102  5.1.4. C o m p a r i s o n o f Invasiveness o f the M u t a n t s  104  5.1.5. V i r u l e n c e o f Mutants i n T y p h o i d M o u s e M o d e l  105  5.2. D i s c u s s i o n  108  5.2.1. D 1 1 H 5 (ssaR) 5.2.2. A I M (sigD/sopB) 5.2.3.  108 and E 1 2 A 2 (pipB )  G 7 H 1 (iicA)  5.3. O v e r a l l C o n c l u s i o n  109 I l l 112  References  114  A p p e n d i x : D N A a n d P r e d i c t e d Protein Sequences S u r r o u n d i n g Insertional M u t a t i o n s  152  viii L i s t of  Tables  Table 1: Primers for Inverse P C R  37  Table 2: Bacterial strains  41  Table 3: Bacterial plasmids  43  Table 4: Induction of expression of reporter enzyme fusions by intracellular S. dublin  71  Table 5: Growth of Bacteria Over Time in Various Conditions  103  Table 6: Relative Invasion of Bacterial Mutants Into Cultured Cells  104  Table 7: Bacterial dose affects kinetics of mortality of mice  107  ix List of Figures Figure 1: Schematic diagram of the type I, type II, type III, and type IV secretion systems of gram-negative bacteria Figure 2: Schematic representation of Salmonella pathogenicity islands  12 18  Figure 3: Schematic representation of the Salmonella typhimurium chromosome, which is divided into 100 centisomes  27  Figure 4: The effect of the detergents Triton X-100 (1.0%) and SDS (0.1%) on the recovery of intracellular S. typhimurium from HeLa cells  57  Figure 5: Plasmid maps of pFF14 and pSPLUX  59  Figure 6: Expression of spvB::lacZ by bacteria inside non-phagocytic cells  60  Figure 7: Comparison of two different methods to detect bacterial light production: Luminograph LB980 versus X-ray film Figure 8: Effect of aldehyde concentration on bacterial viability and light production  64 66  Figure 9: The action of different concentrations of gentamicin on the luciferase activity within bacteria  69  Figure 10: Detection of light production in Salmonella pSPLUX in an intracellular versus an extracellular environment  70  Figure 11: Schematic of the luciferase reporter gene cassette inserted within the bacterial chromosome, and the orientation of the primer pairs used for inverse PCR and subsequent D N A sequencing  77  Figure 12: Luminograph images of L B plates with S. typhimurium colonies transfected with both pTF421 and pFUSLUX plasmids  79  Figure 13: Luminograph images of bacteria expressing light from a single sample 96-well plate of mutants  83  Figure 14: Comparison of the luciferase activity from the P22-transductional mutants with the original S. typhimurium insertional mutants Figure 15: D N A bands resulting from inverse PCR are visualized on a 1% agarose gel  86 91  X  Figure 16: Position of luciferase gene insertions within known S. typhimurium genes  92  Figure 17: S. typhimurium mutants show increased light production inside mammalian cells  96  Figure 18: Comparison of light production of luciferase-expressing bacterial mutants exposed to different environmental conditions  97  Figure 19: Relative luciferase activity by extracellular and intracellular Salmonella mutants Figure 20: Virulence of S. typhimurium mutants in typhoid mouse model  100 106  xi List of Abbreviations  amp  ampicillin  BALB.BM1  cultured bone-marrow-derived macrophages from B A L B / c mice  BALB/c  inbred Bagg albino mice; used for models of various bacterial infections  cAMP  cyclic-adenosine monophosphate  CDC42  cell-division-cycle; small GTP-binding protein  cfu  colony-forming unit  cm  chloramphenicol  DMEM  Dulbecco's modified Eagle's medium  DMSO  dimethyl sulfoxide  dNTP  deoxy-nucleotide tri-phosphate  EDTA  ethylene-diamine tetra-acetic acid  EEA1  early endosomal antigen 1  FBS  fetal bovine serum  FDG  fluorescein-di-galactopyranoside  HeLa  human cervix epitheliod cell line  ID50  bacterial dose at which 50% of cells or animals become infected  IgA  immunoglobulin A  in vitro  (Latin: in glass) here, to mean within labware or within cultured cells  in vivo  (Latin: in the living organism), here, to mean within the host animal  IP  intraperitoneal  rv  intravenous  J774A. 1  cultured macrophage-like cells from B A L B / c mice  JNK  c-Jun N-terminal kinase  LAMP  iysosomal-associated membrane protein  LB  Luria-Bertani; rich bacterial media  LD50  bacteria] dose at w h i c h 5 0 % o f animals die  LPS  bacterial lipopolysaccharide  MDCK  cultured M a d i n - D a r b y canine kidney cells  MEM  minimal Eagle's medium  MHC  major histocompatibility complex  MOI  multiplicity o f infection  N A P S Unit  N u c l e i c A c i d and Protein Services U n i t at U n i v e r s i t y o f British Columbia  NP-1  neutrophil peptide defensin 1  Nramp  natural resistance-associated macrophage rjrotein  ORF  open reading frame  PBS  phosphate-buffered  PCR  polymerase chain reaction  PETG  phenyl-ethyl-thio-galactoside  PMN  p o l y m o r p h o n u c l e a r cell or neutrophil  polymyxin-CAP  cyclic-antibacterial peptides  PVC  saline  polyvinylchloride  SCV  5a/mone//a-containing vacuole  SDS  s o d i u m d o d e c y l sulfate  SOC  r i c h bacterial m e d i a containing tryptone, yeast extract and glucose  str  streptomycin  TE  buffered solution containing T r i s - H C l and E D T A  tet  tetracycline  UBC  University of British Columbia  xm List of Bacterial Genetic Abbreviations  A1A1  S. typhimurium mutant w i t h insertion i n sopB/sigD gene  agf  thin aggregative fimbrial gene  ahp  a l k y l h y d r o p e r o x i d e gene  AIDA-l  adhesin i n v o l v e d w i t h diffuse adherence i n E P E C  atr  acid tolerance response gene  brk  gene encoding Bprdetella resistance to complement  cys  gene e n c o d i n g a protein component i n the cysteine pathway  D l 1H5  S. typhimurium S L 1 3 4 4 w i t h insertion i n ssaR gene  E12A2  S. typhimurium S L 1 3 4 4 w i t h insertion i n pipB gene  emrR  E - m u l t i d r u g resistance gene; i n v o l v e d w i t h low-energy shock adaptation  envZ  histidine kinase; sensor component o f two component regulatory system OmpR/EnvZ  EPEC  enteropathogenic E. coli  fhlA  formate hydrogenlyase  fim  type 1 fimbrial gene  fliA  flagellar gene; specifically an alternative s i g m a factor  flgM  flagellar gene; specifically an anti-sigma factor  fur  ferric uptake regulator gene  G+C  total guanosine plus cytosine content o f the D N A  G5D5  see: G 7 H 1  G7H1  S. typhimurium S L 1 3 4 4 w i t h insertion i n iicA gene  G8B1  see:G7Hl  iic  gene i n d u c e d intra-cellularly  inv  i n v a s i o n gene  ipa  i n v a s i o n p l a s m i d antigen gene  XIV kat  gene encoding catalase  lacZ  gene e n c o d i n g B-galactosidase, from lactose operon  Ipf  l o n g polar fimbrial gene  luxAB  genes encoding bacterial luciferase  marR  multiple antibiotic resistance gene  mgt  magnesium transport gene  mutS  mutator gene i n v o l v e d i n methyl-directed mismatch repair  ompR  regulator component o f two component regulatory system O m p R / E n v Z  orf  open reading frame  P22  Sa/mone/Za-specific bacteriophage  pag  PhoP-activated gene  pef  plasmid-encoded fimbrial gene  phoP  regulator component o f two component regulatory system P h o P / Q  phoQ  histidine kinase; sensor component o f two component regulatory system PhoP/Q  pip  pathogenicity island-encoded protein  pmr  p o l y m y x i n resistance gene  prg  P h o P - r e p r e s s e d gene  rho  gene e n c o d i n g transcription terminator factor R h o  rpoS  gene encoding R N A polymerase alternative s i g m a factor 38  sap  genes corresponding to sensitivity to antimicrobial peptides  sec  amino-terminal secretion signal o f a protein  selC  selenocysteine t R N A gene; selenium metabolism  sif  Salmonella-induced filament gene  sig  Salmonella i n v a s i o n gene  sip  Salmonella i n v a s i o n protein gene  sod  superoxide dismutase gene  XV  sop  Salmonella outer protein gene  spa  surface presentation of antigen gene  SPI  Salmonella pathogenicity island  spi  Salmonella pathogenicity island encoded gene  spv  Salmonella plasmid virulence gene  ssa  Salmonella secretion apparatus gene  sse  Salmonella secretion effector gene  ssr  Salmonella secretion regulator gene  yop  Yjersinia outer protein gene  xvi Acknowledgements I would like to thank the many individuals who have helped me during my studies: : thank you to my supervisor: Dr. B. Brett Finlay - for his continued support, : thank you to the Finlay Lab - for help above and beyond the call of science... both present: Sharon Ruschkowski, Gwen Lowe, Elizabeth Frey, Olivia Steele-Mortimer, Rebekah DeVinney, Jean Celli, Sandra Marcus, Michelle Zaharik, Carrie Rosenberger, John Brumell, Leigh Knodler, Mitchell Uh, Ben Goh, Wanyin Deng, Myriam de Grado, Ursula Heczko, Annick Gauthier, Danika Goosney, Derek Knoechel, Yuling Li and past: Francisco Garcia del Portillo, Han Rosenshine, Ka Leung, Murry Stein, Scott Mills, Patrick Tang, Dan Wilson, Agneta Richter-Dalfours, Akio Abe, Jose Puente, Annette Siebers, Vida Foubister, Markus Stein, Dieter Reinsheid, Maryse St. Louis, Brendan Kenny, Graciella Pucciarelli, Oliva Ortiz-Alvarez, Carol Cordeiro : thank you to the departments of Microbiology and Immunology, and The Biotechnology Laboratory for the many learning opportunities, : thank you to the members of my supervisory committee: Dr. R. A. J. Warren, Dr. G. Spiegelman, and Dr. W. R. McMaster. My studies were made possible by operating grants to B. B. Finlay from the Medical Research Council of Canada.  Dedication  This thesis is dedicated to a number of special people:  to my husband, Tom Pfeifer - for all his understanding and loving support,  to my parents, Ken and Myrtle Morrison - who helped me believe in myself,  to my grandma, Anne Borgeson - who always thought education was important.  Chapter 1 C h a p t e r 1: 1.1.  Salmonella  1  Introduction  a n d salmonellosis  Salmonella are Gram-negative bacteria w i t h i n the family Enterobacteriaceae,  tribe  S a l m o n e l l a e , genus Salmonella, and (according to the E w i n g classification scheme) are d i v i d e d into three species:  typhi, choleraesuis, or enteritidis (168).  S. typhi is solely a human  pathogen, a n d is the causative agent o f t y p h o i d fever (169, 252). H u m a n s are the reservoir for this bacterium, and spread is most often through the c o n s u m p t i o n o f water contaminated w i t h h u m a n feces.  S y m p t o m s o f t y p h o i d fever m a y take f r o m one w e e k to one month  after  ingestion o f the bacteria to manifest and are characterized b y a sustained h i g h fever, bacteremia f o l l o w e d by infection o f the bilary system and other tissues, and an initial constipation period f o l l o w e d b y diarrhea, p o s s i b l y b l o o d y (169). f r o m 2 - 1 0 % and a 2 0 % rate o f relapse.  T h e disease is often severe, w i t h a death rate  E v e n after the person has apparently  recovered,  bacteria m a y s u r v i v e i n the gall bladder and be shed for up to a year (even w i t h antibiotics and surgery) or l o n g e r (168, 169, 2 7 4 ) . S. choleraesuis is able to cause disease i n both animals and humans, w i t h the reservoir being farm animals, i n c l u d i n g s w i n e .  In humans it causes a severe disease i n the f o r m o f a  p r o l o n g e d bacteremia characterized b y fever, c h i l l s , and anorexia.  Gastroenteritis  is not  c o m m o n , h o w e v e r due to its systemic nature, focal lesions m a y develop i n any tissue to cause osteomyelitis, p n e u m o n i a , p u l m o n a r y abscesses, meningitis or endocarditis. depressed  immune  systems  (e.g.  due  to  AIDS,  organ  In patients w i t h  transplantation,  or  cancer),  S. choleraesuis is able to cause a severe t y p h o i d - l i k e disease (168, 2 7 4 ) . S. enteritidis  is the most c o m m o n o f the three species, c o n s i s t i n g o f over  2000  serotypes according to the K a u f f m a n n - W h i t e antigen classification scheme, and are almost ubiquitous i n the environment (14, 2 0 ) . T h e y are found associated w i t h many different types of animals a n d f o o d products ranging f r o m radish sprouts and eggs (227) to c h i c k e n and beef (14,  168, 2 7 4 ) .  Most  often  the bacteria  are referred  to b y their c o m m o n  serotype  Chapter 1 nomenclature, i.e. S. typhimurium  rather than S. enteritidis serotype typhimurium (20).  2 Of  the more than 2000 serotypes existing, only 40% of them account for over 95% of all the clinical isolates (168) and some clinically relevant serovars include: S. dublin, and S. paratyphi  S.  typhimurium,  A.  In humans, the ingestion of food or water contaminated with S. enteritidis  results in  gastroenteritis (274). Symptoms appear within 6 to 24 hr after ingestion and last up to a week, with severity varying from person to person, depending on serotype and dose ingested. The disease is characterized by initial nausea and vomiting followed by abdominal pain and diarrhea, with or without fever. After recovery, a person may continue to shed the bacteria in their feces for up to three months.  Interestingly, antibiotic treatment of uncomplicated  gastroenteritis has been reported to prolong the earner state (168). Most often the disease is self-limiting and localized to the intestinal tract, but in a small number of people, especially young children or immunocompromised patients, the infection may become systemic and thereby more severe (227).  In rare cases, S. enteritidis  serotypes have been implicated in  systemic disorders, including arthritis (313), pancreatitis (8), endocarditis (168), pericarditis (168), and mediastinitis (234).  S. enteritidis  serotypes are also able to cause disease in a  number of animals, resulting in symptoms which range from barely detectable to severe typhoid-like resulting in death. This has allowed for the development of animal models. For example, infection with either S. typhimurium  or S. dublin causes gastroenteritis in humans,  yet result in a severe typhoid-like disease in BALB/c mice (10, 156). introduction  describes  S. typhimurium  research  or S. dublin.  concerning  Salmonella enteritidis  The remainder of this species,  Many of the aspects described below  specifically  also apply to  S. choleraesuis and S. typhi (218), although the differences will not be expounded on. 1.2.  M o d e l Systems for the S t u d y of B a c t e r i a l  Pathogens  The study of bacteria and their interaction with the environment and hosts is done through the use of different model systems, all of which have their advantages and their limitations.  Chapter 1 3 1.2.1.  Bacterial culture  The growth of a pure bacterial culture in the lab can yield a wealth of information regarding the physiology of the organism. This is considered one of the most basic and essential aspects of bacteriology, and in the case of pathogenic bacteria, it is required to fulfill Koch's postulates to determine the cause of a disease (274). Specific biochemical and genetic information regarding the bacteria can only be obtained through the use of a pure culture. Furthermore, environmental cues can be controlled either individually or in combination, and thereby the specific bacterial responses to them determined. Some examples of parameters which can be tested include bacterial responses to changes in temperature, pH, osmolarity, nutritional requirements, resistance to complement, oxidative radicals, heavy  metals,  antibiotics, the production of toxins, and growth phase (115, 119, 295). However, the growth of a single culture in the lab does not reveal all aspects of how bacteria are able to survive within an environment or cause disease. Often, the synergism and competition with other living organisms in the environment cannot be duplicated in a test tube (in vitro). Furthermore, species or tissue tropisms associated with a particular bacterial disease cannot be deduced. It is the rule rather than the exception that clinical isolates of bacteria display very different phenotypes than those passaged many times over in the lab. It is clear that while bacteria are free-living entities, they are greatly influenced by their surroundings and spend a great deal of their time reacting to and altering their environment (115). Salmonella are able to survive in a wide range of temperatures and pHs. They are not only able to react to environmental cues (e.g. pH, temperature, organic and inorganic substrates, oxidative radicals, etc.) (295), but are able to interact with and actually communicate at the cellular level with animate entities such as other bacteria, and even plants and animals (97, 250).  It is this  interaction and communication (or 'argument') between bacteria and host that is the basis of disease in salmonellosis, and most other bacterial diseases.  The chemical signaling between  bacteria and other organisms is very complex and is extremely difficult to reproduce in the lab with a pure bacterial culture. For example, the bacteria are able to survive in contaminated  Chapter 1  4  water and food products. Once ingested, they are subjected to the salivary enzymes of the mouth and then the bile salts and low pH of the stomach. After the shock of the stomach, the bacteria pass through into the small intestine where they encounter the peristaltic movement of the intestines and the mucus continually moving along the ciliated brush border lining the intestinal surface. Furthermore, the ingested bacteria encounter many other microorganisms that are already resident in the intestine. The Salmonella are able to bypass these barriers, to interact with the intestinal cell surfaces and ultimately engineer their own uptake into these cells. Within the host cells, Salmonella remain inside a vacuolar space, where they must adapt in order to survive. 1.2.2. Cell culture To avoid confusion throughout this discourse, bacterial cells will not be referred to as "cells" but as simply "bacteria"; while host cells will be referred to as "cells". The interactions of bacteria with host cells can be studied more closely with the use of cultured mammalian cells. There are a number of host cell types available for study, from various tissues, and from various types of animals. Examples of epithelial cells include HeLa cells which originated from a human cervical tumor and MDCK cells which originated from immortalized canine kidney cells. Examples of phagocytic cells include J774A.1 and BALB.BM1 cells, which are both mouse macrophage-like cells originating from BALB/c mice. The discovery of numerous Salmonella  virulence factors has been made through the controlled infection of cultured host  cells (22), specifically many factors required for bacterial invasion of cells and for survival' within cells. The discovery of a new bacterial protein secretion system, the type III secretion system (TTSS), is especially noteworthy since bacterial effectors are secreted into host cells predominantly upon contact of the cells with the bacteria (188, 208). Salmonella  were long thought to produce disease from extracellular locations within the  body for a number of reasons. The bacteria could be isolated from fecal and blood samples, and in EM studies, the destruction of S. typhimurium  within host polymorphs and  macrophages was seen (160). Moreover, the bacteria were able to replicate in extracellular  Chapter 1 5 spaces, such as the peritoneal cavity (160). Over the past decade, there has been a shift, and many researchers now believe that the ability of Salmonella  to persist intracellular^ greatly  influences the extent and severity of the disease (51, 76, 81, 85, 195, 247, 263). The intracellular stage appears to be especially important as mutants which either cannot invade cells or survive within cells are attenuated (76, 195, 247). However, the bacteria are able to move via flagella (171) and adhere to cells via various fimbrial adhesins and pili (183, 324). They ultimately engineer their own uptake into the intestinal cells, targeting the M cells of Peyer's patches (165, 288). During invasion Salmonella induces selective aggregation and internalization of host cell surface proteins, such as M H C class I heavy chain, fibronectinreceptor, CD-44 and B -microglobulin (109). 2  The bacteria also trigger host cell signaling  pathways in a CDC42-dependent manner (42, 172), (although uptake is Fc receptor independent (205)), that lead to cytoskeletal and nuclear responses resulting in the uptake of the bacteria (5, 90, 91, 267, 272). Salmonella  The mechanisms required for the invasion of cells by  and for their subsequent survival within cells are complex and require the  coordination of a number of bacterial factors (see virulence factors section below) (79). In culture, Salmonella are able to enter every mammalian cell type tested (81, 328). However, in order to separate the bacteria-mediated invasion from phagocytic uptake, invasion studies have used non-phagocytic epithelial cells (79). There is evidence that similar events take place in phagocytes and that the bacteria mediate their own uptake into these cells as well rather than relying on Fc-receptor or complement-receptor mediated uptake (110).  Salmonella  do not bind intimately to the cell surface in order to enter into cells, rather the bacteria initiate a splash or membrane-ruffling event which causes the cell to macropinocytose everything within the immediate vicinity (90, 105, 169). The bacteria are thus taken up into a large spacious vacuole, where they can be seen to actively move around (4, 5, 84). Once the bacteria have been engulfed, the cell surface returns to normal (84). Inside the cell, the Sa/moneZ/a-containing vacuole (SCV) does not traffic in the same manner as would a regular vacuole and the intracellular environment of the S C V is actively  Chapter 1  6  m o d i f i e d b y the bacteria (31, 108). W h i l e the p H o f the vacuole is l o w e r e d , it o n l y falls to around p H 5.0, rather than to p H 4 . 0 ( 3 , 108). There is evidence that w h i l e the S C V s do accumulate some o f the markers o f the regular trafficking pathway ( E E A 1 indicating fusion w i t h early endosomes;  l y s o s o m a l glycoproteins and L A M P s  indicating fusion  with  late  endosomes (219, 2 4 0 , 241) and N r a m p l (220, 284)), they prevent the accumulation o f other markers i n any great amount such as cathepsin D or the mannose-6-phosphate receptor.  It w a s  earlier reported that the mannose-6-phosphate receptor was not associated w i t h the S C V at all (106, 2 5 6 ) , but recently more sensitive evidence indicates that as many as 1 0 % o f S C V s may contain this marker; h o w e v e r this is still significantly different from the normal trafficking pattern seen w i t h a cell (219).  A b o u t 4 - 6 hr after i n v a s i o n , the cell is seen to produce  filamentous structures w h i c h extend out from  the S C V , called Sifs  (Salmonella-induced  filaments), w h i c h c o r r e s p o n d to bacterial replication w i t h i n the cells (195, 2 2 1 , 299). T h e Sifs can be labeled w i t h l y s o s o m a l g l y c o p r o t e i n , and are connected to the S C V .  T h e y are not  actually large enough to contain a bacterium although bacterial membrane blebs are found w i t h i n these structures (299). Other stimulated pathways include inositol phosphate signaling pathways (270) and those stimulated b y rapid c a l c i u m fluxes w i t h i n cells ( 6 5 , 2 4 3 ) , although c y c l i c 3':5'-monophosphate ( c A M P ) levels d o not increase (37).  adenosine  T h e result is the secretion o f fluid  into the intestine and the influx o f neutrophils to the site o f infection, resulting i n diarrhea ( 7 8 , 206).  F u r t h e r m o r e , Salmonella are toxic to cells.  Invasion o f the bacteria into macrophages  induces apoptosis, w h i c h can be seen both in vitro (224) and in vivo (263). A l t h o u g h ultimately more c o m p l e x than the study o f single bacterial cultures, the limitations o f u s i n g cell culture are very s i m i l a r , and this model is still considered an in vitro model.  C u l t u r e d cells exist i n an artificial environment where they cannot interact w i t h other  cell types f o r stimulation, cross-feeding, differentiation, o r to get r i d o f toxic wastes.  Often  they are transformed such that they w i l l not have a limited life-span but w i l l be able to be passaged m a n y times, s i m i l a r to the passage o f bacteria. H o w e v e r , as occurs w i t h bacteria, the  Chapter 1  7  more a cell is passaged in a lab setting, the more it is removed from specific contacts, hormones and other chemical gradients, the less likely it is to resemble the tissue from which it was taken. The use of primary cell cultures for study helps to lessen this 'de-differentiation effect', although they are more difficult to work with in the lab and often their availability is limited.  Similarly, combined cell and bacterial cultures are difficult to work with and the  results difficult to interpret because there are many variables, known and unknown. 1.2.3.  Animal models  Ultimately, the best way to determine bacterial virulence and tissue specificity is to infect the host. In place of experimenting on humans, animal models are used to reproduce a given disease and determine whether a specific bacterial factor, or conversely a host factor, may increase or decrease the extent of bacterial pathogenesis.  A good animal model requires  that the bacterial infection can be established reproducibly. However this trait may actually make them less sensitive when assaying for bacterial virulence, as the animals are often deficient in some way or immunologically immature in order to give the bacteria an edge. Furthermore they are fairly crude measures of virulence as they reflect the cumulative effects of many steps including bacterial colonization and the production of symptoms.  This makes it  possible to overlook important virulence determinants. There are a number of ways to determine virulence using an animal model. Traditionally researchers have used a whole animal model, looking at either the infectious dose (ID50) or the lethal dose (LD50) (274). The infectious dose (ID50) is the number of bacteria necessary to infect 50% of the exposed animals. This parameter only reflects the infectivity of the bacteria and not the morbidity of the disease resulting from infection. The lethal dose (LD50) is the number of bacteria necessary to kill 50% of the infected animals. A variation of this involves the determination of the day on which 50% of the animals died. Other parameters also used as indicators of virulence include the measurement of signs of disease such as weight loss, diarrhea, presence of bacteria in animal fluids (e.g. blood, urine, cerebrospinal fluid) or organs (e.g. spleen, liver), or the extent of the immunological response (e.g. P M N influx,  Chapter 1  8  number and size of infection foci). Another model is the ileal loop model, where the intestine within an anesthetized animal is tied off and the bacteria injected directly into it.  Only more  localized effects of the bacterial infection can be determined with this model (i.e.  fluid  accumulation or P M N influx), however it has the advantage in that more than one bacterial strain at a time may be analyzed within the same animal. For salmonellosis, a number of animal models have been used. The most widely used model involves the infection of B A L B / c mice with S. typhimurium which resembles typhoid fever in humans (233, 306).  resulting in a disease  The B A L B / c mice are particularly  susceptible to intracellular bacterial infections, which has been attributed to a defective host resistance locus nrampl  (formerly known as the ity/bcg/lsh locus) (64, 116, 327).  Other  animals used include guinea pigs (160), rabbits (330), chickens (53, 142, 318), pigs (123, 314), sheep (27), and cows (94, 330). In the mouse, Salmonella are able to cause disease when given by various routes.  The  bacteria may be induced orally, which is the route thought to most mimic the natural course of infection (112, 156). A typical L D  is around 1 x 10 bacteria per mouse (195, 299, 306). 6  5 0  The bacteria are forced to contend with not only the acid and gastric juices within the stomach, but also with various cellular barriers. Although Salmonella invade many cell types in vitro, the bacteria appear to be targeted to the membranous cells (M-cells) in vivo (94, 156). M-cells are specialized cells, found in Peyer's patches within the intestine, which are thought to internalize luminal materials for presentation to underlying antigen-presenting cells  (e.g.  monocytes, macrophages, and neutrophils). Salmonella are able to pass through the intestinal cell layer to the sub-layers (83, 85, 170), where they are able to colonize (204) and/or are picked up by waiting macrophages. The bacteria are able to survive within phagocytes (263), and possibly travel to other organs via this intracellular route (57).  Using this route of  infection, factors required for invasion are often detected as their loss results in a loss in virulence. When bacteria are injected intraperitoneally (IP) into mice (10), they no longer have to deal with these particular barriers, nor do they have to compete with the normal intestinal  Chapter 1  9  flora. C o n s e q u e n t l y , the number o f Salmonella required to cause disease is reduced u s i n g I P inoculation, w i t h an LD50 around 100 bacteria per mouse (299).  A s w e l l , bacteria with  mutations i n i n v a s i o n genes are not attenuated for infection resulting f r o m I P i n o c u l a t i o n , but those w i t h mutations  i n genes needed  for subsequent bacterial g r o w t h w i t h i n cells  are  attenuated. Intravenous ( I V ) inoculation o f Salmonella into mice bypasses the most barriers, putting the bacteria into direct contact w i t h circulating macrophage cells and p r o v i d i n g the most direct route to the organ o f choice in w h i c h to establish an infection (e.g.  liver and spleen).  A g a i n , the number o f Salmonella required to cause disease is drastically reduced, w i t h an LD50 around 10 bacteria per mouse (131, 263). 1.3.  Bacterial  1.3.1.  physiology  S e c r e t i o n systems (Gram-negative b a c t e r i a only)  T h e type I sec-independent secretion system i n v o l v e s the three components:  an inner  membrane transport A T P a s e (coined A B C protein for A T P - b i n d i n g cassette), a periplasmicspanning protein w h i c h is anchored i n the inner membrane, and an outer membrane protein w h i c h is secreted v i a a sec-dependent pathway (80,  161, 208).  T h e genes encoding the  secretion apparatus and the secreted protein are usually clustered.  Proteins secreted v i a the  type I p a t h w a y c o n t a i n the signal for secretion in their carboxy terminus, h o w e v e r this signal appears to vary slightly between subfamilies o f the secretion system.  In contrast to the  sec-dependent secretion pathways, this carboxy signal sequence is not cleaved o f f and there is no periplasmic intermediate o f the secreted protein. pathway  include  the  subfamily  of  E x a m p l e s o f proteins secreted v i a this  hemolysins  Escherichia  coli  alpha-hemolysin,  Bordetella pertussis adenylate c y c l a s e , and Pasteurella haemolytica l e u k o t o x i n , as w e l l as the subfamily o f proteases f r o m Pseudomonas aeruginosa and Erwinia  chrysanthemi (80,  161).  There are n o reported Salmonella proteins secreted by this pathway so far, h o w e v e r the outer membrane component o f the secretion apparatus has been interchangeable w i t h the E. coli h o m o l o g ( T o l C ) (305).  described and is functionally  Chapter 1 10 Both the type II and type IV secretion pathways are sec-dependent and involve two separate steps for transport across the inner and outer membranes.  The first step (the  sec-dependent step) is energized by ATP hydrolysis and requires the protein to have an aminoterminal signal sequence which is cleaved off during export to the periplasm. Components of this first step include a number of inner membrane proteins, an inner membrane associated ATPase, a chaperone to bind presecretory target proteins, a periplasmic signal peptidase, as well as a number of accessory proteins (161, 208). Type II secretion is thought to be the main export pathway for gram-negative bacteria and is known as the generalized secretory pathway (80, 208, 273). The second step for translocation through the outer membrane includes a periplasmic chaperone and a number of outer membrane associated proteins which multimerize to form a channel (273). It is thought that the secretion signal for this step may be encoded by multiple segments on the protein (273), and no signal sequence cleavage occurs. Secretion components usually are encoded by about 13-15 clustered genes. The type II secretion pathway is primarily used for the secretion of degradative enzymes. Some examples of proteins secreted via this pathway are Klebsiella oxytoca  pullulanase, P. aeruginosa  Xanthomonas campestris  phospholipase C, exotoxin A , and elastase, and  polygalacturonase (161).  Proteins which use the type IV secretion pathway are able to induce their own transport across the outer membrane, and are therefore also known as auto transporters (80, 149). Once through the inner membrane, the proteins are able to associate with the outer membrane to form a pore and mediate their own passage out of the cell, in the absence of any energy coupling or accessory factors. Once outside, the protein may remain associated with the outer membrane or be cleaved off. Examples of proteins secreted via this pathway include gonococcal IgA protease, Haemophilus influenzae IgA protease, Helicobacter pylori vacuolating cytotoxin, enteropathogenic E. coli EspC, and Bordetella pertussis pertactin and BrkA (80, 149, 161) The type III secretion pathway is especially important for the specific secretion of a number of virulence factors. This secretion system has been found to be conserved amongst a  Chapter 1 11 number of gram-negative pathogens including Salmonella Yersinia  (268), E. coli (80), Erwinia  (80, 98, 268), Shigella  (325), and Pseudomonas  (98, 268),  (311, 325) species. Proteins  of the type III secretion system, as well as many of the proteins to be exported, are contained within large regions of D N A dubbed pathogenicity islands (111, 208). These regions may be chromosomally encoded, as in Salmonella, Yersinia.  or contained on the virulence plasmid, as in  Type III secretion involves more than twenty components, many of which are  homologous to those involved with flagellar assembly, including a multimeric ATPase and various proteins involved in the synthesis of a specialized surface organelle (113, 182). Amino terminal signal sequences target proteins to the secretion machinery, although the secretion sequences are not cleaved (188).  There is some evidence to indicate that some secretion  information is contained within the structural configuration of the mRNA (7), and that at least for some proteins (but not all (96)), secretion and translation may be coupled (188). Each protein to be secreted also associates with a corresponding specific cytosolic chaperone. The whole system is highly regulated and proteins are only secreted upon the bacteria receiving specific environmental cues. The method is sec-independent and proteins are secreted across both the inner and outer membranes in one step. Furthermore, proteins may be secreted from the bacteria directly into host cells, where they can influence host cell signaling. In contrast to other secretion systems, many of the structural components of the type III system are considered to be virulence genes rather than housekeeping genes because their only function appears to be the selective secretion of proteins required for pathogenicity in the host (188). Moreover, mutations within these structural genes often leads to reduced bacterial virulence without greatly affecting bacterial growth in broth culture.  Chapter 1 12  s  CD 0>  a ra •z.  r  •o s c s  8  ii i CV  g ro  01 01 c  i  9  O  -  IX  j  ~  o> o  i a  11 0) X  ex £ ii e g Ol 0> 3 £ 2  s a  & a  01  3 !_ CZ  ra  = s oc  sL  5E 2E  £ $  Cl  o s.  .5 cs *  0  .  CJ  =  I a  Is •>-.  —  • s  t.  3  2  a  5= E 5  — 0> 0) —  a >. *  •  —o  *1  — 01 ^ > 2 CO  0)  E 2  a ro  Ol  E  |  11 «I E |  o==o  CX  •a § 0> • 3 — "O S £  "S &  u  0==0  i_  **  =  d5 <  t 3 IX  LZ  C  E  9  5  01 ~  I2 u  c  11  i t  Chapter 1 13 1.3.2. a.  Virulence  genes  Pathogenicity islands  The definition of a 'virulence' gene is a gene that is required by the bacterium to specifically cause disease in the host or to subvert the host's immune system.  This differs  from a 'housekeeping' gene which is required for maintenance of regular bacterial functions for growth, D N A division, protein production, nutrient acquisition, etc. Generally, if a mutation is made within a virulence gene, the bacteria will show increased susceptibility within the host, but will not be greatly affected during growth in regular rich media (122). While virulence genes can be found scattered throughout the Salmonella chromosome, the latest research has shown the existence of large regions of D N A encoding groups of genes which appear to coordinate various aspects of pathogenesis known as pathogenicity islands (122).  These islands have also been described in other gram-negative  pathogens and  frequently include the genes needed for the type III secretion system, including the L E E (locus of enterocyte effacement) region of pathogenic E. coli (67, 249), the two P A I (pathogenicity island) regions of uropathogenic E. coli (264), the M x i / S p a region of Shigella (161, 268), the Exs/Psc region of P . aeruginosa  (161), the Hrc/Hrp region of Ralstonia solanacearum (161,  325), and the virulence plasmid-encoded L c r / V i r region of Yersinia (161, 325). Pathogenicity islands are clusters of horizontally acquired virulence genes which are generally found inserted at t R N A loci (24) and have a G+C content that is lower than that of the bacterial chromosomal D N A (around 52%). Within Salmonella  there have been five Salmonella pathogenicity islands  described so far (SPI-1, -2, -3, -4, and -5), although only two encode the machinery for type III secretion. The genes within these islands are important for coordinating and secreting other virulence factors encoded outside of these specialized areas on the bacterial chromosome. In S. typhimurium,  SPI-1 is a 40 kb region located at 63 minutes on the chromosome  between mutS and flilA (49, 217). SPI-1 appears to be present in all S. enterica  subspecific  groups (238). The overall G+C content of the gene cluster was found to range from 42-47%. SPI-1 has been shown to be especially important for bacterial invasion of epithelial cells and  Chapter 1 14 M-cells (6, 47, 49, 98, 99, 331). This active invasion of cells by the Salmonella is in contrast to the antiphagocytosis activity of Yersinia species (207). Many of the SPI-1-encoded genes are also required for cytotoxicity in macrophages (43) and for virulence in vivo.  This region  contains genes encoding components of a type III secretion system which encode structural proteins (invA,  invC, invG, invJ, spaO, spaP, spaQ, spaR, spaS, prgH, prgl, prgj, prgK,  and orgA) (50, 99, 174, 277, 304), secreted proteins (avrA, sptP, spaO, invJ, sipA,  sip/sspC, and sip/sspD) (48, 50, 162, 175, 176, 277) and their corresponding (sicA, sicP , invl, invE) (162).  sip/sspB,  chaperones  and prgP)(95, 98), and proteins for regulation of secretion (sip/sspD, and  Proteins required for regulation of gene expression are also encoded within the  SPI-1 region ( H i l A and I n v F ) ( l l , 174).  Additional proteins secreted by the SPI-l-encoded  type III secretion system (e.g. SopB/SigD, SopE, SopD), as well as additional regulatory factors of this region (e.g. PhoP/PhoQ), are located in unlinked chromosomal locations (60, 101, 173). Many of the SPI-1 secreted proteins are translocated directly into the host cell and elicit direct effects on the host cell signal transduction pathways.  SptP has tyrosine phosphatase  activity within host cells which disrupts the actin cytoskeleton, likely freeing up G-actin monomers necessary for the formation of host membrane ruffles during Salmonella invasion (96, 177). SptP also has sequence similarity to the cytotoxins Y o p E from Yersinia spp. (268) and ribosyltransferase  exoenzyme S from P. aeruginosa  (177).  A v r A has  sequence similarity to A v r X , an avirulence protein from the plant pathogen  significant  Xanthamonas  campestris pv. vesicatoria, and to YopJ/P from Yersinia spp. (311, 325). SipB and SipC are also secreted and are thought to affect the host actin cytoskeleton, as deduced by their ability to block Salmonella invasion when they exist as membrane-associated receptor chimeras in the host cell (36). They are further required for secretion of other proteins into host cells via the SPI-1 type III system (96). Without SipB and SipC, bacteria are able to secrete proteins but are not able to translocate them into host cells (339). I n v H also appears to be important for secretion of proteins via the SPI-1 type III system, however its mechanism is unclear. It may  Chapter 1 15 be acting to post-translationally modify proteins to be secreted via the type III system, but it has not been shown to be secreted itself (55). SPI-2 is located at 31 minutes and also encodes components of a type III secretion system (150, 286). SPI-2 is present in most subspecific groups of S. enterica, but not all (238). It is interesting to note that while cross complementation between type III systems of different species can occur, e.g. Salmonella sipB can complement a Shigella ipaB mutant (98, 154) and a Shigella spa24 could complement a Salmonella spaP mutant (121), many of the type III components of SPI-2 do not compensate for mutations in the components of SPI-1 within the same bacterium. However, this may reflect other factors, such as regulation of each SPI, since certain mutations in SPI-2 affect the transcription of SPI-1 genes and the ability of S. typhimurium  to secrete SPI-1 effector proteins and to invade cultured cells as well (60).  The function of the genes within SPI-2 appear to be divergent from those of SPI-1, and are necessary for intra-macrophage survival and dissemination throughout the host rather than invasion. Mutations within various SPI-2 genes resulted in reduced systemic spread of the infection within the mouse, although colonization of the intestinal Peyer's patches was still seen (46). A recent report has further implicated the SPI-2 region as necessary for bacterial replication in the host rather than bacterial survival per se (285). The secretion apparatus (ssa) is encoded within two regions: a 10 kb operon containing the genes ssaK, L, M, V, N, O, P, Q, R, S, T, and U (152) and a second smaller operon containing the genes spiC, A, B (239) (ssaED, C, B) (153). These two regions are separated by a region containing the potential effector (^e)/chaperone genes sseA, B, C, D, E, F, and G (153). The regulatory genes, ssrA and ssrB, are transcribed in the opposite direction to all the other genes within SPI-2. While many of the potential effector proteins have not been shown conclusively to be translocated into host cells, their expression is upregulated by intra-macrophage Salmonella (46, 321, 322). Mutations within various structural genes have been observed to have pleiotropic effects resulting in the increased sensitivity of the bacteria to complement, gentamicin, and polymyxin (60).  Chapter 1 16 SPI-3 (24) is a 17 kb region located at 82 minutes which is inserted within the selC t R N A locus.  It contains at least, two genes which contribute to bacterial survival within  macrophages, mgtCB (24). Although the G+C content of mgtCB is similar to the overall G + C content of 52-54%, they are immediately surrounded by regions with lower G+C content, (39.8 and 49.3%).  These genes are transcribed in an operon and enable Salmonella to  transport magnesium in low M g  2 +  conditions (225).  Although Salmonella contain other  magnesium uptake systems, mgtCB are tightly regulated by the PhoP/PhoQ two-component regulatory system (see below) and required for full virulence in mice (312).  It has been  suggested that M g t B has another function besides magnesium transport that is required by the bacteria inside host cells (290, 291). SPI-4 is a 27 kb region located at 92 minutes on the S. typhimurium  chromosome  which is inserted between ssb (encoding the single-stranded D N A binding protein) and SoxSR loci (encoding a superoxide regulatory gene) at sites encoding a tRNA-like gene (337). There are two 9 kb regions which have lowered G+C contents (37% and 44%), and one 7 kb region with a G + C content of 54%. SPI-4 encodes for 18 putative proteins, including seven with homology to type I secreted toxin proteins including Pasturella haemolytica leukotoxin and Bordetella  pertussis  adenylate cyclase-hemolysin.  This  region is also important for  intramacrophage survival. A n insertional mutation in this region renders the bacteria unable to grow within macrophages (13) SPI-5 is an 8 kb region located at 25 minutes in S. typhimurium  and in S. dublin  (159, 338). It is also present in other Salmonella serovars but not in Shigella sonnei, E. coli, or Yersinia pseudotuberculosis.  This island appears to consist of six genes: pip D,  orfX)  sigD/sopB, sigE /pipC, pip B, and pip A, which are flanked by the t R N A serT on one side and copS on the other side. O f these genes, sigD/sopB  is secreted by the SPI-1 type III secretion  apparatus and sigE /pipC is thought to be its specific chaperone (101, 159).  In S.  dublin,  SopB has been shown to be an inositol phosphate phosphatase which acts to increase chloride secretion by the host cell. Activities of SopB include the hydrolysis of phosphatidylinositol  Chapter 1 17 3,4,5-trisphosphate,  an inhibitor o f calcium-dependent chloride secretion and o f inositol  1,3,4,5,6-pentakisphosphate, an indirect i n h i b i t o r o f phosphatidylinositol 3,4,5-trisphosphatedependent c h l o r i d e secretion (235). Mutations w i t h i n the S. typhimurium  h o m o l o g s , sigDE,  are reported to reduce bacterial  i n v a s i o n by ten-fold (159), h o w e v e r this is not observed w i t h the S. clublin pipC.  N o n e t h e l e s s , the enteropathogenicity o f S. dublin  models w i t h mutations the sopB  and all the pip  genes, sopB  and  is greatly reduced i n calf ileal l o o p  genes,  although systemic disease appears  unaffected i n a m o u s e m o d e l (338). b.  O t h e r v i r u l e n c e genes S o p E is secreted by the type III secretion system o f the S P I - 1 ( 1 4 0 , 3 3 9 ) , yet it is  encoded outside o f centisome 63 w i t h i n a cluster o f genes from a cryptic P 2 - l i k e prophage (141).  T h e sopE gene was not f o u n d to be present in all S. enterica  number o f S. typhimurium S. choleraesuis  d i d not.  ^-dependent  manner  rearrangements  isolates, as w e l l  as  S. typhi  serovars:  contained the  S. dublin,  gene,  a  whereas  T h e S o p E protein is secreted into the host cell c y t o p l a s m in a (339)  where  its  actions  include the  along a C D C 4 2 - d e p e n d e n t pathway  (140)  activation  and  the  of  cytoskeletal  activation o f J N K .  Furthermore, S o p E was able to b i n d to several (but not all) members o f the R h o subfamily o f G T P a s e s to promote nucleotide exchange in vitro  (140).  Altogether, S o p E is capable o f  stimulating s i g n a l i n g pathways that can lead to both nuclear and m o r p h o l o g i c a l responses by the host c e l l (140). S o p D (in S. dublin)  is translocated into host cells and is secreted without amino-  terminal p r o c e s s i n g , h o w e v e r direct secretion v i a the type III pathway has not been s h o w n . is located d o w n s t r e a m o f the cysJIH SopD  acts  in  concert  with  It  o p e r o n , outside o f the k n o w n pathogenicity islands.  S. dublin  SopB  (SigD  in  S. typhimurium)  to  cause  enteropathogenicity i n the host, although the nature o f its activity remains obscure (173). A number o f flagellar genes have also been implicated in Salmonella  virulence (82).  W h i l e flagellar mutants are still able to invade cells w i t h high efficiency in vitro,  the ability to  Chapter 1  Chapter 1 19 smooth s w i m has been s h o w n to be advantageous d u r i n g the initial interactions w i t h the cells (171). S m o o t h s w i m m i n g m a y cause the formation o f p r o p u l s i v e bundles and free the majority of the bacterial surface f r o m steric constraints, w h i c h may occur w h e n the flagellar filaments are r a n d o m l y distributed d u r i n g tumbly s w i m m i n g . F u r t h e r m o r e , the s w i m m i n g motion may help the bacteria propel through mucus layers towards the cells. Other flagellar genes i n v o l v e d w i t h regulation, such as the alternative s i g m a factor F l i A and a c o r r e s p o n d i n g anti-sigma factor F l g M , m a y act to coordinately regulate diverse virulence-associated genes (283). M u l t i p l e f i m b r i a l adhesins, i n v o l v e d w i t h the initial adherence o f S. typhimurium to the intestinal m u c o s a , are required for full virulence o f the bacterium i n m i c e (324). least four fimbrial operons k n o w n i n S. typhimurium: (plasmid-encoded fimbriae) ( 3 2 4 ) , fim  There are at  Ipf (long polar finbriae) (324), pef  (type 1 fimbriae) (70, 82, 2 4 8 , 324), and agf (thin  aggregative fimbriae) (265, 307, 324). M u t a t i o n s i n any one o f these operons o n l y moderately affect v i r u l e n c e , but a combination o f mutations w i t h i n all four operons results i n more than 2 0 - f o l d attenuation o f the bacteria w h e n given orally to m i c e . H o w e v e r , there is evidence o f other fimbrial structures and/or adhesive organelles w h i c h may a l l o w for colonization o f the intestinal cells in the absence o f the fimbriae encoded by the k n o w n operons (324).  A cell-  contact-stimulated formation o f filamentous appendages has been i m p l i c a t e d in the triggering o f bacterial entry into host cells (258) and these appendages have been termed "invasomes" ( 1 1 3 , 340).  T h e formation o f these contact-stimulated filaments is thought to be secreted v i a a  type III secretion s y s t e m , h o w e v e r they are formed in the absence o f the S P I - 1 t y p e III secretion system, and their presence is not itself sufficient to trigger bacterial i n v a s i o n (258). Resistance to cationic antimicrobial peptides is mediated either directly or indirectly through P h o P / P h o Q regulation o f a number o f genes i n c l u d i n g pagB-pmrAB, L, M, and various L P S biosynthetic genes (117, 120, 245).  sapE, G, H, J,  T h e expression o f these genes  increases Salmonella resistance to the p o l y m y x i n - C A P family o f peptides (216), and may have effects on other systems as w e l l , e.g.  expression o f pmrE also results in modifications to the  l i p i d A portion o f L P S (134, 136, 148).  A gene not regulated by P h o P , sapM, is i n v o l v e d  Chapter 1 20 with resistance  against the neutrophil peptide (NP-1) defensin  (135).  Resistance  to  antimicrobial peptides may be effected via an energized efflux pump system (117), biochemical alteration/inactivation of the peptide (117), or the prevention of peptide binding to the bacterial surface by L P S (319). Salmonella resistance to complement includes a number of other factors such as many of the SPI-2 genes encoding type III secretion apparatus, the virulence plasmid-encoded Rck protein (45, 145), as well as L P S structure.  L P S oligosaccaride side chains act as a physical  barrier to complement such that the components are deposited away from the membrane and the pore-forming complex cannot penetrate the bacteria (82, 223). Reactive oxygen intermediate-mediated damage, such as lipid peroxidation and amino acid carboxylation, is countered by Salmonella in a number of ways. A bacterial copper- and zinc-cofactored superoxide dismutase SodC (mapping between centisome 23.8 and 29.6) prolongs bacterial survival in the presence of superoxide in vitro and within a mouse model (75). SodA, a manganese-cofactored superoxide dismutase, may confer resistance to the early oxygen-dependent microbiocidal mechanisms of phagocytes, especially in the absence of iron (316). Resistance to alkyl peroxides is conveyed via an alkyl hydroperoxide (ahp ) (93), while resistance to hydrogen peroxide is conferred by catalase (katG and katE), as well as by a 59 kD protein (303). Reactive nitrogen intermediates are combated by homocysteine production by Salmonella which may directly interact with S-nitrosothiols such as S-nitrosoglutathione (59). S-nitrosothiols act as nitric oxide donors and have also been shown to have broad-spectrum microbiostatic activities themselves (59).  Glucose 6-phosphate dehydrogenase  has been  shown to protect S. typhimurium against both oxygen and nitrogen intermediates in vitro, and is important during the early stages of salmonellosis (200). L P S and various LPS-biosynthetic genes play an important role in the virulence of Salmonella. Not only do they act to provide resistance to cationic peptides and complement, but the lipid A portion of L P S is toxic to host cells and causes an endotoxic shock in the host.  Chapter 1 21 LPS further is able to act as a chemokine and a macrophage activation factor (16), and may also be involved with the ability of Salmonella to transcytose cell layers (85). Other factors known to be important for the survival of S. enterica species within the host are extra-chromosomal and found to be encoded within a large virulence plasmid (50-90 kb) (21, 128, 242). The Salmonella-plasmid virulence genes, encoded by the operon spvRABCD  (129, 181), are upregulated within cultured cells (77) although they have not been  found to be important for intracellular growth in vitro (128).  These genes are important for  extracellular growth under nutrient-limited conditions however (253).  They are not required  for bacterial invasion into cultured cells (44, 232, 330), but promote survival and growth of the bacteria within animals thereby increasing bacterial virulence (127, 130, 131, 327). The operon is regulated by a number of factors which include nutrient limitation (66, 72), short-chain fatty acids (66), RpoS (66), and SpvR (54, 296). SpvR is the operon regulator and is able to upregulate its own expression, while SpvA downregulates the operon (296). SpvB, C, D are thought to be membrane-associated proteins, although their activities remain obscure.  The virulence plasmid may be involved with the lysis of resident and activated  peritoneal macrophages in vivo which could influence the net growth of Salmonella during infection (124).  1.3.3.  Regulation of virulence  genes  It is known that bacterial pathogens, such as Salmonella, tightly regulate the expression of many of their genes required for virulence. Many of these genes are coordinated in response to very specific environmental conditions, e.g. in response to heat or osmolarity, and often there are multiple regulators for a particular virulence gene such that its expression, and subsequent translation, are in response to a variety of environmental parameters. The expression of Salmonella invasion genes are in response to specific environmental conditions resulting in the production of new proteins immediately prior to bacterial invasion (201).  Chapter 1 22 a.  Environmental regulation T h e environment provides many clues to pathogenic bacteria. Temperature, specifically  the n o r m a l b o d y temperature o f the host, often serves to activate virulence genes, e.g. only m a k e Y o p s at 3 7 ° C , not at 2 6 ° C (211).  Interestingly, the Salmonella  not seem regulated to any great extent b y temperature. Salmonella  Yersinia  virulence genes do  p H serves to activate  numerous  v i r u l e n c e genes (3, 2 1 1 , 257), i n c l u d i n g Fur-regulated genes w h i c h are activated at  l o w p H (317) and m a n y o f the type III secretion genes in S P I - 1 w h i c h are expressed under slightly alkaline conditions (161).  Starvation for different nutrients {e.g.  results i n the activation o f R p o S regulated genes such as spvRABCD  carbon,  (66, 72).  nitrogen)  Environmental  cues k n o w n to affect the expression level o f a number o f virulence genes, i n c l u d i n g those f o u n d i n S P I - 1 , are o s m o l a r i t y (12, 211), o x y g e n tension (12, 7 5 , 91), g r o w t h phase (189), as w e l l as the level o f D N A superhelicity (100,  178).  T h e effects o f s u p e r c o i l i n g on gene  regulation is k n o w n to affect local gene expression, and can even affect the transcription o f regions w h i c h are kilobases away {e.g. promoter relay) (74): b.  Genetic regulation Salmonella  encodes  two-component  systems  for  the  g l o b a l regulation  of  genes,  i n c l u d i n g both v i r u l e n c e and housekeeping genes. E x a m p l e s o f these two-component systems are encoded by the genes phoP/phoQ  , pmrA/pmrB,  of a sensor protein and a regulator protein.  and ompR/envZ.  These systems consist  T h e sensor protein ( P h o Q , P m r B or E n v Z ) is a  histidine kinase w h i c h spans the bacterial membrane w i t h its extracellular d o m a i n acting to sense external signals and the intracellular d o m a i n acting as a kinase (329). the sensor protein undergoes  autophosphorylation  regulator protein ( P h o P , P m r A or O m p R ) .  and is then  U p o n stimulation,  able to phosphorylate  the  T h i s phosphorylation changes the ability o f the  regulator protein to b i n d to specific D N A sequences, a l l o w i n g it to act as a transcriptional activator and/or repressor (12, 80, 102, 103). autoregulated,  and  include  regulation  In addition, these genes are often positively  via two  separate  environmentally-sensitive and one w h i c h is not (135, 2 9 2 ) .  promoters,  one  of  which  is  M o r e o v e r , the two-component  Chapter 1 23 regulatory  systems  can interact to process  multiple environmental signals i n a complex  h i e r a r c h i c a l system (118, 135, 294). T h e P h o P / P h o Q regulatory system (located at 27.4 centisomes (167) has been s h o w n to modulate the e x p r e s s i o n o f over 4 0 genes w i t h i n S. typhimurium  i n c l u d i n g the  phoPQ  operon itself, resulting in the activation or repression o f these genes in response to external magnesium and c a l c i u m concentrations (103, 118, 2 1 1 , 2 9 3 , 3 1 2 ) , p H (12, 15, 118, 2 1 1 ) , o s m o l a r i t y (12), o x y g e n tension (18), and starvation for both phosphate and c a r b o n .  There is  evidence for the direct involvement o f P h o P to all the conditions (329), h o w e v e r , P h o Q appears to respond specifically to m a g n e s i u m and c a l c i u m (102,  118).  The  pathogenic  properties w h i c h are regulated b y the P h o P / P h o Q system include intra-macrophage  survival  (118, 215), resistance to a n t i m i c r o b i a l peptides, such as p o l y m y x i n and N P - 1 defensins ( 1 0 4 , 135,  137, 2 1 2 ) , the formation o f spacious vacuoles (5), adaptive mutagenesis  to g r o w t h -  dependent mutations (139), and the down-regulation o f the ability o f phagocytic cells to present bacterial antigens  to T - c e l l s (332).  Genes  activated by this system  are referred  to  as  PhoP-activated genes (pag) and m a n y virulence pag's promote intracellular s u r v i v a l . E x a m p l e s o f virulence genes activated b y P h o P include mgtCB (293), pmrAB p o s s i b l y pagC (214, 304), pagD, pagj, pagK, and pagM  (135), pagP (137) and  (19, 132-134, 136).  Genes which  are repressed are k n o w n as P h o P - r e p r e s s e d genes (prg), and many are i n v o l v e d w i t h bacterial i n v a s i o n and early s u r v i v a l w i t h i n the p h a g o l y s o s o m e (213, 332). genes repressed b y P h o P include S P I - 1 - e n c o d e d genes hilA, prgHIJK, and orgA (246).  E x a m p l e s o f virulence sip/sspA,  sipC, invF  A balance between activation and repression is needed for full virulence in  vivo, since constitutive expression o f P h o P attenuates Salmonella virulence and s u r v i v a l (215). M a n y genes r e s p o n d i n g to regulation by P h o P / P h o Q are co-regulated b y other systems, such as activation b y S i r A (167), or may be responding to other regulators w h i c h also respond to P h o P , such as H i l A (12, 18, 167) or P m r A (103, 120, 294). T h e P m r A / P m r B regulatory system has been s h o w n to modulate genes directly in response to m a g n e s i u m concentrations and to p H (120, 294), as w e l l as indirectly i n response  Chapter 1 24 to regulation b y P h o P / P h o Q (135).  Furthermore, many genes w h i c h are regulated by p H are  coordinately regulated b y both P h o P / P h o Q and P m r A / P m r B (15, 103, 2 9 4 ) .  The P m r A / P m r B  system confers bacterial resistance to a number o f cationic antimicrobial peptides, such as p o l y m y x i n (but not N P - 1 defensin) through regulation o f the pmrE/pagA and pmrF loci (120). T h e O m p R / E n v Z regulatory system (located at 75 to 80 centisomes) (230) has been s h o w n to regulate the formation o f spacious vacuoles i n macrophages, and affect the ability o f the bacteria to k i l l these cells (199), resulting in bacterial attenuation (61).  The O m p R / E n v Z  system controls the expression o f Salmonella outer membrane proteins, such as O m p F , O m p C , and the tripeptide permease T p p B (40, 198, 199), and also affects the formation o f Salmonellainduced filaments w i t h i n host cells (221).  T h e acid tolerance response also is dependent on  this t w o c o m p o n e n t regulatory system during bacterial growth in m i n i m a l m e d i a (89). S i r A (Salmonella i n v a s i o n regulator) is a transcriptional activator w h i c h similar to the two-component response-regulator F i x J / U v r Y subfamily (167).  F i x J o f Rhizobium meliloti,  has been s h o w n to activate target gene expression after phosphorylation i n response to changes i n environmental o x y g e n (167).  A l t h o u g h it is located outside o f any identified pathogenicity  i s l a n d at 42.4 centisomes, S i r A is k n o w n to activate a number o f pathogenicity island-encoded genes, i n c l u d i n g S P I - 1 encoded hilA  and S P I - 5 encoded sigDE.  It is unclear whether its  effects on these genes are direct or indirect (167). H i l A is encoded w i t h i n S P I - 1 (190) and is a transcriptional activator related to O m p R (11), although it contains neither a p h o s p h o r y l acceptor nor a membrane-spanning d o m a i n .  It  is required for the activation o f m a n y SPI-1 encoded invasion genes i n c l u d i n g the inv-spa—prg operon  and invF  ( w h i c h further  promotes  sipBCDA  expression).  The  hilA  locus  is  coordinately regulated b y both P h o P (12, 167) and S i r A (167), although its expression does not necessarily affect the transcription o f other P h o P or S i r A regulated genes, e.g.  sigDE  transcription is unaffected by H i l A but is dependent on S i r A (159). S l y A , o r i g i n a l l y reported to be a c y t o l y s i n (197), is actually a transcriptional regulator o f S. typhimurium  w h i c h is upregulated w i t h i n macrophages  and b y entry into stationary  Chapter 1 25 phase (29, 2 6 9 ) . Other members o f this family o f proteins include M a r R and E m r R w h i c h p r o v i d e resistance to a w i d e range o f toxic c o m p o u n d s  b y regulating the expression o f  membrane efflux systems. S l y A is required for bacterial resistance to oxidative products such as h y d r o g e n p e r o x i d e , but not to nitric o x i d e products!  W h e n this gene is put into E. coli, it  activates the c r y p t i c h e m o l y s i n clyA, although the presence o f a C l y A - l i k e t o x i n i n Salmonella has not been s h o w n (29). Mutations w i t h i n Salmonella slyA  result i n attenuation i n mice b y  o r a l , I P , a n d I V routes o f i n f e c t i o n , a n d this gene has been implicated for intracellular survival rather than for the initial i n v a s i o n or c o l o n i z a t i o n steps (56). F u r (ferric uptake regulator) is a gene repressor (located at 16.9 centisomes  (230))  w h i c h is inactivated w h e n iron availability is l o w a l l o w i n g for the activation o f genes i n v o l v e d in iron acquisition (88, 3 1 7 ) . It is required for intra-macrophage s u r v i v a l (317).  It is further  required for a transient Salmonella acid-tolerance response (89, 1 9 1 , 3 3 3 ) , although this effect may be both direct and indirect as p H has a varied effect on different iron-regulated genes ( 8 8 , 317).  T h e F u r repressor m a y also act on genes required for resistance to reactive o x y g e n  intermediates, such as sodA (316). T h e role o f F u r regulation i n bacterial virulence is unclear (107). R p o S (sigma-38) is an alternative s i g m a factor virulence.  w h i c h is required for Salmonella  T h e avirulence o f L T 2 strains o f S. typhimurium  results (at least i n part) from a  defective rpoS gene ( 3 0 8 , 3 3 4 ) . R p o S is used to transcribe genes required d u r i n g times o f carbon and nitrogen starvation (73), d u r i n g the accumulation o f metabolic products (such as short-chain fatty acids) (66), and w h e n the bacteria enter stationary phase (71, 335).  W h i l e the  expression o f rpoS has been s h o w n to increase after i n v a s i o n into cells (41) and rpoS is thought to be e x c l u s i v e l y required for systemic infection ( 1 2 5 , 3 3 0 ) , more recently it has been s h o w n to play a role i n the colonization o f Peyer's patches (231).  A n example o f virulence  genes regulated b y R p o S include the spv plasmid-encoded genes, w h i c h are required for long term s u r v i v a l o f Salmonella i n the host. phase use R p o S .  H o w e v e r , not all genes upregulated  i n stationary  F o r example, the increase i n slyA expression d u r i n g stationary phase is  Chapter 1 26 independent o f R p o S (29), and i n d u c t i o n o f S p v R by R p o S occurs d u r i n g exponential g r o w t h but not d u r i n g stationary phase (66). R p o S is also required for a sustained, l o w - p H - i n d u c i b l e a c i d tolerance response i n S. typhimurium that occurs i n stationary phase (88, 191, 192). It is not required for the acid tolerance response  in logarithmic phase g r o w t h , although it may  co-regulate the atrB locus w h i c h is required (191, 333). S p v R is a virulence plasmid-encoded transcriptional activator w h i c h is upregulated d u r i n g stationary phase (54, 296, 335), as w e l l as during logarithmic phase in m i n i m a l salts media (but not rich media) (54, 335), and u p o n entry into host cells (77, 2 6 1 , 2 6 2 ) . p o s i t i v e l y regulates an operon immediately downstream e n c o d i n g genes spvABCD 323).  SpvR  (287, 2 9 6 ,  These genes are required for both gastroenteritis and systemic infection by the bacteria  w i t h i n animal models (196), and more recently they have been s h o w n to be essential for replication o f Salmonella  w i t h i n macrophages in the host (131).  S p v R is acted upon by a  number o f different factors, i n c l u d i n g the upregulation by R p o S and autoregulation (335). It is downregulated b y h i g h levels o f S p v A (296, 323), by iron (296), as w e l l as ( c A M P ) and the c A M P receptor protein ( C R P ) ( 7 1 , 236).  cyclic-AMP  S p v R has been s h o w n to be  regulated b y a two-step m o d e l o f transcription activation in a similar manner to L y s R o f the Vibrio luciferase operon (287). 1.4.  Reporter  systems  f o r b a c t e r i a l gene  expression  Reporter enzymes m a k e it possible to visualize the expression o f bacterial genes w h o s e products are not readily assayable. T h e expression o f a specific gene can be monitored by first fusing that gene w i t h a promoterless reporter gene and then measuring activity o f the reporter gene product.  H o w e v e r , the reporter enzyme must meet at least t w o criteria.  F i r s t , it must  have an activity that is distinct f r o m endogenous cellular or bacterial enzymes in the system used. S e c o n d , (relative to the studies here), the reporter enzyme assay must be very sensitive, as numbers o f i n v a s i v e bacterial are often l o w and bacterial gene expression may be moderated.  Chapter 1  o  _  cu  o  cs E c/l O  © U  E  o C/l O  E  o  sX cu  3  ©  cu JS •w  c © c sh oge nicity isl an ds ar  o  a a  ph  5 •2 °C  s _© sen  S3  cu i-l  a  CU  u _cu '& cs E CU  Xi  CU C€  CU  u  3  X -*H  C/3  e  • CU —•  x Hi  c o a ©  X! C/l cu u es u ©  CU L. CU fl  cu  "3 JIA  O  cs  =H-  ©  CU X  a c  Kn  CU  Xi ^  CU  o  s  cs E  itiso  Salmo  *».  ide.  o  CU T3 w C/1 Hi  gulat  <u  rice gei  ?  cu CU X  um  'hie  JS  E CU CU  o o  JU  xi  CU  icu  Chapter 1 28 1.4.1. a.  Reporter  genes  B galactosidase z  6-Galactosidase has traditionally been the most w i d e l y used bacterial reporter enzyme and is a sensitive reporter o f gene expression (38, 89, 163, 2 8 9 , 2 9 3 ) .  It is encoded by a  single gene, lacZ, w h i c h may be fused to a gene o f interest as either a translational f u s i o n , resulting in a h y b r i d protein, or a transcriptional fusion, in w h i c h both reporter and target genes are expressed under one promoter but the reporter protein is separate f r o m the c o d i n g region (38). B-galactosidase h y d r o l y z e s a number o f c o m m e r c i a l l y - a v a i l a b l e substrates w h i c h may be assayed  colorimetrically  chemiluminescently (28).  (28,  163),  fluorescently  (28,  77,  108,  251,  289)  or  T h e activity o f the enzyme may be detected under a w i d e range o f  b i o c h e m i c a l conditions (on agar plates, i n s o l u t i o n , or w i t h i n host c e l l s ) , and does not require any co-factors for its activity (e.g.  o x y g e n or bacterial energy).  H o w e v e r , enzymatic activity  persisting in a k i l l e d bacterium can not be differentiated f r o m the activity in a viable bacterium and may p o s s i b l y result i n an apparent higher activity per bacterium than F u r t h e r m o r e , the bacterial and host cell membranes  are not permeable  to  really exists. B-galactosidase  substrates and must be l y s e d in order to quantify the enzymatic activity in s o l u t i o n .  Finally,  w h i l e Salmonella do not produce an intrinsic B-galactosidase, host cells do have a l o w level endogenous activity (28). b.  B a c t e r i a l luciferase  B a c t e r i a l luciferases may be found in many different bacteria and are generally encoded by t w o genes, luxAB, w h i c h result i n a heterodimeric enzyme (210), and are transcribed as part o f an o p e r o n (222), w h i c h also encodes gene products necessary for the production o f an aldehyde substrate (210).  In the absence o f the. substrate synthesis genes (e.g.  substrate must be added to the sample (138).  luxAB  only),  the  In addition to an aldehyde substrate,  the  luciferase requires the presence o f o x y g e n (143) and the bacterial energy source, F M N H  2  (17,  186), but does not require metals or cofactors (210). T h e product o f e n z y m a t i c reaction is light in the v i s i b l e range between 4 7 8 - 5 0 5 n m (17, 266). M o s t eukaryotic luciferases (luc firefly or  Chapter 1 29 beetle luciferases (28)) are encoded b y a single gene and require A T P as an energy source. T h e y are more susceptible to changes in p H , ionic strength,  and temperature than are the  bacterial luciferases, and also require a metal cofactor (266). O f the hundreds o f different strains o f luminescent bacteria, the most w i d e l y used reporter  genes c o m e f r o m  Vibrio fischeri (69, 92, 210), V. harveyi (187, 193, 210),  Photorhabdus (Xenorhabdus) luminescens (52, 86, 210), Photobacterium phosphorum (210), or  P. leiognathi (210).  T h e most d i s t i n g u i s h i n g feature between the different luciferases is  their o p t i m a l temperature for activity. Luciferase from V. fischeri is stable and active between 20"C and 3 0 " C but not at 3 7 " C , whereas luciferases f r o m stable at 37"C or higher (209, 2 5 1 ) . over other reporters.  V. harveyi and P. luminescens are  T h e use o f bacterial luciferases offers several advantages  T h e luciferase aldehyde substrate, n-decanal, is volatile, amphipathic,  and readily crosses membranes  (138,  155, 2 1 0 ,  244,  substrates [Garcia-del P o r t i l l o , 1992 #22; (28, 77).  276), u n l i k e most  B-galactosidase  Therefore, luciferase activity can be  assayed without the need for bacterial or host cell l y s i s , and potentially, expression c o u l d be assayed w i t h i n the same sample over time (186, 251).  It is also possible to monitor bacterial  gene expression f r o m w i t h i n a w h o l e animal (35, 52).  A s w e l l , u n l i k e B-galactosidase, most  bacteria and tissue culture cells have no endogenous  luciferase activity (52).  p r o d u c e d is the direct result o f luciferase activity. indicator o f bacterial viability (180, 3 0 1 , 302). mononucleotide  A n y light  Furthermore, light production is also an  In the absence o f a sufficient supply o f flavin  (FMNH2) w i t h i n the bacteria, light is not p r o d u c e d even i f luciferase is  present, resulting i n measurement o f activity f r o m viable organisms o n l y (52). c.  Other reporter  genes  It has been possible to demonstrate the upregulation o f various genes b y tissue culture cells u s i n g various other reporter genes. fimbriae) (262), (28, 157),  Salmonella inside  E x a m p l e s include  papA  (type I  cat (chloramphenicol acetyl-transferase) (297), phoA (alkaline phosphatase)  gusA (B-glucuronidase) (28), and galK (galactokinase) (300).  Chapter 1 30 1.4.2.  Other  detection methods  In Vivo Expression Technology (IVET) is a technique which enables researchers to positively select for Salmonella genes which are expressed upon infection of a host animal (147, 202, 203, 289).  The bacteria are engineered with a promoter trap in which bacterial  promoters drive the expression of a gene cassette. This cassette contains a gene which is required for growth in the host but can be supplemented for in vitro (e.g. either pur A (auxotrophy) or car (antibiotic resistance)) and the reporter lacZ gene (146, 202, 203). The final gene fusions are generated on chromosomal DNA, rather than on a plasmid, using gene duplication in a manner that avoids gene disruption unlike regular transposon insertional fusions. Since the genes are contained on the chromosome and not on a multi-copy plasmid, it is likely that the genes are regulated under the natural promoter environment. IVET identifies genes which are expressed upon infection of mice rather than upon invasion of a particular cell type.  Therefore, bacterial genes that are necessary at any time during infection will be  identified using this technique, providing the specific bacterial clones survive within the host until they can be recovered. IVET does not indicate at which point the bacterial genes are required, i.e. are they transiently induced or expressed in vivo all the time, and a large number of the identified bacterial genes are not specific for virulence (i.e. housekeeping).  As well,  genes which may be inhibitory or refractory to recombination events in the bacteria may be under-represented. Furthermore, complementation in the animal may demand high levels of expression relative to growth on laboratory medium, indicating that small increases in gene expression may be overlooked. Signature-tagged mutagenesis (STM) is a negative selection technique that is also based on in vivo selection of bacterial mutants (147, 151). Transposons carrying gene tags, which consist of 40 bp of unique DNA flanked by 20 bp regions of DNA common to all the tags, are randomly integrated into the Salmonella chromosome. A pool of tagged insertion mutants is then used to infect a host animal and those bacterial mutants which cannot be recovered from the animal are deemed to carry insertions within genes necessary for infection. The presence or  Chapter 1 31 absence o f a particular bacterial mutant  is assessed  by dot blot h y b r i d i z a t i o n , w i t h  hybridization signals f r o m the recovered p o o l compared w i t h those f r o m the input p o o l .  the The  c o m p l e x i t y or size o f the pools must be restricted such that each mutant is present in sufficient numbers so that avirulent mutants are not falsely identified and that the h y b r i d i z a t i o n signals do not become too w e a k to identify. Differential display also utilizes an in vivo mouse m o d e l , h o w e v e r it l o o k s for gene expression i n the absence o f bacterial mutagenesis, i.e. without the use o f reporter genes. requires the subtractive hybridization o f t w o bacterial c D N A  It  libraries, w i t h the resulting  c D N A s are used as probes to search for in v/vc-expressed bacterial genes (147).  One c D N A  library is made f r o m bacteria g r o w n on laboratory m e d i u m and represents the p o o l o f 'housekeeping' genes. T h e other c D N A l i b r a r y is made f r o m bacteria recovered f r o m infected tissue, and represents those genes required for bacterial virulence. T h e ' h o u s e k e e p i n g ' genes are subtracted or r e m o v e d f r o m the p o o l o f genes expressed u p o n infection, and the remaining genes i n the 'infected' p o o l are then used to screen the bacterial genome in search o f genes required specifically for virulence. T h e crucial step o f this technique lies i n the ability to isolate and stabilize the bacterial m R N A s in order to m a k e a representative c D N A library. U n l i k e most eukaryotic m R N A s , bacterial m R N A s do not have a p o l y - A tail. Differential fluorescence induction ( D F I ) (320) is another technique utilizing a D N A promoter trap, where bacterial genes are selected on the basis o f their expression within cultured macrophages rather than a w h o l e animal (147, 3 2 1 , 322).  T h e gene e n c o d i n g the  green fluorescent protein ( G F P ) is transcriptionally fused w i t h Salmonella genes and then, with the use o f a fluorescence-activated cell sorter  (FACS),  host cells containing intracellular  bacteria w i t h genes expressing h i g h levels o f G F P are detected and sorted.  A n advantage o f  this technique  within  is the ability to determine the bacterial gene expression  a single  macrophage (321). A disadvantage o f this technique is that gene fusions to G F P are contained on a m u l t i - c o p y p l a s m i d , rather than i n the c h r o m o s o m e . o f fluorescence obtained f r o m the various gfp  fusions,  T h i s was done to increase the range i m p l y i n g that l o w levels o f  gfp  Chapter 1 32 expression may be missed with this technique. Furthermore, regulation of the genes may not have reflected the true nature of the expression within the chromosomal environment, e.g. repressors and/or activator proteins may have been titrated out by the multiple copies of the gene promoters, and there would be no gene regulation in cis or by promoter relay. A second disadvantage arises from the use of pooled clones, where competition between bacteria during growth (either intracellular or extracellular expansion of the pools) may result in the loss of some fusions. Using two-dimensional protein gels, it has been shown that bacteria inside macrophage cells express different protein profiles than those grown outside cells (1, 30, 34, 254). With this technique, radiolabelled proteins in bacteria and/or cells can be separated into a discrete spots on a gel by separating them on both their isoelectric point and their molecular size. More recently, a technique involving the use of radiolabelled diaminopimelate (a lysine precursor specifically used by bacteria) has been used to identify the patterns of intracellular bacterial protein expression, without contamination by host proteins (33, 34, 147). These techniques are very powerful, however many of the proteins have yet to be identified. 1.5.  Summary  of  thesis  In the past six years there has been a veritable explosion of data regarding the mechanisms of virulence for not only Salmonella but many other gram-negative bacteria as well. When this study was initiated, pathogenicity islands and type III secretion systems were unknown. At that time, it had been shown that Salmonella expressed an unique set of genes inside macrophages, that were not expressed in the presence of other stresses (heat, low p H , starvation, oxidative stress) either singly or in combination (1, 30).  Moreover, this gene  expression differed slightly depending on the isolate of Salmonella used as well as the type of cultured cells used.  It was determined that over 100 bacterial genes were upregulated by  intracellular S. typhimurium,  with over 40 genes deemed unique to the intra-macrophage  environment. However, only a handful of these genes could be attributed to those already known (30). It had furthermore been determined that the ability of Salmonella to invade cells  Chapter 1 33 and to g r o w intracellularly were separate events and each were required for virulence in animal models (78).  Therefore, the goal o f this study was to develop a system to search  for  Salmonella genes w h i c h were o n l y expressed inside cells, and thereby u n c o v e r genes w h i c h may be essential for intracellular survival and perhaps virulence. T h e first section describes the development o f a light-based reporter system by w h i c h to detect genes expressed f r o m intracellular Salmonella. made to the Salmonella  Initially, reporter gene fusions  p l a s m i d virulence genes spvRAB,  were  w h i c h had p r e v i o u s l y been s h o w n  to be required for Salmonella virulence in a mouse m o d e l . T h e spv  genes were also k n o w n to  be induced b y carbon and nitrogen starvation and d u r i n g stationary phase g r o w t h .  U s i n g the  enzyme 6-galactosidase (lacZ), the spv genes were s h o w n to be upregulated after Salmonella i n v a s i o n into epithelial cells (77).  S i m i l a r results o f induction were c o n f i r m e d b y t w o other  studies, where the genes were s h o w n to be upregulated w i t h i n phagocytic cells as w e l l (262). Nevertheless, the use o f 6-galactosidase as a reporter was not ideal, and therefore bacterial luciferase was tested as alternate reporter o f intracellular bacterial gene e x p r e s s i o n .  The  luciferase genes were fused to the spv genes i n order to be able to directly compare between the t w o reporter systems.  T h e results indicated that w h i l e luciferase was also not a  reporter, it was as sensitive as 6-galactosidase  'perfect'  and offered a number o f advantages as a  reporter, n a m e l y the ability to monitor gene expression w h i l e the bacteria were inside the cells and the ability to differentiate between the activity f r o m l i v e and dead bacteria (251). T h e second section describes the construction o f a library o f Salmonella mutants to be used to search for genes upregulated by intracellular bacteria.  A l s o described i n this section is  the initial screening procedure used to look for genes w h i c h were o n l y upregulated  once  Salmonella had b e c o m e intracellular. T h e promoterless luciferase reporter genes were inserted as single r a n d o m insertions throughout  the c h r o m o s o m e ,  using a modified two-plasmid  competition system (138). B a c t e r i a were then tested for their ability to produce little to no light outside m a m m a l i a n c e l l s , w h i l e p r o d u c i n g light f r o m the intracellular environment.  This  Chapter 1 34 a l l o w e d for the identification o f genes w h i c h were repressed d u r i n g g r o w t h i n rich m e d i a , but were activated o r upregulated d u r i n g Salmonella growth w i t h i n cells. T h e final section describes the characterization o f the Salmonella mutants w h i c h were f o u n d to contain upregulated gene fusions.  T w o o f the genes identified i n this screen were  f o u n d to be part o f S P I - 5 , sigD/sopB and pipB one gene w a s f o u n d to be encoded within S P I - 2 , ssaR; and the fourth gene w a s completely n o v e l , iicA.  N o o b v i o u s difference i n  phenotypes c o u l d be identified w h i l e the bacteria grew in vitro w h e n c o m p a r i n g the insertional mutants to the w i l d t y p e bacteria. T h e bacterial mutants retained their ability to invade and g r o w w i t h i n cultured cells (at least d u r i n g short time periods).  Interestingly, all four gene fusions  were not o n l y upregulated w i t h i n phagocytic cells, but were upregulated w i t h i n non-phagocytic cells as w e l l .  T h e insertions w i t h i n all o f these genes reduced the in vivo virulence o f  S. typhimurium to v a r y i n g degrees, except f o r iicA w h i c h retained its virulence i n the mouse model. W i t h i n this study, a reporter system was described w h i c h was specifically able to detect the gene e x p r e s s i o n from bacteria residing inside mammalian c e l l s .  M o r e o v e r , the activity  c o u l d be correlated to viable bacteria. B y incorporating this system into the bacterial genome, several Salmonella virulence factors were identified and characterized. That not all the genes upregulated b y intracellular bacteria were f o u n d to be virulence factors indicates the increasing c o m p l e x i t y o f a n i m a l models over c e l l culture models.  Chapter 2 35 C h a p t e r 2: 2.1.  Media and  2.1.1.  Methods  chemicals  Chemicals and  Detergents  Materials and  Assay  Reagents  T r i t o n X - 1 0 0 and s o d i u m d o d e c y l sulfate ( S D S ) were purchased  from  S i g m a - A l d r i c h C a n a d a , L t d . ( O a k v i l l e , O N ) and made up as 10% stock solutions i n sterile water, w h i c h were stored at r o o m temperature. T r y p s i n ( G i b c o L i f e Technologies; B u r l i n g t o n , O N ) was stored at - 2 0 ° C and used undiluted during the passage o f epithelial c e l l s . D i m e t h y l sulfoxide ( D M S O ) ( B D H Inc.; T o r o n t o , O N ) was used as a cryoprotectant cultured c e l l s , at a final concentration o f 10%. bromide,  agarose,  ethanol,  methanol,  w h e n freezing  M i s c e l l a n e o u s chemicals such as ethidium  phenol,  chloroform,  sodium  chloride,  h y d r o x i d e , h y d r o c h l o r i c a c i d , etc. were purchased from a number o f different  sodium  companies  i n c l u d i n g : S i g m a - A l d r i c h C a n a d a , L t d , B D H I n c , V W R Scientific C a n a d a , L t d . ( L o n d o n , O N ) , Amersham Canada, L t d . (Oakville, O N ) , Baxter-Canlab ( V W R - C a n l a b ; Mississauga, O N ) , D i f c o L a b s - F i s h e r S c i e n t i f i c (Ottawa, O N ) , and G i b c o - B R L ( B u r l i n g t o n , O N ) . n-Decanal ( 9 9 % ; S i g m a ) was used for luciferase assays and kept stored at 4 ° C in an airtight container. rc-decanal  A range o f aldehyde concentrations  were made b y first adding  1 pi  into 1, 2, 4, or 10 m l o f a solution o f 7 0 % (v:v) ethanol:8% (v:v) methanol.  One  milliliter o f this solution was then added to 4.5 m l concentrations  of 0.02%,  0.011%, 0.0055%,  of M E M +  10% F B S , resulting in  and 0 . 0 0 2 2 % ( v : v ) n-decanal,  respectively.  T w o further concentrations o f aldehyde were made by adding 1 pi n-decanal to 100 or 2 0 0 p i ethanol:methanol, and then adding 100 p i o f this solution to 1 m l o f tissue culture media, resulting i n aldehyde concentrations  of 0.099%  and 0 . 0 4 9 5 %  (v:v) respectively.  alcohol a l l o w e d the even dispersal o f the aldehyde in the solutions. alcohol was dilutions.  Use of  T h e concentration o f  14.2% (v:v) for the four lowest d i l u t i o n s , and 7 . 8 % (v:v) for the t w o higher  D u e to their long-term instability in suspension, aldehyde solutions were kept in  airtight containers, at r o o m temperature, for no longer than 4 h . (Note: the final concentration  Chapter 2 36 of the aldehyde in the luciferase assay well is another 10-fold diluted: see "luciferase assay" below.) (251) For fluorescent B-galactosidase assays,  the fluorescent substrate fluorescein-di-  galactopyranoside (FDG) (25) and the B-galactosidase inhibitor phenyl-ethyl-thio-galactoside (PETG) (58) were used (Molecular Probes; Eugene, OR) (77, 108). F D G and PETG were initially dissolved in DMSO, and then diluted and stored at a concentration of 50 m M at -20°C for up to 1 month. The final concentration of DMSO in the stock solutions was 25% (v:v). For chemiluminescent B-galactosidase assays, the Galacto-iSTA/? ™ assay kit was used according to manufacturers instructions (Tropix - Perkin Elmer; Bedford, M A ) .  2.1.2.  Antibiotics  All antibiotics were purchased from Sigma.  Ampicillin (amp; 100 pg/ml (w:v)),  tetracycline (tet; 15 pg/ml (w:v)), streptomycin (str; 25 pg/ml (w:v)), and chloramphenicol (cm; 30 pg/ml (w:v)) were used as selection agents for plasmid maintenance and bacterial growth. Gentamicin was used to inhibit extracellular bacterial growth during invasion assays (310) at a concentration of 100 pg/ml (total weight per volume) for the first four hours and at 10 pg/ml for incubations longer than four hours.  2.1.3. a.  Molecular  Biology  Reagents  Reagents  Restriction enzymes Haelll,  Rsal, and BamHI  were purchased from both New  England Biolabs (Mississauga, ON) and Boehringer Mannheim Canada (Laval, QC). T4 DNA ligase and deoxynucleotides (dNTPs) were purchased from Boehringer Mannheim Canada. The dNTPs were made as a 2.5 m M stock solution and stored at -20°C; all four deoxynucleotides were in equal proportions.  The polymerase AmpliTaq (Perkin-Elmer  Applied Biosystems; Norwalk, CT) was used for inverse polymerase chain reactions (inverse PCR).  For sequencing reactions, ABI's AmpliTaq Dye Terminator Cycle Sequencing  chemistry with FS Taq was used as recommended by the Nucleic Acid and Protein Services Unit (NAPS Unit) at the University of British Columbia. Centrisep spin columns (Princeton  Chapter 2 37 Separations; Adelphia, NJ) were used to remove unused dyed dinucleotides from the sequencing reactions, b.  Primers  DNA primers were made by the NAPS Unit and are listed in Table 1. The primers LUX76 and LUX340 were made to regions within the luxA gene; all other primers were made to regions on the S. typhimurium  chromosome. Primer set LUX76:LUX340 was used to  amplify the regions upstream from the luxA gene in all six clones initially identified.  Primer  sets E-MINUS:E-PLUS and E3615:E3805 were used to amplify the region around the insertion site of E12A2; chromosomal DNA from both E12A2 and SL1344 was used. Primer set G-MINUS:G-Plus was used to amplify the region around the insertion site of G7H1; chromosomal DNA from both G7H1 and SL1344 was used. All primers except for LUX340 were used for sequencing.  Table 1: Primers  Primers for Inverse PCR Length  Sequence (5' to 3')  LUX76  18 nt  CAA  GCG  ACG  TTC  LUX340  18 nt  TGC  CGC  ACA  T C T A T T AGG  E-PLUS  20 nt  CAG  TTT  TCC AAT  TAC  E-MINUS  22 nt  TTC  TGG  AGG  ATG  TCA ACG  GGT  G  E3615  24 nt  ACA  GCG  TGT  AGA  TTT  GCA  CAA  CAC  E3805  21 nt  GAC  AGG  TAG  TCA ACA  TAC  CCC  G-MINUS  20 nt  GGA  GGA  ATG  CAC  ACC  TTT  AG  G-PLUS  19 nt  TAG  TCC  CTA ACC  CCC  ATT  G  ATT  CAC  CTC  CC  Chapter 2 38 2.1.4.  Media  Tissue culture cells were grown in either minimal essential media (MEM #410-1500; Gibco Life Technologies) or Dulbecco's modified Eagle medium (D-MEM #430-2800; Gibco Life Technologies), both of which were supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies). DMEM++ refers to basic D - M E M supplemented with both 10% serum and 20 m M HEPES (pH 7.4).  Phosphate-buffered saline containing calcium and  magnesium (PBS++) was used for washing and dilutions. Bacteria were grown in a number of different media. Luria-Bertani (LB) broth and agar plates, MacConkey plates, and SOC broth, were used for routine bacterial culture, and recipes can be found in Sambrook et al. (275) and Ausubel et al. (9). Special "green plates" were used to isolate phage-free bacteria after transductions and made as previously described (68, 300). The green plates contain the dyes Alizarin Yellow and Aniline Blue, and infected colonies appear dark green in color while uninfected colonies remain pale. Bacteria were also grown in DMEM++ where indicated. 2.1.5.  Buffers  A number of different buffers were used throughout this study.  Phosphate-buffered  saline (PBS— or PBS++) was used as a diluent for bacteria and cells where indicated. PBS consisted of 0.2 g/L KCI, 0.2 g/L K H P 0 , 8 g/L NaCl, and 2.16 g/L N a H P 0 . 7 H 0 and 2  4  the final pH was adjusted to 7.4 with HC1.  2  4  2  P B S - contained no calcium or magnesium,  whereas PBS++ contained 130 m M calcium and 200 m M magnesium. HEPES buffer (pH 7.4) was used as an extra buffering agent in DMEM++. T A E buffer was used for making and running agarose gels (185). T E buffer was used for resuspending preparations of DNA (275), and 50 m M T E (i.e. 50 m M Tris pH 8.0 + 50 m M EDTA) was used with chromosomal DNA preparations, while a 5 m M TE concentration was used with plasmid D N A samples (i.e. 5 m M Tris pH 8.0 + 5 m M EDTA). Tris-HCl buffers (made in a range from pH 7.0 to 8.0) were used for molecular biological manipulations and made according to Sambrook et al. (275). P22 buffer was used during the dilution and storage of the bacteriophage P22 (300).  Chapter 2 39 2.2.  Equipment H o r i z o n m i n i - g e l apparatus ( B R L ; B u r l i n g t o n , O N ) were used in conjunction w i t h an  F B 1 0 5 p o w e r source (Fisher Scientific; P i t t s b u r g h , P A ) was used for r e s o l v i n g D N A on agarose gels as p r e v i o u s l y described (275).  D N A gels were stained w i t h ethidium bromide  (275) and v i s u a l i z e d u s i n g a long-wavelength transilluminator (Fisher Scientific).  Pictures  were taken u s i n g a P o l a r o i d camera and 667 f i l m ( P o l a r o i d , L t d . ; St. A l b a n s , Hertfordshire, E n g l a n d ) . T h e G e n e P u l s e r ™ f r o m B i o R a d ( R i c h m o n d , C A ) was used to electroporate D N A into competent bacterial c e l l s .  T h e E r i c o m p T w i n b l o c k S y s t e m thermocycler ( E r i c o m p Inc.;  S a n D i e g o , C A ) was used for P C R reactions (both inverse P C R and sequencing reactions). S e q u e n c i n g gels were made and run b y the N A P S determined u s i n g an automated 373 D N A Sequencer.  U n i t and the D N A sequences  were  Sequences were further analyzed u s i n g  N C B I ' s B L A S T p r o g r a m and the G e n B a n k database. Three different sets o f apparatus were used for determining light production from bacterial samples.  F i r s t , microtiter plate formats were exposed to X - r a y f i l m ( X - O M A T by  Eastman K o d a k C o m p a n y ; Rochester, N Y ) and the resulting spots were quantitated w i t h a c o m p u t i n g densitometer ( M o l e c u l a r D y n a m i c s ; S u n n y v a l e , C A ) . S e c o n d , light f r o m tubes o f bacteria was measured u s i n g the 1250 L u m i n o m e t e r ( L K B - W a l l a c ; F i n l a n d ) .  Third,  the  L u m i n o g r a p h L B 9 8 0 ( E G & G B e r t h o l d ; G e r m a n y ) (26) was used to measure light from a number o f formats i n c l u d i n g agar plates and microtiter plates.  Fluorescence was monitored in  9 6 - w e l l microtiter F l u o r i c o n assay plates ( I D E X X C o r p o r a t i o n ; W e s b r o o k , M E ) w h i c h were read on a P A N D E X F l u o r e s c e n c e Concentration A n a l y z e r ( I D E X X ; Portland, O R ) . C u l t u r e d cells were routinely expanded in sterile, flat-bottomed, tissue culture flasks (Becton D i c k i n s o n Canada; M i s s i s s a u g a , O N ) , w h i c h were 25 c m , 75 c m , or 125 c m 2  size.  2  D u r i n g passage o f the cultured macrophages, the cells were scraped  2  in  w i t h 25 c m  disposable c e l l scrapers ( C o r n i n g - C o s t a r , F i s h e r Scientific; P i t t s b u r g h , P A ) . F o r the invasion and reporter assays,  cells were cultured overnight in 9 6 - w e l l plates,  completely clear ( F a l c o n M i c r o t e s t III  tissue  culture  plates;  which  were  Becton Dickinson  either  Canada;  Chapter 2 40 Mississauga, ON), or contained clear wells with white grids between each well (Immunoware 8-Well EIA strip plates; Pierce, Rockford, IL), or contained black wells with clear bottoms (Costar; Fisher Scientific). The EIA plates were sterilized with 70% ethanol prior to seeding with cells; all other tissue culture plates used were previously sterilized by gamma irradiation. For the B-galactosidase assays, samples containing chloroform were transferred to Falcon Microtest III Flexible Assay Plates (Becton Dickinson Canada) for incubation, since the polyvinylchloride (PVC) plastic was more tolerant to chloroform than the polystyrene plastic of the other plates.  Bacterial optical density  was  determined using an Ultraspec Plus  Spectrophotometer (Pharmacia Biotech; Uppsala, Sweden). Miscellaneous equipment such as Eppendorf tips, disposable pipettes, and sterilizing filters were purchased from VWR Scientific Canada, Ltd. (London, ON), Millipore Canada, Ltd. (Nepean, ON) and Amicon Division, W.R. Grace & Co.- Conn. (Beverly, MA). 2.3.  Strains and  2.3.1.  Cells  Plasmids  lines  Non-phagocytic cell lines used included Madin-Darby canine kidney cells M D C K (ATCC number: CCL-34) and the human epithelial cells HeLa  (ATCC number:  CCL-2).  Phagocytic cells used included the BALB/c mouse macrophage-like cells J774A.1 (ATCC number:  TIB-67) (255), and the mouse bone marrow-derived macrophage cell line  B A L B .BMI. For the initial studies, the M D C K and HeLa cells were grown in M E M at 37°C in an atmosphere of 95% air-5% CO2, however for later studies, all cells were grown in DMEM++. For most studies, the cell lines were used between passages 5 and 20, although for the results obtained in Chapter 3 the cells may have been passaged up to 30 times. 2.3.2.  Bacteria  The bacteria used in this study are listed in Table 2.  The bacterium S.  typhimurium  SL1344 (158) was used throughout for all manipulations. All Salmonella strains were tested by agglutination to antiserum specific for S. typhimurium 12 (Difco Labs).  group B, O-antigen factors 1, 4, 5,  Chapter 2 41  Table 2:  Bacterial strains  Strain  Bacterial Type  Antibiotic Resistance  Other Features  DH5a  Escherichia coli  N a l a d i x i c acid  r-m+, recA  S. dublin  C m (<6 p g / m l )  W i l d type; virulence p l a s m i d  S. dublin L a n e  C m (<6 p g / m l )  C u r e d o f virulence F a n g etal. 1991 plasmid  typhimurium WRAY from S2337/65 parent  Str  U s e d as w i l d type; H o i s e t h & S t o c k e r hisGA6; virulence 1981 plasmid  A1A1  SL1344  Tet & Str  sigD:duxAB  This work  D11H5  SL1344  Tet & Str  ssaR::luxAB  This work  E12A2  SL1344  Tet & Str  sigF::luxAB  This work  G7H1  SL1344  Tet & Str  UcAr.luxAB  This work  S.  dublin Lane  LD842  SL1344  2.3.3.  S.  References  S a m b r o o k et 1989  ai,  G u i n e y et al.., 1990  Bacteriophage T h e methods for i s o l a t i n g , storing, and u s i n g the bacteriophage P 2 2 are described in  (62, 6 3 , 226, 2 7 8 - 2 8 2 , 3 0 0 , 336). T h e phage P 2 2 H T m f was used as a v e h i c l e to transfer both plasmids and c h r o m o s o m a l insertions f r o m one Salmonella to the next, w h i l e P 2 2 H 3 was used for cross-streaking experiments i n order to determine whether bacteria were (true  transductants  transduction.  with  no  remaining  phage  lysogens)  or  resistant  phage-sensitive  (lysogens)  after  Chapter 2 42 a.  P r e p a r a t i o n o f p h a g e P22  stock  Bacteria were grown overnight in L B broth at 37°C shaking at 200 rpm. To 1 ml of this culture was added 4 ml of a P22 lysate containing 5 x l 0 plaque-forming units (pfu) per 6  ml. This mixture was further incubated with aeration overnight at 37°C. The next day, a few drops of chloroform were added to the culture and incubation continued for another 10 min. The cells and debris were removed with centrifugation (e.g. 10 min at 10000 rpm). The clarified supernatant was then placed in a sterile tube and stored at 4°C. A couple drops of chloroform were added to this phage lysate stock to prevent bacterial growth during long-term storage. Original phage P22 stocks of P22HTmr and P22H3 were made using the host Salmonella  strain SL 1344. The phage P22HT z'rcf stock containing the plasmid pFUSLUX  was then made in a similar way by making a lysate from the host bacteria SL1344 pFUSLUX, into which the pFUSLUX had originally been electroporated. The phage P22HTint  was used  because it cannot lysogenize within the host bacterium and also has the ability to mobilize plasmid D N A . The stocks typically contained 1 0 pfu/ml, which was diluted one-hundred 10  fold for use during transduction. b.  T r a n s d u c t i o n o f Salmonella  with phage  P22  Bacteria were grown overnight in L B broth at 37°C shaking at 200 rpm. From the overnight culture, a 1:100 dilution of the bacteria was made into fresh L B media and grown to approximately l - 5 x l 0 colony-forming units (cfu) per ml. One volume of bacteria was then 8  mixed with a one-tenth volume of the appropriate P22HTint phage (at 1x10 pfu/ml), and held s  at room temperature for 10 min. One hundred microliter aliquots were then plated onto L B plates, containing the appropriate antibiotics, and the plates incubated overnight at 37°C. The resulting colonies were then purified on "green plates" to ensure they were not undergoing active infection, nor contained lysogenized phage.  As stated earlier, on these  specialized plates the infected bacterial colonies turned dark green, while uninfected colonies  Chapter 2 43 remained a pale green c o l o r . T h e pale green colonies were further tested b y cross-streaking them against a line o f P 2 2 H 3 phage lysate, w h i c h resulted i n infected dark green colonies only i f the o r i g i n a l c o l o n y was lysogen-free. 2.3.4.  Plasmid  Preparation  Table 3: Bacterial plasmids Plasmid  Antibiotic Resistance  Other Features  References  pACYC184  C m (>12 u g / m l ) &Tet  L o w c o p y number p l a s m i d  C h a n g & C o h e n 1978  pFF14  C m (>12 u g / m l ) s/?vZ?.-:/acZtranslational fusion  F a n g etal. 1991  (spvRAB::lacZ) pSPLUX  C m (>12 u g / m l )  spvB::luxAB transcriptional fusion (spvRAB::luxAB::lacZ)  P f e i f e r & F i n l a y 1995  pTF421  Amp  R N A 1 overproduction  G u z z o & D u B o w 1991  pFUSLUX  Tet  Tn5::luxAB::tet gene cassette; G u z z o & D u B o w 1991 C o l E I o r i g i n o f replication  T h e p l a s m i d s used i n this study are listed i n T a b l e 3. T h e p l a s m i d p F F 1 4 containing an spvBr.lacZ translational fusion w a s p r e v i o u s l y described b y F a n g et al. ( 7 2 ) . T h e p l a s m i d p S P L U X w a s m a d e b y inserting the luxAB gene cassette (251) into the BamHI site o f p F F 1 4 , as s h o w n i n F i g u r e 11, to create a transcriptional fusion between spvB and luxA.  Both pFF14  and p S P L U X are l o w c o p y number plasmids derived from p A C Y C 1 8 4 ( 3 9 ) . T h e plasmids p T F 4 2 1 a n d p F U S L U X were used i n concert (as described b e l o w ) to r a n d o m l y insert into the Salmonella c h r o m o s o m e (138).  luxAB  Chapter 2 44 2.4.  Molecular 2.4.1. a.  Biology  D N A Isolation  Plasmid Preparation  Plasmid preparations were made using the alkaline lysis technique described in Sambrook et al. (275), with an extra phenolxhloroform step added. Bacterial plasmid DNA preparations were made from 2 ml overnight cultures. resuspended in either 5 m M TE or sterile water.  Isolated plasmid D N A was  Qiagen plasmid kits (Qiagen Inc.;  Mississauga, ON) were also used to isolate plasmids from bacterial preparations, according to manufacturer's directions. b.  Chromosome  Preparation  A cesium chloride preparation of chromosomal DNA from S. typhimurium  SL1344  was made previously by Dr. Murry Stein. Chromosomal DNA preparations from Salmonella mutants were made as described below (9). First, a stock solution of protease (Streptomyces greisus Type X X I ) was made at 10 mg/ml in distilled H2O and incubated at 37°C for 1.5 h.  Then 2 ml of overnight bacterial  culture was pelleted at full speed in an Eppendorf microfuge (Fisher Scientific) for 5 min. The pellet was resuspended in 300 pl 50 m M TE using the flat end of a sterile toothpick, and the bacteria were then mixed with 100 pl of 20 mg/ml lysozyme for 15 min at room temperature. After this incubation, 20 ul of 10% SDS was added and the tube was mixed by gently inverting it.  Then 100 pl of the predigested protease were added and the mixture was  incubated at 37°C for 1 h with occasional mixing. In the next step, 400 pl phenol was added and the tube was again mixed by inverting it. This was incubated at 37°C for 1 h with occasional mixing. After 1 h, 600 pl chloroform was added, gently mixed and the phases separated by centrifuging for 5 min at 8000 rpm. The upper aqueous phase containing the D N A was transferred into a new Eppendorf tube and respun. To transfer the upper phase, a modified 1 ml Eppendorf tip was used. This tip had about 1/4 cm of its end chopped off to enlarge the tip opening and thereby minimize shear  Chapter 2 45 forces on the DNA. After the second centrifugation, the DNA was again transferred to a new tube and a one tenth volume of 3 M sodium acetate was added to the sample. The tube was then filled with -20°C 95% ethanol (about 1 ml or 2.2 volume of ethanol). The sample was not mixed, but instead the D N A globs were fished out with a hooked glass rod (melted Pasteur pipette). The 'glob' was briefly transferred to a new tube containing 1 ml 95% ethanol and then moved to an empty tube. The sample was dried at 37°C for about 1 h.  The D N A was  resuspended in 150 pi 50 mM T E and left to stand at 4 ° C overnight to achieve complete rehydration of the DNA. The next day, the purity of the D N A was checked by absorbance measurement with OD260 OD280 ratio. ;  2.4.2.  Basic Method of D N A  Precipitation with  Ethanol  Ethanol-precipitation of D N A has been previously described (23).  Briefly, a one-tenth  volume of 3 M sodium acetate was added to the sample containing the D N A and then 2.5 volumes of 100% ethanol was added to the sample. This mixture was allowed to cool for a minimum of 15 min at -70°C, and then spun at 13000 rpm for 20 min. The pellet was washed once with 70% ethanol. The resulting pellet was then dried and the D N A was rehydrated in 5 mM T E .  2.4.3.  Isolation of D N A from  Agarose  Three separate methods were used to isolate bands of DNA from agarose gels. All three methods were equally effective and resulted in the recovery of more than 80% of the D N A in a band. a.  Sephaglas Bandprep K i t  The Sephaglas™ Bandprep Kit from Pharmacia Biotech was used to extract DNA from agarose gels, as directed by the manufacturer. b.  Freeze Squeeze M e t h o d for Isolating D N A Fragments  The D N A band of interest was cut out from the agarose gel, then wrapped in parafilm and set on dry ice or in -70°C to freeze (about 20 min). Once frozen, the liquid was squeezed  Chapter 2 4 6 out from parafilm as band melted leaving a residue of agarose in the parafilm pocket. The DNA was then recovered using ethanol precipitation and resuspended in 5 mM TE or sterile distilled H 0 . 2  c.  S p i n - C o l u m n M e t h o d for Isolating D N A Fragments  A hot straight wire was used to poke a tiny hole in the bottom of a 500 pl Eppendorf tube. A small plug of loosely wound glass wool (about 0.5 0.75 cm deep) was then inserted into the end of the small Eppendorf tube (i.e. the column). This small tube was then placed into a larger 1.5 ml Eppendorf tube (i.e. the collection unit). The agarose gel plug containing the DNA was then placed on top of the glass wool in the small tube and the tubes were centrifuged for 45 sec at 6000 rpm in microfuge. This allowed maximum DNA recovery with little agarose contamination. The DNA was then recovered using ethanol precipitation and resuspended in 5 mM TE or sterile distilled H 0 (144). 2  2.4.4. a.  Electroporation of bacteria  Preparation of electrocompetent bacteria  Bacteria were grown overnight in LB broth at 3 7 ° C shaking at 200 rpm. From the overnight culture, a 1:100 dilution of the bacteria was made into fresh LB media and grown to an OD600 of 0.3-0.5. Once bacteria reached proper O D , the culture flask was chilled on ice for 30 min and all subsequent steps were performed on ice or in refrigerated units. The bacteria were transferred to centrifuge bottles and spun at 10,000 rpm for 10 min in a Beckman Model J2-21 centrifuge (Beckman Instruments (Canada) Inc.; Mississauga, ON). The pellet was resuspended in 1 volume of chilled 10% glycerol for first wash. recentrifuged as before.  The sample was then  Subsequent washing of the bacteria was carried out in reduced  volumes of 1 0 % glycerol (1:2, 1:50, 1:100, and 1:500) in order to decrease the ionic strength of the sample and concentrate the bacteria to about 1 0 to 1 0 9  1 0  bacteria per 4 0 pl aliquot.  Aliquots were placed into Eppendorf tubes and flash frozen using dry ice and ethanol. They were stored at - 7 0 ° C for up to 6 months.  Chapter 2 47 b.  Electroporation Electrocompetent bacterial aliquots were thawed on ice o n l y immediately before u s i n g .  O n e to t w o m i c r o l i t e r s o f D N A preparation was added to the thawed bacteria and the m i x was incubated o n ice for about 1 m i n . T h e sample was then transferred to a 0.2 c m electroporation cuvette and u s i n g the G e n e P u l s e r ™ f r o m B i o R a d ( R i c h m o n d , C A ) , the sample was p u l s e d at 2.5 k V w i t h a 25 p F capacitance and 4 0 0 Q parallel resistance. T h e samples were immediately resuspended in 1 m l o f S O C broth containing no antibiotics and incubated for 1 h at 3 7 ° C , shaking 200 rpm.  After this recovery p e r i o d , the sample was diluted and plated out  onto  selective m e d i u m to quantitate the transformants per pg o f D N A . 2.4.5.  T w o plasmid competition  system  A m o d i f i e d v e r s i o n o f a t w o - p l a s m i d competition system (138) was used to obtain random  insertions  chromosome.  o f a promoterless  luciferase gene cassette throughout  Competent S. typhimurium  the  Salmonella  S L 1 3 4 4 bacteria were initially transformed using  electroporation w i t h either the p l a s m i d p T F 4 2 1 or p F U S L U X .  R e s u l t i n g transformants  selected o n L B plates containing either ampicillin or tetracycline, respectively. P 2 2 H T m ? lysate was then made o f S L 1 3 4 4 p F U S L U X ,  were  A phage  and this lysate used to  transfect  S L 1 3 4 4 p T F 4 2 1 . T h e p S P L U X p l a s m i d was transferred into S L 1 3 4 4 p T F 4 2 1 about 100 fold more efficiently by the phage P 2 2 c o m p a r e d w i t h electroporation.  S L 1 3 4 4 bacteria transfected  w i t h both p l a s m i d s were then g r o w n for an extended period o f time on L B plates containing both a m p i c i l l i n and tetracycline.  The plasmid p F U S L U X  has a C o l E l origin o f replication  (184) and contains a gene cassette e n c o d i n g a promoterless tetracycline resistance (Figure 6) (138, 166).  luxAB  gene operon  and  gene surrounded b y insertional sequences f r o m the transposon  a  Tn5  T h e p l a s m i d p T F 4 2 1 carries an ampicillin resistance gene and also  encodes for the production o f R N A 1 , w h i c h inhibits the replication o f plasmids w i t h C o l E l origins (131).  T h i s extended incubation a l l o w e d the bacteria to enter a hypermutability state  that a l l o w e d for  the  random  insertion  o f the  Z«xA5-containing  gene cassette into  the  Chapter 2 48 chromosome of the bacteria. Previous testing with E. coli showed that single chromosomal insertions resulted from this technique (138). 2.4.6.  Inverse  PCR  Inverse PCR was used for amplifying the regions either upstream or downstream from the site of reporter gene insertion (164, 237, 315).  Chromosomal D N A was first cut with  either the restriction enzyme Haelll or Rsal at 37°C for a minimum of 1 h.  After cutting, the  restriction enzymes were removed using phenol:chloroform extraction and ethanol precipitation as described previously, and the D N A resuspended in 20 pl distilled H2O. Five microliters of this cut D N A was placed in a 500 pl microfuge tube containing 1 pl ligase (5 U), 10 pl 5X ligase buffer and 34 pl distilled water, and incubated for 10 min in a sonicating waterbath set between 12°C and 16°C. Inverse PCR reaction tubes contained: 5 pl ligated D N A , 5 pl of each of two primers (10 pmol stock), 5 pl 25 m M magnesium sulfate, 5 pl 10X AmpliTaq buffer, 4 pl dNTPs (2.5 m M stock), 20 pl distilled sterile water, and 1 pl AmpliTaq or AmpliTaq Gold (5 U ) . The PCR program consisted of an initial cycle of 94°C for 5 min and then 30 cycles of 94°C for 45 sec, 60°C for 45 sec, and 72°C for 2 min 30 sec. The reaction was then subjected to electrophoresis on a 1% agarose gel with I X T A E buffer. 2.4.7.  Sequencing  For sequencing, 90 ng purified template D N A was added to 3.2 pmol of primer and mixed with 8 pl of the FS Taq terminator premix reagent for a final volume of 20 pl. Each sequencing reaction consisted of 25 cycles of 96°C for 30 sec, 50°C for 15 sec, and 60°C for 4 min. Unused terminators (fluorescently pre-dyed dNTPs) were removed using Centrisep columns, as directed by the manufacturer. (UBC, Vancouver, BC).  Sequencing gels were run by the NAPS Unit  Sequences were further analyzed using NCBI's B L A S T program  and the GenBank database .  Chapter 2 49 2.5.  Invasion and  Survival  Assays  F o r the studies i n v o l v i n g the development o f the intracellular reporter system, and for the initial screening o f bacteria w i t h cultured cells, the Salmonella were g r o w n overnight in L B broth at 3 7 ° C , w i t h no s h a k i n g (159). mutants, S. typhimurium  F o r later studies i n v o l v i n g the characterization o f the  S L 1 3 4 4 and mutants A 1 A 1 , D 1 1 H 5 , E 1 2 A 2 , and G 7 H 1 were first  g r o w n i n 1 m l L B broth i n culture tubes overnight, w i t h s h a k i n g at 2 0 0 r p m . T h e next day the bacteria were subcultured at a 1:100 dilution i n p r e w a r m e d , pre-equilibrated (CO2  buffered)  D M E M + + and g r o w n for another 3 h, w i t h s h a k i n g . I n v a s i o n assays were done u s i n g a m o d i f i e d version o f the gentamicin protection assay described by T a n g et al. (310).  Sterile 9 6 - w e l l plates were seeded w i t h cultured cells 18 h in  advance w i t h 100 pi o f 0 . 5 - 1 . 0 x l 0 c e l l s / m l (depending on cell type), in order to obtain 9 0 % 5  confluency b y the next day.  T h e cultured cells were then infected w i t h 2 p i  cultures (multiplicity o f infection ( M O I ) was, 5 0 - 1 0 0 bacteria per c e l l ) .  o f bacterial  Bacteria were a l l o w e d  to invade p h a g o c y t i c cells for 30 minutes and non-phagocytic cells for 1 h. (Note that bacterial i n v a s i o n o f host cells took place in the presence o f serum.)  F o l l o w i n g internalization o f the  bacteria, the cells were w a s h e d w i t h P B S + + and incubated w i t h 100 p i D M E M + + containing 100 p g / m l  gentamicin.  W h e r e bacterial g r o w t h was  studied for longer  than  4 h,  the  gentamicin concentration was reduced to 10 p g / m l at 4 h i n order to reduce any toxic effects of gentamicin on both the cells and intracellular bacteria. containing 1% T r i t o n X - 1 0 0 + 0 . 1 % S D S . then plated onto L B agar. dilutions and p l a t i n g .  C e l l s were then l y s e d i n 2 0 p i P B S  D i l u t i o n s o f bacteria were made in P B S — and  Bacterial c o l o n y - f o r m i n g units (cfu) were enumerated  A l l counts  by serial  were obtained f r o m duplicates w e l l s w i t h i n triplicate  experiments, and error bars represent standard error o f the means, P<(0.5) (228).  Chapter 2 50 2.6.  Reporter  gene  assays  T h e reporter assays were set up in the same manner as the i n v a s i o n assays.  Both  bacterial and cell cultures were set-up the day p r i o r to the assay and g r o w n as indicated. B a c t e r i a were a l l o w e d to invade the cells as described in section 2.5,  and intracellular bacteria  were differentiated from extracellular bacteria by their resistance to gentamicin w h i l e inside the host cells. E a c h experiment was performed i n triplicate or quadruplicate, and each mutant was tested i n duplicate in each experiment. P<(0.5)  E r r o r bars represent  standard error o f the  means,  (228).  2.6.1.  B-galactosidase  assays  F o r determination o f bacterial B-galactosidase activity, assays were done in 9 6 - w e l l microtiter plates.  Activity from  extracellular bacteria was  determined f r o m  the  bacteria  r e m a i n i n g i n the supernatant above the cells, prior to the addition o f gentamicin. A c t i v i t y from intracellular bacteria was determined after treatment o f the cells w i t h gentamicin. N o t e that the cells were w a s h e d once w i t h P B S + + to remove gentamicin p r i o r to assaying for enzyme activity. F o r B-galactosidase assays, separate w e l l s were used for the determination o f enzyme activity and for v i a b l e bacterial counts. O v e r the course o f this study, t w o different substrates were  used  to  determine  B-galactosidase  activity from  bacteria:  a  fluorescent  and  a  c h e m i l u m i n e s c e n t substrate. a.  fi-galactosidase assay  using fluorescent  substrate  T o each s a m p l e , 20 p l o f 0.1% S D S was added and incubated for 5 10 m i n at 3 7 ° C . T h e w e l l s were made up to approximately 100 p l w i t h P B S + + , m i x e d w e l l , and the contents transferred to a 9 6 - w e l l P V C ( p o l y v i n y l c h l o r i d e ) plate.  One drop (-10 pl) c h l o r o f o r m was  m i x e d w i t h the 100 p l sample in flexible P V C plate and the plate was put on ice for at least 5 m i n . ( T r i t o n X-100 was not used for lysis since the t w o detergents ( T r i t o n X-100 and S D S ) f o r m e d a precipitate f o r m e d w h e n were c o m b i n e d w i t h the substrate.)  T w o microliters of a  50 m M stock o f F D G were then added to each w e l l and the plate was incubated at 3 7 ° C for 1 h . T o stop the reaction, the plate was again put on ice and 2 pl o f a 50 m M stock o f P E T G  Chapter 2 51 were added to each w e l l . T h e reagents were light sensitive, therefore all sample manipulations were done w i t h m i n i m a l exposure to light. T o determine the e n z y m e activity, the samples were transferred to a F l u o r i c o n assay plate and the fluorescence at 5 3 5 n m excitation) was  measured  in a P A N D E X  emission (485 n m  Fluorescence Concentration A n a l y z e r  (JDEXX;  P o r t l a n d , O R ) . Duplicate w e l l s for bacterial counts were run under the same conditions as w e l l s for the 6-galactosidase assay, and v i a b l e bacteria were enumerated b y serial dilutions and plating.  Fluorescence was then correlated w i t h viable counts to calculate  6-galactosidase  activity as fluorescent units/cfu. b.  fi-galactosidase assay  using chemiluminescent  substrate  F o r the chemiluminescent assay, the bacteria and cultured cells were treated w i t h S D S and c h l o r o f o r m as above, h o w e v e r the G a l a c t o S t o r ™ K i t ( T r o p i x P e r k i n E l m e r ) was used to detect B-galactosidase activity. One hundred microliters o f detection reagent (diluted 1:50 o f stock solution as per instructions) was added to the 100 p l o f sample, and then incubated at r o o m temperature for 3 0 m i n .  T h e chemiluminescence (or light output) o f the sample was  determined u s i n g the L u m i n o g r a p h L B 9 8 0 photon imager.  Duplicate w e l l s were used for  viable counts, and B-galactosidase activity was correlated as photons/cfu. 2.6.2.  Luciferase  assay  F o r the luciferase assay, the aldehyde substrate was added directly to the sample o f either intact cells or bacteria alone, and then viable bacterial counts were obtained f r o m the sample w e l l . A c t i v i t y from extracellular bacteria was determined from the bacteria remaining in the supernatant above the cells, p r i o r to the addition o f gentamicin. A c t i v i t y f r o m intracellular bacteria was determined after treatment o f the cells w i t h gentamicin. In order to m i n i m i z e any toxic effects d u r i n g the assay, the media containing gentamicin was replaced w i t h fresh media before adding aldehyde solution. T o determine light production (i.e.  luciferase activity) f r o m bacteria, 10 p l  o f the  aldehyde s o l u t i o n was added to the 100 p l sample. Immediately the light p r o d u c t i o n was read over the course o f 1 m i n at m a x i m u m sensitivity on the L u m i n o g r a p h L B 9 8 0 . L i g h t emissions  Chapter 2 52 were obtained as p h o t o n s / w e l l . L i g h t production was also assessed in microtiter plates w i t h a 1 min  exposure  densitometer.  to  X-ray  film,  and  the  results  were  quantitated  using  a  computing  After the light production was determined, the host cells were l y s e d w i t h  1% T r i t o n X - 1 0 0 and 0 . 1 % S D S , and the bacteria plated out onto L B plates as indicated for the i n v a s i o n assays. L u c i f e r a s e activity was defined as photons/cfu (when u s i n g the L B 9 8 0 ) . 2.7.  Screen Outside  2.7.1.  for T r a n s f o r m e d Host  Salmonella  Exhibiting L o w Luciferase  Activity  Cells  Extracellular Bacterial  Screen  T h e Salmonella colonies obtained from the t w o - p l a s m i d competition system (described above) were screened for l o w light production on L B plates.  Approximately 1 . 5 x l 0  bacterial  5  colonies, resistant to both a m p i c i l l i n and tetracycline, resulted from the transformations. colonies were e x p o s e d to vapors o f n-decanal (e.g.  These  substrate was streaked onto the l i d o f the  petri dish) and the resulting light output measured w i t h a L u m i n o g r a p h L B 9 8 0 l o w light video i m a g i n g system (Siemens) (251). C o l o n i e s s h o w i n g little to no light production were then restreaked onto fresh L B plates containing both antibiotics, and retested for both g r o w t h and l o w light p r o d u c t i o n . passing through the second stage o f the screen  were then  transferred  Colonies  to 9 6 - w e l l plates  containing L B broth w i t h both antibiotics; each w e l l contained a separate c o l o n y .  The bacterial  mutants were also streaked onto green plates to ensure that the bacteria were not chronically infected w i t h P 2 2 phage (300). 2.7.2.  Intracellular  versus E x t r a c e l l u l a r B a c t e r i a l  Screen  T h e selected bacterial mutants were further tested for l o w light production outside cells in broth, and tested for an induction o f light production from w i t h i n c e l l s .  Bacteria were  initially g r o w n i n 100 p i o f L B broth in 9 6 - w e l l plates sealed w i t h p a r a f i l m , at 3 7 ° C s h a k i n g at 150 r p m overnight. (Costar)  were  used  to g r o w  and  Sterile 9 6 - w e l l plates w i t h black w a l l s and clear bottoms tissue culture c e l l s .  W e l l s were  seeded  with  100 p i  of  Chapter 2 53 lxlO  5  c e l l s / m l i n order to obtain 9 0 % c o n f l u e n c y by the next day. B o t h macrophage cell lines  ( J 7 7 4 A . 1 and B A L B . B M 1 ) were used for the initial screen.  T h e overnight bacterial plates  were used to inoculate a l l other plates, and served as the extracellular stationary phase bacterial controls.  F o r the intracellular test plates, 2 p l o f each bacterial mutant f r o m the stationary  phase plates were used to inoculate the plates containing the cell lines. Bacteria were a l l o w e d to invade the cells for 1 h and then were w a s h e d o f f w i t h P B S and the media replaced with D M E M + + containing l O O p g / m l  gentamicin.  L i g h t from the intracellular bacteria was first  determined at 2 h p o s t i n o c u l a t i o n and again at 4 h. T h e 2 h time point a l l o w e d the gentamicin to act on extracellular bacteria for at least 1 h, w h i c h has been f o u n d to be sufficient time to k i l l the bacteria and eliminate light p r o d u c t i o n . Once the activity at the 2 h time point had been determined, the aldehyde-containing media was r e m o v e d and replaced w i t h fresh D M E M + + w i t h gentamicin for another determined again.  2 h.  A t the 4 h  time point, the intracellular activity was  A t 4 h , the viable bacteria were plated out on L B plates to determine  bacterial n u m b e r s / w e l l .  D u r i n g the screen for bacterial genes  intracellularly, the c f u ' s were not determined for each sample.  which  were  upregulated  Instead, t w o to three samples  per plate were used as an estimate o f bacterial numbers and applied to the rest o f the plate for convenience. F o r characterization o f the mutants in later studies, actual c f u ' s were determined for each sample F o r the extracellular l o g a r i t h m i c phase control plates, 2 pl from each o f the w e l l s i n the stationary phase plates was first diluted i n 2 0 0 p l P B S (1:100 dilution) and then 2 p l o f this was added to 100 p l D M E M + + i n a new plate.  T h e plates were then incubated under similar  conditions to the tissue culture cells, i.e. not s h a k i n g at 3 7 ° C in 5 % CO2. extracellular  bacteria  (both  stationary  postinoculation o f the l o g phase plate. luciferase activity was determined.  and  l o g phage  bacteria)  was  T h e light from the detected  at  4 h  V i a b l e bacteria were plated out f r o m the w e l l s after  Chapter 2 54 2.11.  Mouse  2.11.1.  Studies  Typhoid  Mouse  Model  Salmonella suspensions were g r o w n at 3 7 ° C i n L B broth overnight, w i t h s h a k i n g at 200 r p m . T h e next day, bacteria were diluted 1:100 into fresh L B broth and incubated w i t h shaking. A f t e r 4 h , bacteria were w a s h e d once w i t h P B S and resuspended i n P B S c o n t a i n i n g 2 % glucose. C o n t r o l m i c e were g i v e n P B S w i t h glucose o n l y . B A L B / c female m i c e , aged 6 to 10 w e e k s , were i n o c u l a t e d o r a l l y w i t h bacterial suspensions (195, 299), after b e i n g deprived of water for 4 h . In the first experiment, the i n o c u l a t i o n size was 25 p i . T h e dose o f w i l d - t y p e S L 1 3 4 4 was 2 . 5 x 1 0 cfu/mouse, (approximately t w i c e the reported LD50 (195, 299)). T h e 6  Salmonella mutants were g i v e n at 200 times this dose ( 5 x l 0 cfu/mouse). A c t u a l counts per 8  mouse (per 25 pi) were as f o l l o w s ( ± 1 0 % error): for D I 1 H 5 , 4 . 2 x l 0 c f u ; for A l A l , 8  4.1xl0  8  c f u ; for E 1 2 A 2 , 3 . 3 x l 0 c f u ; and for G 7 H 1 , 5 . 0 x l 0 cfu. F o u r m i c e were used per 8  8  group. In the s e c o n d experiment, three doses were used o f a p p r o x i m a t e l y l x l O , l x l O , and 8  lxlO  6  7  cfu/mouse, and the i n o c u l a t i o n size was 10 p i . . A c t u a l counts per m o u s e (per 10 pi)  were 10-fold d i l u t i o n s o f the f o l l o w i n g ( ± 10% error): 7 . 6 x l 0 cfu o f S L 1 3 4 4 ; 9 . 6 x l 0 cfu o f 7  A 1 A 1 ; 1 . 3 x l 0 o f E 1 2 A 2 ; and l . O x l O cfu o f G 7 H 1 . 8  8  7  F i v e m i c e were u s e d per group. M i c e  s u r v i v i n g after 28 days were sacrificed, and their livers and spleens were harvested, the organs h o m o g e n i z e d and the resulting slurry spread onto M a c C o n k e y plates to obtain bacterial c o l o n y counts. T h e day at w h i c h 5 0 % o f the m i c e i n each group had d i e d was determined u s i n g the statistical calculations o f R e e d and M e u n c h (259,260) and the m e d i a n s u r v i v a l time (228).  Chapter 3 55 C h a p t e r 3:  Development of an Intracellular Reporter  System  Chapter 3 describes the development o f an assay system for gene expression f r o m intracellular bacteria.  S p e c i f i c a l l y , the benefits and challenges o f w o r k i n g w i t h t w o different  reporters, B-galactosidase and luciferase, are discussed. T h e ability o f Salmonella  to s u r v i v e and g r o w w i t h i n cells is crucial to its ability to  cause disease w i t h i n a host (27, 199).  M a n y mutants w h i c h are defective i n their ability to  invade or replicate w i t h i n cells are also avirulent in the host (4, 4 9 , 7 6 , 85). o f cell-mediated i m m u n i t y in addition to a humoral i m m u n e response  T h e development against Salmonella  infection, is also indicative o f a host response to intracellular pathogens ( 2 7 1 , 326). Salmonella to propagate w i t h i n the intracellular environment, the bacteria must  For  adapt by  regulating the expression o f proteins necessary for g r o w t h w i t h i n that environment.  This  global regulation has been observed by u s i n g two-dimensional protein gels, where bacteria have been s h o w n to regulate the expression o f proteins upon infection o f cultured macrophages (1, 30, 3 3 , 34). H o w e v e r , to study the expression o f individual genes, fusion o f the gene o f interest to a reporter gene provides a s i m p l e r method to m o n i t o r expression. T h e detection o f reporter gene expression often uses bacteria g r o w n as colonies on agar plates or g r o w n to h i g h density in broth cultures.  T h e number o f Salmonella w i t h i n host cells  after i n v a s i o n w i l l be 10-100 f o l d l o w e r , therefore the suitability o f u s i n g specific reporter genes to determine gene expression from bacteria w i t h i n host cells was e x a m i n e d . 3.1.  Results  3.1.1.  I n v a s i o n assay  to d e t e r m i n e  intracellular bacterial  numbers  A gentamicin-protection assay (310) was used to define the bacteria w h i c h had invaded a host cell and thus become intracellular.  Salmonella were added to m e d i u m containing  cultured cells and a l l o w e d to invade the cells for a set p e r i o d o f time. T h e extracellular bacteria were then r e m o v e d , the cells washed, and the new m e d i u m containing the antibiotic gentamicin was added.  B a c t e r i a residing inside the host cells were protected f r o m the k i l l i n g effects o f  gentamicin, w h i l e extracellular bacteria were k i l l e d (310).  T o enumerate the intracellular  Chapter 3 56 bacteria, the infected host cells were then l y s e d w i t h 1% T r i t o n X - 1 0 0 , w h i c h destabilized the membrane o f the host cell without i m p a i r i n g the bacterial membrane, a l l o w i n g the bacteria to be released f r o m the c e l l .  T h e bacteria were then diluted and c o l o n y - f o r m i n g units (cfu) were  determined f o l l o w i n g g r o w t h on agar plates.  C e l l u l a r 'ghosts'  resulting f r o m host cell lysis  were observed under the m i c r o s c o p e , and bacteria were seen s w i m m i n g through the lysis buffer. A p o s s i b l e concern w i t h the method was that some o f the bacteria might c l u m p or associate w i t h cellular debris, or that the intracellular vacuoles might not lyse w i t h the same efficiency as the outer cell membrane. of intracellular bacteria.  T h e result w o u l d be an underestimation o f the number  Therefore, the addition o f the ionic detergent S D S was added to the  lysis buffer i n an effort to disrupt any r e m a i n i n g cell/bacterial interactions. Further experiments s h o w e d that 0 . 1 % S D S was not d a m a g i n g to Salmonella g r o w n i n m e d i a alone, i.e. cfu's were not reduced significantly by the presence o f 0.1 % S D S . A combination o f 1 % T r i t o n X - 1 0 0 and 0 . 1 % S D S was tested and resulted i n the recovery o f 10-fold more bacteria f r o m infected cells than w i t h T r i t o n X - 1 0 0 alone (Figure 4).  A s the estimation o f intracellular bacterial  numbers is v e r y important for the determination o f specific activity o f a reporter e n z y m e , the combination o f 1% T r i t o n X - 1 0 0 and 0 . 1 % S D S  in the lysis buffer  enumeration o f intracellular bacteria in all further experiments.  was  used for  the  Chapter 3 57  F i g u r e 4: T h e effect o f the detergents T r i t o n X - 1 0 0 (1.0%) and S D S (0.1%) o n the recovery of intracellular S. typhimurium f r o m H e L a cells.  T h e experiments were done i n triplicate and  the error bars represent the standard deviation o f the means.  Chapter 3 58  3.1.2.  B - g a l a c t o s i d a s e as a r e p o r t e r  of intracellular bacterial  expression  T h e p l a s m i d p F F 14 (11.2 k b p ) is a p A C Y C 1 8 4 - b a s e d (39) l o w - c o p y number p l a s m i d containing a translational fusion between the S. dublin spvB gene and the lacZ reporter gene (72, 77) (Table 2; F i g u r e 5).  It w a s p r e v i o u s l y demonstrated that the c o p y number o f the  p l a s m i d p F F 1 4 remained constant throughout the g r o w t h cycle o f S. dublin ( 7 2 ) , indicating that an increase i n the expression o f genes encoded on this p l a s m i d w a s not due to g r o w t h c y c l e but to a change i n regulation. T h e spv operon has p r e v i o u s l y been s h o w n to be regulated by S p v R (72, 7 7 , 129), a n d spvB::lacZ expression increased m o r e than ten-fold u p o n bacterial entry into stationary phase o r under conditions o f carbon o r nitrogen starvation (72, 1 2 5 ) . N o t e that the regulation o f the spvB gene remained under the control o f S p v R w i t h both the p F F 1 4 a n d p S P L U X constructs. T h e p l a s m i d p F F 1 4 w a s used to examine the production o f 6-galactosidase resulting from expression o f the spvBr.lacZ  f u s i o n , f r o m Salmonella w i t h i n epithelial cells ( M D C K and  H e L a cells). A f t e r incubation o f bacteria and host cells together as described above, the sample was treated w i t h 0 . 1 % S D S and c h l o r o f o r m to fully release the reporter e n z y m e .  Activity of  B-galactosidase w a s assayed u s i n g a fluorescence assay based o n the substrate F D G , w h i c h had p r e v i o u s l y been s h o w n to be more sensitive than the colorimetric assay based on the substrate O M P G (28, 108). After a set p e r i o d o f incubation, the reaction w a s stopped and the fluorescence o f the sample w a s measured u s i n g a Pandex fluorimeter.  T h i s reading w a s  converted into specific activity (fluorescence/cfu) i n combination w i t h the number o f c o l o n y f o r m i n g units (cfu) o r viable bacteria recovered f r o m a parallel sample.  N o t e that c h l o r o f o r m  was not added to samples u s e d for d e t e r m i n i n g cfu's. T h e data s h o w n i n F i g u r e 6 A represents the expression f r o m the spvBr.lacZ gene fusion f r o m S. dublin  w h i c h were either extracellular  (i.e. w i t h i n the culture supernatant taken from above the cultured cells) or intracellular (i.e. inside cells and thus protected from gentamicin).  T h e amount o f 6-galactosidase activity  increased per bacterium w h e n the bacteria were intracellular as compared to those w h i c h were  Chapter 3 59  F i g u r e 5: P l a s m i d maps o f p F F 1 4 a n d p S P L U X . T h e 11.3 k b p p l a s m i d p F F 1 4 (circular map) w h i c h contains  spvR,  B-galactosidase studies.  spv A,  and a translational  spvB::lacZ  fusion,  w a s used  for the  T h e 14.55 k b p p l a s m i d p S P L U X w a s made b y inserting a 3.25 k b p  promoterless luxAB gene cassette into the BamHI site between spvB and lacZ, thus placing luxAB under the transcriptional control o f the spvB gene.  T h e p l a s m i d p F F 1 4 w a s used for  the B-galactosidase studies, w h i l e p S P L U X was used for the luciferase studies.  Chapter 3 60  1E+0  1E-1  c/j  u  < ••!=: c cn ro  O  CD  1E-2  CD  cn CD  1E-3  Extracellular  Intracellular  HeLa  MDCK  B 10  £> 23 6 w  CO  .2 € =3 CO  CC  CD  o  4  CO  -  2  F i g u r e 6: E x p r e s s i o n o f spvBr.lacZ by bacteria inside non-phagocytic cells. A ) 6-galactosidase activity f r o m bacteria w h i c h are either intracellular (inside H e L a or M D C K cells) or extracellular (remaining i n the supernatant above the cells).  B ) R a t i o o f induction  s h o w n i n ( A ) where intracellular activity is divided by extracellular activity.  Chapter 3 61 extracellular.  A s s h o w n in F i g u r e 6 B , this induction i n activity was greater than  five-fold  w i t h i n both epithelial cell types ( M D C K and H e L a ) tested. A l t h o u g h B-galactosidase was deemed to be a sensitive reporter o f intracellular bacterial gene e x p r e s s i o n , there were p r o b l e m s associated w i t h its use. First, the host cells had v a r y i n g b a c k g r o u n d levels o f activity. T h i s reduced the sensitivity o f the assay w h e n u s i n g these cells and made comparisons between cell types more difficult.  S e c o n d , both the host cells and the  bacteria needed to be completely l y s e d to accurately assay the enzymatic activity.  Since  c h l o r o f o r m was u s e d i n combination w i t h l o w levels o f detergent, the samples had to be transferred to special P V C plates as the c h l o r o f o r m reacted w i t h the polystyrene components o f regular m u l t i w e l l plates. A s w e l l , higher amounts o f detergent c o u l d not be used for lysis as it formed a precipitate i n the substrate buffer, fluourescence i n the sample.  which  interfered w i t h the determination o f  T h i r d , the F D G substrate was sensitive to light and  therefore  samples had to be kept i n the dark as m u c h as p o s s i b l e . F o u r t h , it was i m p o s s i b l e to separate l i v e bacteria (i.e. metabolically active bacteria that were able to be cultured) f r o m those that had been k i l l e d by the host c e l l , therefore B-galactosidase activity resulted f r o m both live and dead bacteria w i t h i n a sample.  T h i s was especially important for the determination o f specific  e n z y m e activity (defined as the number o f fluorescent units per viable bacterium).  T h e state o f  the Salmonella w i t h i n cells has been reported to be in a constant flux w h i c h can be d i v i d e d into t w o p o p u l a t i o n s , one w h i c h is d y i n g and the other w h i c h is g r o w i n g (2, 32). A n o t h e r reporter, bacterial luciferase, was k n o w n to be detectable  f r o m o n l y live  bacteria due to its requirement for the bacterial energy F M N H 2 for activity. It was  therefore  tested to determine whether it c o u l d be used as a sensitive reporter o f intracellular gene expression.  A promoterless 3.25 k b p luxAB  gene cassette f r o m Vibrio harveyi (138) was  ligated into the BamHI o f the p l a s m i d p F F 1 4 creating an spvB::luxAB resulting i n the i s o g e n i c p l a s m i d p S P L U X (14.5 kbp) (Figure 5) (251).  transcriptional f u s i o n ,  Chapter 3 62  3.1.3. a.  L u c i f e r a s e as a r e p o r t e r  Measurement  o f i n t r a c e l l u l a r b a c t e r i a l gene expression  of light production.  T h e product o f luciferase is light, w h i c h is produced specifically at a wavelength o f 4 9 0 n m f r o m V.  h a r v e y i  luciferase (210). N o t e that the bacterial substrate, n-decanal, was able to  diffuse across both bacterial and host c e l l membranes, therefore lysis o f bacterial and host cells was unnecessary  for the determination o f enzyme activity.  Furthermore,  the number o f  bacteria c o u l d be determined on the same sample f r o m w h i c h the enzyme activity was detected (180). These details are discussed more fully i n the f o l l o w i n g sections. L i g h t production from bacteria was detected and quantitated w i t h the use o f different systems,  i n c l u d i n g a tube  luminometer, X - r a y f i l m c o m b i n e d w i t h a densitometer, or a p h o t o n - i m a g i n g camera c o m b i n e d w i t h a computer processor.  T h e sensitivity and linearity o f the different  light-detection  methods was a n a l y z e d . A single tube luminometer was initially used for determining light p r o d u c t i o n f r o m bacteria free in culture, h o w e v e r , difficulties arose in determining light production from intracellular bacteria. First, the host cells c o u l d not be g r o w n in the luminometer tubes, so the invasion portion o f the assay had to be performed i n a separate d i s h and then the host cells l y s e d in order to free the intracellular bacteria for transfer to the light assay tube.  This  introduced another variable into the system and raised the concern that detergents and other buffer components may alter the activity o f the e n z y m e . It also resulted in the assaying o f live bacteria w h i c h were no longer intracellular, and w h i c h c o u l d p o s s i b l y adapt their gene expression.  O n l y the host cells were completely l y s e d and the bacteria were exposed to not  o n l y l o w levels o f detergents but to host cell contents proteins, etc.  containing l y s o s o m a l  degradative  S i n c e there was often a delay o f 5-10 m i n during cell l y s i s , there was enough  time for the bacteria to react to the 'new extracellular' environment, and perhaps exhibit an altered f o r m o f gene expression f r o m that seen f r o m truly intracellular bacteria.  T h i s differed  f r o m the assay for 6-galactosidase activity where both bacterial and host cells were completely  Chapter 3 63 l y s e d at the same time. F i n a l l y , the luminometer c o u l d o n l y measure one sample at a time and was not c o n d u c i v e to screening m a n y samples. X - r a y f i l m was tested and found to be capable o f determining bacterial light production from both extracellular and intracellular bacteria.  B o t h bacteria and host cells c o u l d be g r o w n  i n 9 6 - w e l l microtiter plates, w h i c h were then exposed directly to the X - r a y f i l m for a set period o f time, and the spots appearing on the X - r a y f i l m quantitated u s i n g a densitometer. a l l o w e d for the testing o f many samples at one time. encountered.  This  H o w e v e r , a new set o f problems was  First, regular 9 6 - w e l l microtiter plates were made o f clear plastic w h i c h a l l o w e d  the light to s p i l l over into n e i g h b o u r i n g w e l l s , altering the true reading o f those w e l l s .  The  cross-contamination o f light between w e l l s was eliminated w i t h the use o f 9 6 - w e l l white plastic g r i d plate w i t h 8 - w e l l strips w h i c h c o u l d be snapped I m m u n o w a r e 8 - W e l l E I A Strip Plates).  into place w i t h i n the grid  (Pierce  T h e second p r o b l e m was that w h i l e light c o u l d be  detected from over a 1000-fold range, it was o n l y linear over a 10-fold range as determined by densitometry scans o f the X - r a y f i l m . T h i r d , due to the small numbers o f bacteria inside cells, the light produced f r o m intracellular bacteria was often at the l o w e r limit o f detectable activity. . W i t h the L u m i n o g r a p h L B 9 8 0 photon-imaging camera/computer system it was possible to detect and quantitate light production over a 10000-fold range, w h i c h was linear w i t h i n a 100-fold range (about 1 . 0 x l 0 - 1 . 0 x l 0 3  5  photons/well).  screening i n a number o f different formats, e.g. plates.  T h i s system was also amenable  to  9 6 - w e l l microtiter plates or colonies on agar  It was f o u n d to be more sensitive than X - r a y f i l m by at least f i v e - f o l d (Figure 7 ) .  H o w e v e r , as s h o w n i n F i g u r e 7, the white plastic from the microtiter plates produced a l o w level phosphorescence and was h i g h l y reflective. T h i s p r o b l e m was later reduced w i t h the use of  black microtiter plates w i t h  clear  bottoms  (Costar).  The  black  plastic  was  not  phosphorescent and less reflective, w h i l e the clear bottoms o f the w e l l a l l o w e d for the analysis o f the host cell m o n o l a y e r . polystyrene  was  about  T h e b a c k g r o u n d level from the g r i d plates made o f white  1.0-4.0xl0  3  p h o t o n s / w e l l , w h i l e f r o m the plates made o f black  p o l y s t y r e n e , it was reduced to about 2 . 0 - 5 . 0 x 1 0 p h o t o n s / w e l l . T h i s l o w b a c k g r o u n d level o f 2  Chapter 3 64  Bacteria Dilutions  Method of Light Detection  1/5  |  w  ii  1/125  1/25 m  1/625  w  LB980 Luminograph  X-ray Film  F i g u r e 7:  C o m p a r i s o n o f two  1 different  L u m i n o g r a p h L B 9 8 0 versus X - r a y f i l m . to induce spvB::luxAB  methods  to detect bacterial light production:  S. dublin p S P L U X were g r o w n to stationary phase  expression and then diluted i n P B S w i t h i n 9 6 - w e l l gridded microtiter  plates. T h e substrate rc-decanal was added to a final concentration o f 0 . 0 0 2 2 % .  (Note that in  order to directly compare the two methods, the picture obtained from the l u m i n o g r a p h is s h o w n as a negative image w i t h the light output appearing as a dark image o n a light background.)  Chapter 3 65 light (i.e.  from  static electricity, dark noise f r o m  the camera)  was  subtracted f r o m  the  calculations. b.  Effects of aldehyde concentration on bacteria a n d luciferase activity. A s mentioned p r e v i o u s l y , to produce a blue-green light (490 n m ) , bacterial luciferase  required an energy source (reduced flavin mononucleotide - FMNH2), o x y g e n , and a l o n g chain aldehyde (210).  Therefore, w h e n live bacteria were used, o n l y the aldehyde substrate  had to be added e x o g e n o u s l y .  P r e v i o u s studies indicated that h i g h aldehyde concentrations  may be i n h i b i t o r y to light production (194), therefore a range o f concentrations was prepared to determine the optimal aldehyde concentration needed to measure light output f r o m intracellular and extracellular Salmonella.  both  T h e final concentration o f rc-decanal i n the assay  w e l l s ranged f r o m 0 . 0 0 0 2 2 % to 0 . 0 0 9 9 % . The effects o f different aldehyde concentrations o n both extracellular and intracellular S. dublin L D 8 4 2 p S P L U X are s h o w n i n F i g u r e 8. T h e results u s i n g 5\ typhimurium  SL1344  p S P L U X were s i m i l a r . In F i g u r e 8 A , a decrease i n total light output f r o m the w e l l s was seen for  the  two  highest  concentrations  o f n-decanal  (0.00495%  and 0 . 0 0 9 9 % ) .  Further  investigation revealed that these concentrations o f aldehyde were actually toxic to bacteria ( F i g u r e 8 B ) . V i a b l e counts (cfu's) were reduced b y 5 to 10 f o l d i n the presence o f the t w o highest concentrations o f aldehyde, whereas cfu's were unaffected for aldehyde concentrations o f 0 . 0 0 2 2 % or less. T h e aldehyde concentrations w h i c h caused toxicity were similar for both intracellular and extracellular bacteria.  Interestingly, the decrease i n the number o f viable  bacteria was greater than the decline i n light production per w e l l .  Therefore, the light output  per v i a b l e b a c t e r i u m appeared to rise as the aldehyde concentration increased (Figure 8 C ) .  Chapter 3 66  F i g u r e 8: Effect o f aldehyde concentration o n bacterial viability a n d light p r o d u c t i o n . cells were g r o w n to confluency i n 9 6 - w e l l microtiter plates and infected w i t h S. S L 1 3 4 4 p S P L U X as stated i n the methods.  MDCK  typhimurium  Bacteria remaining w i t h the cell m o n o l a y e r after  washing and gentamicin treatment were termed intracellular, whereas bacteria r e m o v e d w i t h the supernatant before w a s h i n g were termed extracellular.  T h i s figure depicts the i n d i v i d u a l data  points f r o m one experiment, a n d the lines represent the means o f the data points. production as photons per 100 p i w e l l ; L i g h t production  as photons/cfu:  extracellular bacteria.  (A) Light  ( B ) V i a b l e bacterial counts per 100 p i w e l l ;  (closed s y m b o l s )  intracellular bacteria;  (open  (C)  symbols)  Chapter 3 68 Since the purpose o f u s i n g the luciferase reporter was to correlate the expression o f a gene to e n z y m e activity w i t h i n a single live bacterium, it was crucial that the enzyme substrate not be toxic to that bacterium.  Therefore,  i n subsequent experiments  a final  aldehyde  concentration o f 0.0022% was used, as this concentration produced the highest amount o f light without a reduction i n bacterial numbers. T r y p a n blue e x c l u s i o n studies were used to determine whether tissue culture cell death occurred (9).  N o n e o f the aldehyde concentrations were toxic to the m a m m a l i a n cells, even  over extended periods o f co-incubation o f up to one hour (data not s h o w n ) .  It was  further  determined that gentamicin-killed bacteria d i d not produce light ( F i g u r e 9) and therefore, light produced from intracellular bacteria was not contaminated by k i l l e d bacteria remaining outside the cells. T h i s p r o v i d e d further support that bacteria had to remain alive in order for luciferase activity to be detected; i f they were k i l l e d b y the c e l l , they w o u l d most likely cease to produce light. T h e i n d u c t i o n o f the spvB::luxAB transcriptional fusions is s h o w n in F i g u r e 10, equal numbers o f bacteria were present i n the extracellular and intracellular w e l l s . lacking the luxAB  fusions (S. dublin  or S. typhimurium)  d i d not produce light.  where  Bacteria Wells  containing extracellular Salmonella p S P L U X also d i d not produce m u c h light since the bacteria were in logarithmic phase g r o w t h ,  and not entering into stationary  phase.  Figure  10  furthermore demonstrates that there is no light-producing activity detectable f r o m the host cells. H o w e v e r , intracellular Salmonella p S P L U X expressing the spvB::luxAB s h o w e d increased light p r o d u c t i o n .  transcriptional fusion  Chapter 3 69  Gentamicin Concentration (Mg/ml)  S. dublin S. typhimurium S. dublin S. typhimurium pSPLUX pSPLUX  1000  100  10  0  m •  A •  *  Figure 9: The action of different concentrations of gentamicin on the luciferase activity within bacteria. Salmonella were incubated for 45 min in the presence of DMEM++ plus gentamicin, at 37°C without shaking, prior to determining luciferase activity. Concentrations of gentamicin which killed more than 90% of the bacteria within the sample (>100 pg/ml) reduced the light output of the samples to near background levels. X-ray film was used to determine light output.  Chapter 3 7 0  SL1344 Extracellular  SL1344 pSPLUX  LD842 pSPLUX  Hi HH  Intracellular  DL  F i g u r e 10: Detection o f light production in Salmonella p S P L U X i n an intracellular versus an extracellular environment.  M D C K cells were seeded into 9 6 w e l l microtiter plates w i t h grids  and infected w i t h Salmonella, as described in the methods.  After 2 h , extracellular bacteria  were r e m o v e d and placed into w e l l s containing no M D C K c e l l s . T h e samples were such that all w e l l s contained equal numbers o f bacteria (approximately  lxlO  5  adjusted  cfu/well).  No  light w a s p r o d u c e d i n the absence o f the luxAB genes. A l o w level o f light w a s detected from the extracellular bacteria containing the p l a s m i d p S P L U X conditions).  {e.g.  under  spvB  repressing  Increased light production was detected i n w e l l s containing intracellular bacteria  w i t h p S P L U X , indicating an increased expression o f the spvB::luxAB T h i s figure w a s obtained u s i n g the L u m i n o g r a p h L B 9 8 0 .  transcriptional f u s i o n .  Chapter 3 71 3.1.4. Comparison  of  B-galactosidase  and  luciferase  as  reporters  of  intracellular bacterial gene expression.  Table 4:  Induction of expression of reporter enzyme fusions by  intracellular S. dublin.  Reporter Enzyme  Bacterial Location  B-Galactosidase  Extracellular  0.0084 +/- 0.001  Intracellular  0.1872 +/- 0.174  Extracellular  0.0157 +/- 0.015  Intracellular  0.3820 +/- 0.130  Luciferase  a  3  Specific  Relative  Activity  Increase  15  0  22  24  Extracellular location refers to the M D C K cells culture medium, 2 hr after initial infection  by S. dublin. Intracellular location refers to the M D C K cells, 4 hr after initial infection. b  Units for specific enzyme activity.  B-galactosidase units are expressed as F D G  fluorescence/cfu; luciferase units are expressed as photons/cfu. The error bars represent the standard deviation of the means. c  Relative increase for each enzyme is the specific activity of intracellular bacteria divided by  specific activity of extracellular bacteria.  The  sensitivity of luciferase as an intracellular reporter was compared to that of  B-galactosidase through the use of the isogenic plasmid fusions to spvB (126). Activities were first normalized to activity per bacterium (cfu) for each respective enzyme. Then the relative increase in activity after invasion was determined by dividing the intracellular activity by the extracellular activity.  In side-by-side experiments, the increase in luciferase activity was  similar to the increase in B-galactosidase activity (24 versus 22 fold), as shown in Table 4. Therefore the luciferase was as sensitive as B-galactosidase for the detection of intracellular gene expression.  Chapter 3 7 2 F o r both assays, the m i n i m u r n number o f bacteria required i n order to detect the spvB expression varied as the gene w a s induced or repressed. 0.5-1.0xl0  5  U n d e r repressing  bacteria were required, w h i l e o n l y about l . O x l O  3  conditions,  bacteria were required w h e n  spvB w a s i n d u c e d . It w a s p r e v i o u s l y s h o w n (72) that the induction o f spv gene expression was not the result o f an increased p l a s m i d c o p y number w i t h i n the bacteria.  3.2.  Discussion Bacterial luciferases have been used i n previous studies as reporters  o f intracellular  bacterial gene e x p r e s s i o n , h o w e v e r , many o f those studies were performed under conditions w h i c h were not p h y s i o l o g i c a l f o r mammalian cells (e.g. l o w e r temperatures ranging from 22-30°C).  S o m e examples include the m o n i t o r i n g o f gene activation d u r i n g plant-microbe  interactions under conditions optimal f o r plant g r o w t h (187), a n d the induction o f hydrogen peroxide-stimulated genes i n 5. typhimurium u p o n interaction w i t h mouse macrophages at 3 0 ° C ( 9 2 ) . T h i s w a s o f concern since temperature is the basis for induction o f numerous virulence-associated genes, over a broad range o f pathologenic bacteria ( 1 , 3 0 , 8 0 , 2 1 1 ) . M a n y virulence factors are optimally expressed at 3 7 ° C .  T h e luciferase f r o m V. fischeri  is  inadequate for temperature-dependent studies since it is inactivated above 3 0 ° C ( 6 9 , 9 2 , 155), h o w e v e r the luxAB gene cassette f r o m V. harveyi encodes a heterodimeric luciferase w h i c h remains active at 3 7 ° C ( 6 9 , 155, 2 1 0 ) . I n the w o r k reported here, bacteria a n d tissue culture cells were g r o w n a n d assayed at p h y s i o l o g i c a l temperatures ( 3 7 ° C ) , w i t h m i n i m a l disruption to interactions o c c u r r i n g between  the intracellular bacteria  and the m a m m a l i a n  host  cells.  A l t h o u g h an alternative luciferase f r o m Xenorhabdis luminescens remains thermostable up to 4 5 ° C (155, 2 1 0 ) , it has a l o w e r specific activity than V. harveyi luciferase (309).  A s well,  V. harveyi luxAB genes were contained w i t h i n a convenient 3.25 k b p BamHI gene cassette w h i c h d i d not contain its o w n endogenous promoter.  N o t included w i t h i n this cassette were  the aldehyde biosynthetic genes luxCDE, encoded b y an extra 4 k b p segment o f D N A .  While  the presence o f substrate synthesizing genes may appear to be an advantage, a fusion w i t h high expression promoters  w o u l d result i n the h i g h production o f aldehyde,  and potentially  Chapter 3 73 increased Salmonella mortality. E v e n though the aldehyde substrate had to be exogenously supplied, previous studies have indicated that recombinant lux products exhibit h i g h activity in the presence o f externally-added d e c y l aldehyde (210, 309). Langridge  et  al.  (186)  had  previously found  that  vapors  from  high  decanal  concentrations ( 1 0 % or more) resulted in increased levels o f mortality a m o n g y o u n g plantlets. Therefore, the effects o f aldehyde concentration on both bacteria and M D C K  cells was  addressed. N o n e o f the aldehyde concentrations tested appeared to harm the m a m m a l i a n cells, but higher aldehyde concentrations (>0.0022%) were t o x i c to the bacteria (Figure 8 B ) . It was not determined whether this toxicity was a direct or indirect effect o f the aldehyde. T h e drop in numbers o f c o l o n y - f o r m i n g bacteria was greater than the drop in total light output per w e l l (Figures 8 A , B ) , indicating that higher aldehyde concentrations elicited more light production f r o m the r e m a i n i n g viable bacteria (Figure 8 C ) . It may be that the concentrations o f aldehyde tested d i d not reach the substrate saturation range o f the luciferase e n z y m e , or c o n v e r s e l y , that the e n z y m e activity rendered them unculturable.  T h e use o f higher amounts o f aldehyde  therefore m a y have resulted i n more efficient activity o f the e n z y m e .  H o w e v e r , since the  m e c h a n i s m o f aldehyde toxicity was u n k n o w n , the concentration o f aldehyde  (0.0022%)  w h i c h permitted the highest light production b y luciferase without concomitant bacterial death was used. T h e use o f bacterial luciferase as a reporter o f gene expression d i d pose some other problems.  A n article b y F o r s b e r g et al. (87) reported that an intrinsically c u r v e d segment o f  D N A i n the 5 ' c o d i n g end o f the luxA gene may influence promoter activity o f the target gene. H o w e v e r , the luciferase activity correlated w i t h the p r e v i o u s l y established B-galactosidase data, indicating that the 5 ' end o f the luxA gene d i d not interfere w i t h spv G o n z a l e z - F l e c h a and D e m p l e (114) reported  that luciferase  regulation.  activity (in the  Likewise, absence  of  n-decanal) was associated w i t h an increase in oxidative radicals w i t h i n the bacteria over time. H o w e v e r , since the spv  operon is not influenced by the redox state o f the bacteria (72), it  seems u n l i k e l y that either the luxAB genes or the luciferase enzyme activity interfered with  Chapter 3 7 4 spvB gene e x p r e s s i o n . F u r t h e r m o r e , it h a d been suggested b y M e i g h e n (210) that changes in intensity o f luminescence in vivo m a y depend not o n l y o n the amount o f functional luciferase available, but also o n the availability o f substrates (FMNH2, aldehyde, and O2).  T h i s implied  that luciferase w o u l d be an inaccurate reporter i n situations where either o x y g e n or energy were lacking.  It appeared that the environment Salmonella encounters u p o n invasion o f epithelial  cells contained enough o x y g e n to support luciferase activity ( F i g u r e s 8 a n d 10; and Table 4 ) . A s w e l l , intracellular Salmonella remained viable, p r o v i d i n g the aldehyde concentration w a s not too h i g h . T h e s e results indicated that use o f luciferase for Salmonella studies w o u l d not be l i m i t e d b y either the lack o f o x y g e n or bacterial energy available to the intracellular bacteria. F r a n c i s a n d G a l l a g h e r (92) s h o w e d that luciferase activity in response to oxidative stress  w a s variably  expressed  within  infected  cells,  suggesting  that  the intracellular  environment m a y differ somewhat between individual cells or w i t h i n a cellular subpopulation. F u r t h e r m o r e , there is a constant struggle between the bacteria and the host cells taking place d u r i n g an infection (2, 32). A s a result o f the d y n a m i c invasion process o f the bacteria, there are t w o populations o f Salmonella w i t h i n cells, one static (and p o s s i b l y decreasing in number) and the other rapidly d i v i d i n g . These variables together m a y account for the variation i n Table 4. N o reporter system is ideal for a l l situations and each system has its advantages and disadvantages.  I n this study, the use o f luciferase as a reporter o f intracellular bacterial gene  expression w a s assessed u s i n g conditions optimal for Salmonella i n v a s i o n o f non-phagocytic mammalian cells.  T h e bacterial luciferase, encoded b y promoterless luxAB reporter  system  to  6-galactosidase,  genes  V. harveyi,  p r o v i d e d an alternative  advantages.  L u c i f e r a s e w a s an accurate and sensitive reporter o f intracellular Salmonella spv  gene e x p r e s s i o n , as c o n f i r m e d b y data u s i n g 6-galactosidase assays ( 7 7 ) .  with  from  several  M o r e o v e r , the  luciferase assay w a s faster and easier to perform than the 6-galactosidase assay.  F i r s t , there  was n o need to lyse either the cells or the bacteria, and activity in the sample w a s measured immediately after substrate addition. F o r the 6-galactosidase assay, it w a s necessary to first  Chapter 3 75 lyse the bacteria and then incubate the sample in the presence of the substrate for a set period of time. It was further necessary to protect the fluorescent 6-galactosidase substrate, FDG, from light. With luciferase, it was not only possible to determine bacterial gene expression from live extra-cellular bacteria but from intra-cellular bacteria as well.  Another advantage of using  luciferase was the absence of background activity. Neither the cells nor the bacteria had any endogenous luciferase activity and therefore any light detected resulted from the  luxAB  constructs, whereas endogenous 6-galactosidase activity could be detected from mammalian cells. The ability to monitor luciferase activity without physically disrupting either the bacteria or the cells would also allow for the monitoring of bacterial-cell interactions over time. Furthermore, the product (light) did not build up within the sample, and low dose applications of the aldehyde substrate were found to be non-toxic to both bacteria and tissue culture cells. Collectively, the results demonstrate that luciferase gene fusions are a sensitive way to monitor gene expression of bacterial pathogens found within mammalian host cells.  Chapter 4 76 C h a p t e r 4:  Development of a Screen for B a c t e r i a l Genes  Chapter 4 describes a screening system developed to search for Salmonella  genes  i n d u c e d after bacterial i n v a s i o n o f host c e l l s . Luciferase was used as the reporter o f bacterial gene e x p r e s s i o n . S. typhimurium  T h i s chapter first describes transfer  o f the luxAB  reporter genes to the  c h r o m o s o m e , and the selection o f the resulting bacterial mutants.  Genes  w h i c h were differentially expressed were then identified, specifically those genes induced by intracellular bacteria and repressed by extracellular bacteria. 4.1.  Results 4.1.1.  Transformation  o f Salmonella  and Screen  for U p r e g u l a t e d  Bacterial  Genes A t w o p l a s m i d competition system, described b y G u z z o and D u B o w (138), was used to obtain r a n d o m insertions o f the promoterless w i l d - t y p e S. typhimurium  S L 1 3 4 4 chromosome  reporter gene cassette luxAB (Figure  11).  The  plasmid  w i t h i n the pFUSLUX  contained a C o l E l o r i g i n of replication and a T n 5 - t o A 5 - t e t r a c y c l i n e resistance cassette, w h i l e the p l a s m i d p T F 4 2 1 encoded for a m p i c i l l i n resistance and the production o f R N A 1 w h i c h acted to i n h i b i t the replication o f p F U S L U X . B y g r o w i n g the transformed bacteria in the presence o f both tetracycline and a m p i c i l l i n , the luxAB inhibited p F U S L U X  gene cassette was forced f r o m the replication-  p l a s m i d to integrate into the bacterial  tetracycline resistance c o u l d be maintained b y the bacteria. o w n promoter; h o w e v e r , the luxAB  chromosome,  such  that  the  T h e tetracycline gene contained its  genes were not expressed unless the cassette integrated  such that it was under the control o f an active bacterial promoter.  Chapter 4 77  Tn5L  luxA  luxB  tet Tn5R  Figure 11: Schematic of the luciferase reporter gene cassette inserted within the bacterial chromosome, and the orientation of the primer pairs used for inverse PCR and subsequent D N A sequencing.  Chapter 4 78  Initially, the t w o plasmids were sequentially transferred to S. typhimurium  SL1344  u s i n g electroporation. F i r s t the p F U S L U X p l a s m i d was transferred into a S L 1 3 4 4 bacterium. P u r i f i e d p T F 4 2 1 p l a s m i d was  then electroporated  into  SL1344 p F U S L U X .  However  transformation o f the second p l a s m i d by electroporation was inefficient and o n l y a few hundred colonies w i t h dual antibiotic resistance resulted f r o m multiple attempts. Therefore, the phage P 2 2 H T int was u s e d to transfer the p l a s m i d p T F 4 2 1 into S L 1 3 4 4 p F U S L U X . was m u c h more efficient and approximately 15,000 S. typhimurium  T h i s procedure  colonies resulted f r o m  the transductions. T o initially identify extracellular bacteria expressing the integrated reporter genes, the L u m i n o g r a p h L B 9 8 0 photon imager was used to examine the transduced Salmonella  colonies  on agar plates for light p r o d u c t i o n . T h e substrate was added to the colonies b y streaking the aldehyde onto lids o f the plates and a l l o w i n g the vapors to penetrate the c o l o n i e s . p r o d u c i n g h i g h relative amounts o f light o n the plates were discarded since they  Those  represented  extracellular bacteria expressing h i g h amounts o f luciferase, w h i l e those p r o d u c i n g little to no light on plates were retested ( F i g u r e 12).  E a c h c o l o n y to be retested was streaked out onto a  fresh plate to ensure the c o l o n y was truly resistant to both antibiotics and was also a single clone.  O v e r 3 5 0 0 colonies (about 2 . 4 % o f the original number o f transformants) d i s p l a y i n g  little to no luciferase activity were detected i n this manner. E a c h o f these was further tested on green plates to ensure that they d i d not contain active P 2 2 phage infections (i.e. lytic infections resulted i n dark green colonies rather than light green colonies). B a c t e r i a free o f l y t i c infections were then transferred to broth cultures w i t h i n i n d i v i d u a l w e l l s o f a 9 6 - w e l l plate.  Chapter 4 79  F i g u r e 12: L u m i n o g r a p h images o f L B plates w i t h S. typhimurium  colonies transfected w i t h  both p T F 4 2 1 a n d p F U S L U X p l a s m i d s . B o l d , left-facing arrows point to examples o f colonies p r o d u c i n g light o n L B agar alone, w h i c h were not further screened.  S m a l l e r , right-facing  arrows point to examples o f colonies p r o d u c i n g little to no light o n plates alone, w h i c h were p i c k e d for further screening.  Chapter 4 80  4 «  Chapter 4 81 At this point, the bacteria were stored at minus 70°C in 25% glycerol stocks within the 96-well plates and covered by a 96-well storage mat (Costar). Note that individual colonies were not pooled but placed into separate wells. 4.1.2. S c r e e n i n g f o r B a c t e r i a l G e n e I n d u c t i o n  Inside  Cultured  Macrophages  Each colony expressing low levels of light on agar plates was tested for luciferase activity during growth both outside and inside host cells. 96-well plates containing L B broth were inoculated with bacterial mutants and grown overnight, with shaking. The next day these plates, now considered the 'extracellular stationary phase' bacteria, were used to inoculate another plate of L B broth (the 'extracellular logarithmic phase' bacteria) and two plates of cultured macrophages, J774A.1 or B A L B . B M 1 (i.e. 'intracellular' bacteria). which the bacteria was added to the cells was time zero. S. typhimurium  The time at  typically has a lag  period about 4 hr after it invades cells, during which no increase in cfu's is seen within the cells. This lag period is thought to allow the bacteria to adapt to the intracellular environment, and after this lag, the bacteria are able to grow exponentially, at least within epithelial cells. Therefore, both 2 and 4 hr after invasion, the bacterial samples were tested for the induction of light production, which was indicative of the promoter activity of genes expressed during intracellular growth. Bacteria were incubated with the cells for 1 hr, after which the medium was removed and the cells were washed.  The infected cells were then treated with  gentamicin for 1 to 3 hr in order to kill any bacteria remaining extracellular.  T w o and four  hours after the initial infection of the cultured cells, the aldehyde substrate was added to the wells and the light production measured using the Luminograph L B 9 8 0 .  After the 2 hr  reading, the aldehyde-containing medium was replaced by fresh gentamicin-containing medium and the incubation continued for another 2 hr.  The light production from the plates of  extracellular bacteria (stationary and logarithmic phase) was only read 4 hr after time zero. A n example of this screening procedure is shown in Figure 13, which demonstrates the light production from one 96-well plate of bacterial mutants under the six different conditions described:  two extracellular (stationary  and logarithmic phases of growth)  and  four  Chapter 4 82 intracellular (2 and 4 hr inside either J774A.1 or B A L B . B M 1 cells). Light from each well was quantitated using the Luminograph LB980 and compared to the estimated bacterial count for that well. Bacterial numbers were estimated based on the averages of randomly picked wells within the various conditions, and generally, stationary phase bacteria averaged between 0.5-1.0xl0 cfu per well; logarithmic phase bacteria averaged between 0.5-l.OxlO cfu per 9  8  well; and intracellular bacteria averaged between 1.0-5.Ox 10 cfu per well at both 2 and 4 hr. 5  The thirty-three plates of frozen bacterial mutants were tested under each of the separate conditions at least twice. This was considered round one of the 96-well plate screen. Mutants demonstrating the induction of light inside cells were then assembled into new 96-well plates and frozen as before.  These mutants were again tested in all six conditions  mentioned above, and this was considered round two.  This was done to affirm that the  reporter gene expression by these mutants was indeed induced and that the phenotypes were stably expressed. After screening, the mutants continuing to demonstrate the induction of light inside cells were once again assembled into new 96-well plates for round three of the screen. From this final round of testing the S. typhimurium  SL1344 mutants, 10 mutants  (from the original 3500 mutants) consistently upregulated light production by more than an estimated five-fold at both 2 and 4 hr inside the mouse macrophage-like cell lines (both J774A.1 a n d B A L B . B M l ) when compared to the extracellular controls (both stationary and logarithmic phase).  Chapter 4 83  F i g u r e 13: L u m i n o g r a p h images o f bacteria expressing light from a single sample 9 6 - w e l l plate o f mutants. infection; infection;  Bacterial g r o w t h conditions are as f o l l o w s : B ) inside B M I cells 4 hr  after infection;  A)  inside B M I cells 2 hr  after  C ) inside J 7 7 4 A . 1 cells 2 hr  after  D ) inside J 7 7 4 A . 1 cells 4 hr after infection; E ) extracellular bacteria i n l o g phase  growth; F ) extracellular bacteria i n stationary phase growth.  Chapter 4 84  Chapter 4 85 4.1.3. T r a n s f e r o f G e n e s to E n s u r e I n d u c t i o n P h e n o t y p e is L i n k e d to G e n e Insertions T o be certain that the induction phenotype resulted from the c h r o m o s o m a l insertion o f the gene cassette into a single gene, phage P 2 2 H T int lysates were made from each mutant. These lysates were used to transduce  new S. typhimurium  SL1344,  and the resulting  transduced bacteria were then tested for a pattern o f light induction that matched the original parental mutant. S i x mutants were identified and were named A 1 A 1 , E 1 2 A 2 , D I 1 H 5 , G 5 D 5 , G7H1,  and G 8 B 1 ( F i g u r e 14).  G r e e n plates were once again used to ensure  transductants no longer contained the P 2 2 phage.  that the  T h i s time h o w e v e r , the light green colonies  o f the transductants (i.e. no lytic P 2 2 infection) were also cross-streaked against P 2 2 H 3 .  The  transductants were considered free o f l y s o g e n i c P 2 2 o n l y i f they c o u l d be re-infected by the P 2 2 H 3 phage (300). 4.2.  Discussion Intracellular s u r v i v a l and g r o w t h o f Salmonella are important for the virulence o f the  bacteria, and recent evidence has suggested that many genes required for intracellular survival are not expressed b y the bacteria w h e n they are g r o w n on regular m e d i a i n the absence o f cells. Therefore a screening procedure was designed to detect bacterial genes that were upregulated b y intracellular environments o f m a m m a l i a n cells. A t w o - p l a s m i d competition system wasused to obtain single r a n d o m insertions o f a promoterless luxAB S. typhimurium  c h r o m o s o m e . T h i s ensured the luxAB  reporter gene cassette w i t h i n the  genes, (encoding luciferase), were  under the transcriptional control o f the natural Salmonella promoter environment, rather than from an alternate promoter or from a m u l t i - c o p y number p l a s m i d .  T h e Salmonella genes were  furthermore disrupted w i t h this insertion and were not duplicated.  W h i l e this reduced the  chances o f identifying genes crucial to bacterial v i a b i l i t y (i.e. housekeeping genes), there was a concern that genes crucial to Salmonella s u r v i v a l w i t h i n cells (i.e. have  come  out  o f the  screen.  However,  despite  this  virulence genes) may not  concern,  genes  required  for  Chapter 4 86  F i g u r e 14: C o m p a r i s o n o f the luciferase activity from the P22-transductional mutants w i t h the original S. typhimurium insertional mutants. various S. typhimurium clones. S L 1 3 4 4 (no  A ) 9 6 - w e l l template s h o w i n g placement o f the  C l o n e s w i t h p S P L U X were included as positive controls.  luxAB) was i n c l u d e d as a b a c k g r o u n d level c o n t r o l . Uncharacterized Salmonella  mutants w h i c h either constitutively expressed luciferase or not were further i n c l u d e d as "on" or "off" controls, respectively.  B ) C o r r e s p o n d i n g luminograph images o f the S. typhimurium  clones e x p r e s s i n g light w h i l e under different intracellular a n d extracellular c o n d i t i o n s . T h e transduced mutants displayed luciferase activity in s i m i l a r patterns to the corresponding original parent mutants.  Q_  CO CO  Q. CC  *l_  E o O cC Q.  CO  CD  CM  < CD LO  < CD  < CQ O Q CD  LO  r-O  00  UJ U_ CD  "On" Mutant  CO  "On" SL1344 SL1344 SL1344 Mutant pSPLUX pSPLUX  LD842 LD842 "Off" SL1344 Mutant pSPLUX pSPLUX  CD  "On" SL1344 SL1344 SL1344 Mutant pSPLUX pSPLUX  G8B1 CO  "Off" Mutant  G7H1  G5D5  CD  "Off" Mutant  CD  G8B1  LO  G7H1  G5D5  E12A2 CD  r~ CD  LD842 LD842 SL1344 pSPLUX pSPLUX  CO Q  G8B1  c < O  G8  N-  00 LO  G7H1  G5D5  CC  E12A2  CC  E12  c;  <  E12  CO  E12A2  5  D11H5  CC  D11  O  D11H5  CC  O)  If)  E12  c o  E12  -4—'  D11H5  CO  D11  o •3 ~o CO c  "On" Mutant  "Off" Mutant  G8B1  G7H1  G5D5  E12A2  D11H5  A1A1  1—  D11  c A1A1  CO  A1A1  CM  A1A1  Chapter 4 87  O X  T3  8£  "D CO  CO  2  Cl CO -*-  c c n => O 2  T3 CP O CO  c  CO  CO  c  ro c  O  2 2  Chapter 4 8 8  Intracellular  BALB.BM1 Cells 1  2  3  A B  4  5.  t  »  J774A.1 Cells 6  7  8  9  10 11 12  9  •  *  *  i  t  t  »  i  1  C D  •* »*  t  9  E F  § §  G  *  -  H Original  Original  Transduced  Transduced  Extracellular  Logarithmic 1  2  A  3  4 f1  Stationary 5,  6  7  8  9  10 11 12  •  B C D E F G H  '<#  Oriainal  #•  Transduced  Oriainal  Transduced  Chapter 4 89 intra-macrophage s u r v i v a l were identified (refer to Chapter 5 results and discussion), indicating that the screening parameters were not reliant on the ability o f the bacteria to s u r v i v e within cells. Salmonella were initially g r o w n on agar plates to test for c o l o n i e s not p r o d u c i n g significant amounts o f light outside host cells.  These l o w - l i g h t producers were then retested  inside cells to see whether the amount o f light c o u l d  be  i n d u c e d by  the intracellular  environment. T h e screening effectively eliminated bacteria containing gene insertions that were constitutively expressed or i n d u c e d by m e d i u m or serum alone.  In retrospect,  bacteria  containing insertions w i t h i n invasion genes were most l i k e l y eliminated by default as w e l l since intracellular bacterial numbers w o u l d be too l o w to detect an increase in e n z y m e activity.  For  the initial screening phase, an estimate o f bacterial numbers was used for the calculation o f the specific activity o f the luciferase, but later these numbers appeared to be an overestimation o f the actual bacterial count.  B y l o w e r i n g this threshold, it m a y be p o s s i b l e to observe more  bacterial genes that were upregulated w i t h i n host cells and not just the genes described in this study.  Chapter 5 C h a p t e r 5:  90  Characterization of Genes  Upregulated by Intracellular  Salmonella  In C h a p t e r 5, the S. typhimurium mutants that were identified b y the screen described in C h a p t e r 4 are further characterized.  T h e disrupted bacterial genes were sequenced  and  identified b y c o m p a r i s o n to k n o w n genes. T h e g r o w t h and pathogenicity o f the mutants were also c o m p a r e d to that o f the w i l d - t y p e bacteria. 5.1.  Results  5.1.1.  Inverse  P C R and  Sequencing  Inverse P C R was used to amplify regions immediately upstream f r o m the inserted luxAB genes u s i n g outfacing primers L U X 7 6 and L U X 3 4 0 , w h i c h were specific for the luxA gene sequence, as indicated in F i g u r e 11.  F i g u r e 15 demonstrates the single D N A fragment  resulting f r o m inverse P C R u s i n g c h r o m o s o m a l D N A f r o m each o f the mutants. were seen u s i n g D N A f r o m the w i l d - t y p e bacteria S L 1 3 4 4 .  N o bands  B a s e d on the conditions o f the  inverse P C R used, it was h i g h l y u n l i k e l y that there was more than the one identified insertion o f the luxAB cassette w i t h i n an i n d i v i d u a l bacterial c h r o m o s o m e .  F o r e x a m p l e , u s i n g a four-  basepair cutting restriction enzyme results i n frequent cutting o f the c h r o m o s o m a l D N A , and u s i n g an extension times o f 2 m i n or more i n the P C R s h o u l d be l o n g enough to amplify segments o f 2 to 3 k b p i n length. Amplified  D N A fragments  were  isolated f r o m  the  agarose  gel and  sequenced.  Sequences o f the upstream regions f r o m each o f the six mutants were c o m p a r e d w i t h k n o w n sequences i n G e n B a n k . F o r the mutant E 1 2 A 2 , the p r i m e r set E P L U S / E M I N U S was used to amplify and sequence further upstream and downstream f r o m the insertion site. primer set G P L U S / G M I N U S insertion site.  S i m i l a r l y , the  was used to identify the D N A region around the G 7 H 1  F i g u r e 16 illustrates the positions o f the insertions o f the luxAB gene cassette  w i t h i n the S. typhimurium c h r o m o s o m e .  D I 1 H 5 had the luxAB  cassette inserted w i t h i n the  ssaR gene w h i c h is f o u n d w i t h i n the Salmonella Pathogenicity Island 2 (SPI-2) ((152);  Chapter 5 91  (Kbp) 23.5 9.4 6.6 4.4  2.3 2.0  1.5  0.7  8:1  Figure 15: D N A bands resulting from inverse PCR are visualized on a 1% agarose gel. The chromosomal D N A from each mutant was cut with Haelll, and then the fragments re-ligated prior to inverse PCR reactions using the LUX76:LUX340 primer set. The presence of a single band per mutant was indicative of a single insertion of the luxAB gene cassette within the chromosome.  Chapter 5  F i g u r e 16:  P o s i t i o n o f luciferase gene insertions w i t h i n k n o w n S. typhimurium  Insertions are indicated b y the large black arrowheads. the arrows underneath the genes.  S. typhimurium  gene w h i c h  N u m b e r s u n d e r l y i n g genes correspond to the sequence  is part o f S P T 2  A ) D l 1H5 had an insertion  f o u n d between  centisome  30-31 on the  c h r o m o s o m e ( A c c e s s i o n # X 9 9 9 4 4 ) . T h i s region is part o f an o p e r o n , where  the open reading frames are f o u n d to overlap. within  genes.  Transcription direction is indicated b y  n u m b e r i n g w i t h i n the A c c e s s i o n references for S. typhimurium. w i t h i n the ssaR  92  S P I - 5 located between  near centisome  B)  B o t h A 1 A 1 a n d E 1 2 A 2 h a d insertions  25 o n the S. typhimurium  chromosome  ( A c c e s s i o n # A F 0 2 1 8 1 7 ) . A 1 A 1 h a d an insertion i n sigD/sopB w h i l e E 1 2 A 2 had an insertion w i t h i n a d o w n s t r e a m O R F pipB (S. dublin:  Accession #AF060858) .  genes i n S. dublin  In S. dublin  are indicated i n brackets.  T h e corresponding  the c o r r e s p o n d i n g region w i t h i n  S P I - 5 w a s thought to be transcribed as one continuous m R N A , as indicated o n the diagram. C)  G 7 H 1 h a d an insertion w i t h i n a p r e v i o u s l y uncharacterized region o f the Salmonella  chromosome (Accession #AF164435). potential open reading frame.  T h e insertion appeared to be w i t h i n the 5 ' e n d o f a  Chapter 5 93  4  Chapter 5 Accession #X99944).  94  A 1 A 1 had the gene cassette inserted w i t h i n the sigD gene f o u n d at  centisome 25 o n the c h r o m o s o m e ((159); A c c e s s i o n # A F 0 2 1 8 1 7 ) .  In S. dublin  there is a  h o m o l o g o u s gene called sopB, w h i c h is f o u n d at the corresponding c h r o m o s o m a l p o s i t i o n , w i t h i n a region recently dubbed the fifth Salmonella Accession #AF060858).  In the mutant E 1 2 A 2 ,  d o w n s t r e a m o f the sigD/sopB reading frame.  pathogenicity island ( S P I - 5 )  the luxAB  gene cassette was  ((338); inserted  gene, i n a region p r e v i o u s l y identified as a potential open  T h i s gene was named pipB,  as it was f o u n d to have h i g h h o m o l o g y (>90%  identity at the D N A l e v e l ) w i t h the S. dublin pipB gene, w h i c h is f o u n d downstream o f sopB. T h e mutants G 5 D 5 , G 7 H 1 and G 8 B 1 were f o u n d to have the exact same insertion site, w h i c h was i n a region that has not been identified yet. Therefore the potential open reading frame was named iicA for i n d u c e d intracellularly A ( G e n B a n k A c c e s s i o n # A F 1 6 4 4 3 5 ) . ( F o r the sake o f brevity, G 5 D 5 / G 7 H 1 / G 8 B 1 w i l l be referred to s i m p l y as G 7 H 1 . ) R a p i d m a p p i n g to locate the c h r o m o s o m a l p o s i t i o n o f the insert u s i n g l o c k e d - i n P 2 2 m a p p i n g sets was unsuccessful ( 1 7 9 ) . H o w e v e r , this sequence appears to be present in both S. typhimurium and S. typhi genomes and matched a number o f contigs found i n the unfinished Salmonella sequencing projects at the Genome  Sequencing  Center  at  Washington  University  School  of  Medicine  (http://genome.wustl.edu/gsc/bacterial/salmonella.shtml) ( B _ S T M . C O N T I G . 1 6 0 7 ; B_STMA2A.CONTIG.3097; (http://www.sanger.as.uk/)  B_STMA2A.CONTIG.3068)  at  The  Sanger  Centre  (B_TYPHI2.hb56c04.sl).  See A p p e n d i x A for sequences o f the four mutants: G5D5/G7H1/G8B1.  and  A 1 A 1 , D 1 1 H 5 , E 1 2 A 2 , and  Chapter 5 5.1.2.  95  E x t e n t o f G e n e E x p r e s s i o n b y the M u t a n t s  T h e luciferase activity f r o m both the intracellular and extracellular Salmonella mutants is s h o w n i n F i g u r e 17.  T h e activity f r o m the intracellular bacteria can be seen to be induced  w h e n compared to the extracellular controls.  T h i s image is enhanced for l o w light and  demonstrates the upregulation o f bacterial gene expression w h i c h occurred w i t h i n phagocytic cells, J 7 7 4 A . 1 , as w e l l as w i t h i n the non-phagocytic cells, H e L a and M D C K .  T h e spvB gene  was used as a p o s i t i v e c o n t r o l for intracellular gene expression b y S. typhimurium.  T h e spvB  gene was actually i n d u c e d b y 10-fold inside cells, once light output a n d bacterial numbers had been n o r m a l i z e d as photons/cfu.  F i g u r e 17 differs f r o m F i g u r e 10 i n that the number o f  extracellular bacteria h a d not been adjusted to the same number f o u n d intracellularly.  In  F i g u r e 17, the extracellular bacteria were 2 0 - 2 0 0 f o l d higher than the number f o u n d inside cells, as indicated b y the relative bacteria numbers.  C o m p a r a t i v e l y , the four Salmonella genes  described i n this study d i s p l a y e d l o w e r expression outside o f host cells a n d were more h i g h l y i n d u c e d inside cells than w a s the spvB gene. F i g u r e 18 demonstrates the increase i n luciferase activity b y Salmonella  w i t h i n the  intracellular environment over those remaining extracellular. T h e activity seen at the 1 hr time point represents o n l y bacteria i n the extracellular supernatant, w h i l e the other time points (3-7 hr ) represent o n l y bacteria w h i c h were intracellular. T h e bacteria outside cells d i d not produce any more light than those g r o w n i n media alone, indicating that neither the proteins in the m e d i a and serum nor factors secreted b y the cells were sufficient to induce the genes, as had been f o u n d w i t h some Shigella genes (229).  T h e sigD/sopB  gene d i d not appear to be  induced over time i n m e d i a alone ( F i g u r e 1 8 A ) , as had been reported b y H o n g and M i l l e r , 1998 (159).  Instead sigD/sopB  w a s constantly expressed  at a l o w level outside  cells,  Chapter 5  Gene Insertion  96  Intracellular  ixtracellular 3h  MDCK  HeLa  J774A.1  DMEM  5h 7h  3h  5h  7h  spvB A1A1  (sopB/sigD) D 1 1 H 5 (ssaR)  E12A2  (pipB)  G 7 H 1 (iicA)  Relative Bacterial Numbers  200  F i g u r e 17: S. typhimurium  10  10  10  mutants s h o w increased light production inside mammalian c e l l s .  T h e image w a s taken u s i n g a L u m i n o g r a p h L B 9 8 0 photon detector a n d p r o v i d e s a visual demonstration o f the gene i n d u c t i o n . N o t e that the number o f extracellular bacteria are 2 0 to 2 0 0 - f o l d higher than the number o f intracellular bacteria.  T h e spvB gene is included as a  positive control a n d has p r e v i o u s l y been s h o w n to be induced w i t h i n cells by 5-20-fold compared to extracellular l o g a r i t h m i c a l l y - g r o w i n g bacteria. T h e numbers s h o w n at the bottom of the figure indicate the relative number  o f bacteria w i t h i n the w e l l s to further  c o m p a r i s o n o f bacterial gene expression between the different conditions.  enable  Chapter 5 97  Figure 18: Comparison of light production of luciferase-expressing bacterial mutants exposed to different environmental conditions. photon detector. (sigD/sopB);  Light was measured using a Luminograph LB980  Individual bacterial mutants are represented in the panels:  B) D11H1 (ssaR); C) E12A2 (pipB ); D) G7H1 (iicA).  A) A1A1  The lines indicate  bacteria grown in media alone, shaking while in LB broth and stationary while in DMEM++ media: DMEM++ (filled squares); LB broth (open circles). The bars for the 1 hr time points represent only the activity of the bacteria remaining in the supernatant outside the cells; the bars for the following time points (3, 5, and 7 hr) represent activity from only intracellular bacteria. J774A.1 (open bars); HeLa (hatched bars); MDCK (filled bars). Triplicate experiments were performed, and error bars represent standard error, P<(0.5).  Chapter 5  98  Chapter 5  99  w h i c h was slightly higher than the other three genes described here (pipB, ssaR, iicA).  As  w e l l , the other three genes were not found to be induced in m e d i a alone ( F i g u r e 1 8 B - D ) .  This  was not unexpected, as the screening system was set up to eliminate mutants w h i c h  were  i n d u c e d b y g r o w t h phase. F i g u r e 19 s u m m a r i z e s the i n d u c t i o n ratios o f luciferase activity o f the bacterial mutants in the various c o n d i t i o n s . T h e ratios were determined by d i v i d i n g the activity o f the bacteria w i t h i n each c o n d i t i o n at the different time points ( s h o w n in F i g u r e 18) by the average activity o f each particular mutant i n D M E M + + at 1 hr  .  E x p r e s s i o n o f the genes in L B broth was  s i m i l a r to that seen i n D M E M + + . T h e ssaR gene was upregulated w i t h i n cultured macrophages by about 4 0 - 1 0 0 f o l d , and w i t h i n cultured epithelial cells b y 30-800 f o l d d e p e n d i n g on the cell type ( 3 0 - 9 0 f o l d w i t h i n H e L a cells and 4 0 0 - 8 0 0 f o l d w i t h i n M D C K c e l l s ) .  The  sigD/sopB  gene was i n d u c e d b y 10-20 f o l d inside cultured J 7 7 4 A . 1 macrophages, b y 10-70 f o l d w i t h i n H e L a epithelial c e l l s , and by 2 0 0 - 2 5 0 f o l d  inside M D C K  k i d n e y epithelial cells.  The  d o w n s t r e a m pipB gene was i n d u c e d m o r e h i g h l y w i t h i n J 7 7 4 A . 1 cells (30-100 fold) than was the sigD/sopB gene, whereas i n d u c t i o n patterns w i t h i n epithelial cells were s i m i l a r . T h e pipB gene was i n d u c e d b y 2 0 - 7 0 f o l d inside H e L a cells, and by 140-260 f o l d inside M D C K cells. T h e iicA gene was i n d u c e d by 3 0 - 1 2 0 f o l d inside J 7 7 4 A . 1 cells, by 8 0 - 1 6 0 f o l d inside H e L a c e l l s , and by 2 0 0 - 2 7 0 f o l d inside M D C K cells.  These data re-emphasize that none o f the  bacterial gene fusions were induced b y the media alone, nor by cell-secreted factors; h o w e v e r , they do suggest that the conditions that Salmonella encounters w i t h i n cells vary greatly between cell types.  Interestingly, the gene fusions appear to be continuously induced over time, and  expression o n l y appears to decline once the cells begin to l o o k sick (i.e. start to r o u n d up and lose contact w i t h the m o n o l a y e r ) .  Chapter 5 100  F i g u r e 19:  R e l a t i v e luciferase activity by extracellular and intracellular Salmonella  mutants.  T h e relative light expression is the ratio of the specific activity at each time point d i v i d e d by the specific activity seen b y bacteria g r o w n in D M E M + + for 1 h. B a c t e r i a were g r o w n in A ) D M E M + + m e d i a alone; B ) macrophage-like J 7 7 4 A . 1 cells; C ) epithelial-like H e L a cells; and D ) k i d n e y - l i k e M D C K cells. S y m b o l s represent: D I 1H5 (ssaR) (clear bars); A 1 A 1 (sigD) ( s p e c k l e d bars); E 1 2 A 2 (pipB) (striped bars); G 7 H 1 (iicA) (black bars). F o r panels B , C , and D , the 1 h time points represent the activity o f extracellular bacteria remaining in the supernatant taken f r o m outside the cells; the f o l l o w i n g time points (3, 5, and 7 h) represent activity f r o m the intracellular bacteria. T r i p l i c a t e experiments were performed, and error bars represent standard error o f the ratio o f t w o means.  Chapter 5 101  < o 1  (0  LO  3  o  x c  CO  E  j  !=M  L  CO  o Q  •  Q o O CO  o  o  LO CM  o O CM  uojssajdxg  o LO i-  o o  o  O i-  "*  LO  3Ajie|ay  o o  CM  o o  O  o o  CO  o o  CD  o o  T  o o  CM  uojssajdxg m6\-\ 3Ajie|oy  lO  3 O X  c  CU  E CO  LU Q  < •  o CM  ">  <=>  to  o  uojssajdxg jijBn aAjieiau uojssajdxg juBn 3AUB|au  Chapter 5 102 H e L a cells were the most sensitive to Salmonella infections and w o u l d begin to " l o o k s i c k " 5-6 hr after i n f e c t i o n , w h i l e both M D C K and J 7 7 4 A . 1 cells were more hardy and the cells w o u l d remain m o s t l y intact up to 8-9 hr after infection. 5.1.3.  C o m p a r i s o n o f G r o w t h R a t e o f the  Mutants  T h e reporter gene insertions were targeted to genes w h i c h were upregulated  upon  bacterial i n v a s i o n into cells; however, it was u n k n o w n whether the insertions affected bacterial functions or s u r v i v a l .  Therefore, the relative g r o w t h o f the bacteria was tested i n order to  determine whether the insertional mutations increased or decreased s u r v i v a l .  Bacterial g r o w t h  in m e d i a alone and w i t h i n cells after invasion was e x a m i n e d , w i t h the bacteria being g r o w n in D M E M + + p r i o r to i n v a s i o n .  Table 5 s h o w s the g r o w t h o f bacteria w i t h i n the  conditions at 5 and 7 hr, as compared to the 3 hr time point.  various  The g r o w t h rates o f the four  mutants d i d not appear to differ greatly from the g r o w t h rate o f the w i l d type S L 1 3 4 4 w h e n tested in extracellular media ( L B broth or tissue culture m e d i u m plus serum,  DMEM++).  W i t h i n the epithelial c e l l lines ( H e L a and M D C K ) , the growth patterns o f all four mutants were also similar to the parent strain.  W i t h i n the macrophage cells, the bacteria d i d not appear to  increase in numbers; however, both the parental strain and a l l four mutants were able to survive w i t h i n cultured macrophages.  L o n g e r time periods o f g r o w t h w i t h i n the cell lines  were  attempted, but the bacteria were v i s i b l y cytotoxic to the mammalian cells over longer time periods o f i n c u b a t i o n , i.e. longer than 9 hr (data not shown).  Chapter 5  Table 5:  Growth of Bacteria Over Time in Various Conditions  Environment  J774A.1 Cells  HeLa Cells  MDCK Cells  DMEM++ Broth  LB Broth  Salmonella  Gene  Mutant  Insertion  A1 A1 D11H5 E12A2 G7H1 SL1344  sigD/sopB  A1A1 D11H5 E12A2 G7H1 SL1344  sigD/sopB ssaR pipB  Relative 5hr  Growth 7hr  1.2 1.0 1.3 1.1 1.4  0.9 1.2 0.9 0.9 1.3  -  1.2 1.4 1.5 1.1 1.9  3.4 1.9 2.5 1.9 3.6  A1A1 D11H5 E12A2 G7H1 SL1344  sigD/sopB ssaR pipB iicA -  2.0 2.6 1.9 1.0 1.6  7.0 10.0 12.5 4.2 13.4  A1 A1 D11H5 E12A2 G7H1 SL1344  sigD/sopB ssaR pipB iicA -  3.2 2.8 3.3 2.9 2.6  9.8 9.9 10.3 9.2 10.6  A1 A1 D11H5 E12A2 G7H1 SL1344  sigD/sopB ssaR pipB iicA -  3.9 3.5 4.1 3.2 4.1  6.5 10.1 11.1 6.6 10.3  ssaR pipB iicA  -  iicA  Relative growth is the increase in bacterial  3  numbers over time expressed as the  number of cfu at xhr divided by the number of cfu at 3hr  Chapter 5 104 5.1.4.  Comparison  o f I n v a s i v e n e s s o f the  Mutants  All four mutants, when grown in DMEM++, remained as invasive as the wild type bacteria in the epithelial cells tested (Table 6). Since previous findings indicated that a small deletion in sigD resulted in a ten-fold reduction in invasion (159), the mutants were also tested by growing them in L B broth (both shaking overnight with subculture the next day, and standing overnight) prior to infection of the cells. Bacteria grown in L B broth were found to be about 10 fold more invasive than those grown in DMEM++ (data not shown); however, no significant difference between invasion of the mutants and the parent SL1344 was seen. Note that bacterial invasion took place in the presence of serum. The observation that the ssaR mutant D l 1H5 remained invasive correlates with previous findings where mutations within the ssa region of the SPI-2 did not affect invasion (60) Instead, the SPI-2 genes are thought to be necessary for the long-term survival of the bacteria within the mouse.  Table 6: Cells  Relative Invasion of Bacterial Mutants Into Cultured  Salmonella  Gene  Mutant  Insertion  A1 A1  sigD/sopB  7.3  ±1.2  0.9  ±0.7  1.7  ±2.2  D11H5  ssaR  8.6  ±1.1  1.2  ±0.7  1.4  ±2.2  E12A2  pipB  6.7  ±1.4  0.9  ±0.1  2.1  ±3.3  G7H1  iicA  9.1  ±3.0  1.0  ±0.3  3.0  ±2.5  SL1344  -  7.4  ±0.3  0.9  ±0.7  1.5  ±2.4  %  Invasion  a  J774  in Different HeLa  Cell Types MDCK  % invasion equals the number of intracellular bacteria at 3 hr divided by the total number of bacteria added to the well, then multiplied by 100. DMEM++ prior to invasion.  Bacteria were subcultured into  Chapter 5 105 5.1.5.  Virulence of Mutants in Typhoid Mouse  Model  Having identified genes upregulated by the intracellular environment, experiments were carried out to determine the requirement for these genes in the disease process. The insertional mutations did not affect the growth of the bacteria within cells or media, nor the invasion of the bacteria into cells. The virulence of the S. typhimurium mutants was therefore tested in a mouse infection model. The inbred mouse strain B A L B / c was used for the infection model, as it has previously been shown to be susceptible to infection with a number of bacteria, including Salmonella. When infected with either S. typhimurium or S. dublin, BALB/c mice succumb to a systemic disease very similar to that of typhoid fever in humans, (which is caused by S. typhi). Signs of disease in the mouse were observed first as a lack of grooming and an overall scruffy appearance of the mice. As the disease progressed, the mice would appear more scruffy, their backs would become hunched, and they would begin to shiver uncontrollably. They would also become indifferent to the other mice in the cage. Eventually they would die. Although the disease is more systemic than the gastrointestinal disease caused by S. typhimurium in humans, this mouse model has proven very useful for identifying virulence factors. The results of two experiments where mice were inoculated orally are presented in Figure 20. In the first experiment, the mice given D11H5 (ssaR) showed no detectable signs of disease even though they received 200 times the dose at which the wild type bacteria were 100% lethal (i.e. approximately 1000-fold higher than the reported LD50 for SL1344).  In the second  experiment, insertion into the pipB gene (E12A2) attenuated the bacteria by reducing the mortality to approximately 60% that of wild-type, when given at a dose of 1 .OxlO cfu/mouse. 6  At this dose, the insertion within the sopB/sigD gene (A1A1) was also seen to attenuate virulence of the bacteria to approximately 80% mortality, although this is not statistically significant due to the small numbers of mice used for these experiments. The mutant G7H1 (iicA) remained as virulent as the wild type SL1344 bacteria. The possibility of polar effects caused by these insertions remains (see discussion).  Chapter 5 106  Day  Post-Infection  F i g u r e 2 0 : V i r u l e n c e o f S. typhimurium mutants i n an orally infected t y p h o i d mouse m o d e l . M i c e were g i v e n l x l 0  6  cfu per mouse and disease progression w a s monitored for one month:  S L 1 3 4 4 ( w i l d type) (filled squares);  A 1 A 1 (sigD) (open circles);  d i a m o n d s ) ; G 7 H 1 (iicA) (open triangles).  E12A2  (pipB)  (open  Chapter 5 107 Although mutations in both the sopB/sigD (Al A l ) and pipB (E12A2) regions appeared to cause attenuation, only a disruption of the pipB gene appeared to affect the rate at which disease developed in the mouse (Table 7). Generally, the higher the bacterial dose, the quicker the mice were to develop signs of disease and die. The delay in the time at which 50% of the mice in the group had died was largest when lower doses of bacteria were used (i.e. at a dose of 1x10^ cfu/mouse, 50% mouse mortality was seen on day 21 for E12A2 and on day 13 for wild-type SL1344) and was not as noticeable at higher doses (i.e. at a dose of l.OxlO "!.OxlO , 50% mouse mortality for E12A2 occurred only one to two days after that of 7  8  the wild-type bacteria). A delay in mortality was also observed when the mice were inoculated intravenously with E12A2 (pipB).  Mice inoculated with D11H5 (ssaR) showed no signs of  disease (data not shown).  Table 7: Salmonella Mutant  Bacterial dose affects kinetics of mortality of mice Gene Insertion  Day of 5 0 % M o u s e Mortality at v a r i o u s b a c t e r i a d o s e s 10  SL1344 A1A1 E12A2 G7H1  wild-type  sigD/sopB pipB iicA  6  10  7  10  13 13  10 10  21  11  8  12  10  7  3  8  6 7  Day post-infection at which over half the mice in the group died  After 28 days postinfection, the remaining mice were sacrificed and organ homogenates were prepared from spleens and livers.  Only the mice inoculated with either the A1A1  (sopB/sigD) or the E12A2 (pipB) were tested in this manner as all mice inoculated with G7H1 (iicA) or wild type SL1344 had died. No Salmonella could be cultured from the homogenates, indicating the mice were able to clear the bacteria completely and were not chronically infected (47).  Chapter 5 108 5.2.  Discussion T h e expression o f four specific bacterial genes (ssaR, sigD/sopB,  pipB , and i i c A ) was  s h o w n to be i n d u c e d w i t h i n the host cells, and this induction was not induced b y rich media or b y factors secreted by host cells (Figures 17, 18, and 19).  These genes were also not only  induced at early time points (2-4 hr), but remained upregulated after the bacteria had adapted and begun to g r o w w i t h i n the cells (5-7 hr). Interestingly, reporter activity f r o m all four genes indicated that expression was induced in both phagocytic cells and non-phagocytic  cells.  E x p r e s s i o n f r o m w i t h i n cultured phagocytes continued to escalate over time, w i t h the exception o f the sigD/sopB  gene, whereas intracellular gene expression leveled o f f or declined within  epithelial cells. 5.2.1.  D11H5  T h e ssaR enterocolitica  (ssaR)  gene ( D 1 1 H 5 ) , f o u n d w i t h i n S P I - 2 ,  gene yscR,  was  h o m o l o g o u s to the  Yersinia  w h i c h encodes a membrane-bound subunit o f the type III secretion  system (150, 152). T h e Y s c R forms a critical part o f the secretion apparatus.  T h e ssaR gene  was upregulated after bacterial i n v a s i o n o f both cultured macrophages and epithelial cells, and continued to be expressed throughout the course o f the infection. T h e insertion o f the reporter gene cassette into the ssaR gene abolished virulence o f S. typhimurium model.  i n the t y p h o i d mouse  These findings are novel for the ssaR gene but agree w i t h previous reports  insertions w i t h i n S P I - 2 (46, 3 2 1 ) . apparatus-encoding  A ssaH  r.gfp gene fusion ( w i t h i n another  gene) was induced by 4 0 0 - f o l d w i t h i n macrophages  bacteria avirulent i n mice (321). structural, and effector/chaperone macrophage cells b y 3 to 100 f o l d .  S i m i l a r l y , various other gfp  insertions  the  regulatory,  genes o f S P I - 2 were also f o u n d to be induced w i t h i n Specifically the insertions w i t h i n ssrA  and sscB  reduced the ability o f the bacteria to spread to other organs w i t h i n the mouse (46). argue that the luxAB  secretion  and rendered into  of  insertion w i t h i n the ssaR gene caused a polar effect on  genes  One c o u l d downstream  genes, and therefore this gene is not directly related to virulence i n m i c e . H o w e v e r , a previous report had indicated that an insertion w i t h i n ssaT, a downstream gene w i t h i n the same o p e r o n ,  Chapter 5 109 reduced bacterial i n v a s i o n (152), whereas the mutant D 1 1 H 5 w a s not impaired i n its ability to invade non-phagocytic c e l l s .  T h e insertion is mostly likely h a v i n g a direct effect o n bacterial  virulence, since type I I I secretion has been p r e v i o u s l y implicated as important to bacterial virulence (46), and this gene potentially encodes a structural subunit o f the type III secretion system (152). 5.2.2.  A 1 A 1 (sigD/sopB)  and E12A2  (pipB )  T h e mutant A 1 A 1 had an insertion w i t h i n the sigD/sopB  gene (159), w h i c h is found  w i t h i n S P I - 5 . S P I - 5 is located near centisome 25 i n S. typhimurium  (159).  P r e v i o u s studies  have s h o w n that S i g D is a secreted protein, and have suggested that it is secreted into the host cell v i a the S P I - 1 type I I I secretion system (159). sigD/sopB  Furthermore,  the S.  gene is more than 9 5 % identical at the D N A level to the S. dublin  typhimurium sopB  gene,  w h i c h has recently been s h o w n to act as an inositol phosphate phosphatase inside host cells (235).  N o r r i s et al. (235) s h o w e d that Salmonella  affected the cellular signal transduction  pathways a n d that S o p B w a s able to h y d r o l y z e phosphatidylinositol  3,4,5-trisphosphate,  w h i c h is k n o w n to be a direct inhibitor o f calcium-dependent chloride secretion. SopB  h y d r o l y z e d 1,3,4,5,6-pentakisphosphate  to 1,4,5,6-tetrakisphosphate,  signaling molecule that indirectly acts to increase chloride secretion. activities  during  infection  was proposed  to be increased  chloride  A s well,  which  is a  T h e result o f these secretion  b y the  gastrointestinal cells, ultimately resulting i n diarrhea i n the host (235). T h e mutant E 1 2 A 2 h a d an insertion w i t h i n a p r e v i o u s l y unreported S. gene, pipB. SPI-5.  T h i s gene was located downstream o f sigD/sopB  T h e hypothetical pipB  typhimurium  and was also contained within  gene product w a s h o m o l o g o u s to that o f the S. dublin  pipB  gene product, w h i c h has structural similarity to proteins i n v o l v e d w i t h g l y c o l i p i d biogenesis (338). A s i n d i c a t e d i n A p p e n d i x A , where the S. dublin S. typhimurium However,  region m a y actually be d i v i d e d into t w o open reading frames  the size  S. typhimurium  gene is one O R F , the corresponding  o f the actual  o r S. dublin  pipB.  gene  product  has not been  determined  (ORF).  f o r either  Chapter 5 110 It has been  suggested  that w i t h i n the S P I - 5 o f S. dublin,  the sopB  and genes  downstream {pipC, pipB, and pipA) are contained w i t h i n the same transcriptional unit, based on the analysis o f the size o f m R N A transcripts (338). performed for the S. typhimurium  been sigDE  In the study described here,  the  ( A 1 A 1 ) and pipB ( E 1 2 A 2 ) genes had a similar expression pattern w i t h i n epithelial  cell types; h o w e v e r , sigD/sopB. (Figures  have not  S P I - 5 r e g i o n , although there is evidence that the  genes may be transcribed f r o m a single promoter (159). sigD/sopB  S i m i l a r studies  the downstream pipB  gene was  more  h i g h l y induced than  A s w e l l , the genes were not induced to the same extent w i t h i n 18  and  19),  with  the  expression  o f pipB  continuing to  intramacrophage environment, w h i l e the expression o f sigD/sopB suggests that pipB was transcribed f r o m a separate promoter. promoter was that the insertion w i t h i n the pipB S. typhimurium,  escalate  was  macrophages within  the  leveled o f f over time. T h i s  Further support for a second  gene greatly attenuated virulence o f the  w h i l e the insertion w i t h i n the upstream sigD/sopB gene had o n l y a marginal  effect on v i r u l e n c e . T h e fact that the downstream gene (pipB) had a greater effect o n virulence than the upstream gene (sigD/sopB)  l i k e w i s e argues that the insertion o f the reporter gene  cassette d i d not cause a downstream polar effect o n gene e x p r e s s i o n , but that the attenuation resulted directly f r o m inactivation o f the genes containing the insertions. W i t h i n cultured cell m o d e l s , previous findings (159) indicated that insertions w i t h i n either sigD or sigE resulted i n a ten-fold reduction i n invasion o f epithelial cells w h e n compared to w i l d type bacteria. In the assays described here, i n v a s i o n was not significantly reduced by the insertions w i t h i n either the sigD/sopB or pipB gene as compared to the parental strain. cause o f these differences is difficult to e x p l a i n , although different S. typhimurium and different cell lines were used here.  It is interesting to note that others have  The  isolates reported  variability in i n v a s i o n efficiency o f bacterial mutants depending on the point o f mutation w i t h i n a gene. H e n s e l et al. (152) reported that o n l y one out o f three insertions w i t h i n the gene ssaV (SPI-2) resulted i n a 10 f o l d reduction i n bacterial i n v a s i o n , w h i l e t w o other single insertions w i t h i n this gene d i d not affect the level o f invasiveness.  F u r t h e r m o r e , it was f o u n d that  Chapter 5 111 mutations within the S. dublin  gene homologues did not affect invasion (101, 338,  339).  However, when the sigD/sopB mutant described here was grown overnight and subcultured in L B , shaking at 37°C, and then allowed to invade cells in the absence of serum, there was a 30% reduction in invasion (298). (Note that there was no reduction in invasion by the other mutants (ssaR, pipB, iicA) in the absence of serum.)  Future experiments with deletions of  these genes may help determine their ultimate effect on bacterial invasion and virulence. The insertions within the sigD/sopB S. typhimurium  and pipB  genes resulted in attenuation of  in the typhoid mouse model, but did not totally abolish virulence of the  bacteria as did the insertion within ssaR.  The effect of the insertion within the pipB gene  appeared to delay the onset of the disease allowing the infected mice to live longer, irrespective of dose (although the largest delay was seen with the LD50 dose).  In S. dublin  studies by  Galyov et al. (1997) and Wood et al. (1998), mutations within the SPI-5 region reduced intestinal secretion and inflammatory responses in a calf ileal loop model (101,  338).  However, in a mouse model, the S. dublin mutants were recovered from organs in the same numbers as the wild-type strain (338). Their conclusion was that mutations in SPI-5-encoded genes affect the enteropathogenicity  of Salmonella,  development of systemic disease in mice.  but have no major effect on the  However the study here suggests there was a  difference seen in the development of systemic disease, although the difference was only noticed when lower doses of bacteria were used for inoculation. In the previous S. dublin study, only one dose was reportedly tested on the mice and it could be that at that dose, the mouse immune response was overwhelmed such that no significant difference was seen when comparing the mutant to the wild-type bacteria. 5.2.3.  G7H1  (iicA)  The mutant G7H1 contained an insertion within the previously undescribed region of the S. typhimurium  chromosome which appeared to encode an open reading frame, and was  thus named iicA for induced jntracellularly. This gene fusion displayed upregulated reporter activity by intracellular Salmonella, within both macrophage and epithelial cells. However, the  Chapter 5 112 insertion had no effect on i n v a s i o n or s u r v i v a l o f the bacteria w i t h i n c e l l s , nor d i d it appear to attenuate bacterial virulence i n the mouse m o d e l . N o significant similarity to any other k n o w n gene was f o u n d to this r e g i o n , based on a search o f the available D N A databases, and the function o f the iicA gene and gene product remains to be determined. 5.3.  Overall In  Conclusion  c o n c l u s i o n , four  individual  genes  o f the  S. typhimurium  chromosome  were  identified that were upregulated in response to the intracellular environment o f mammalian cells.  T h e screen described in this report a l l o w e d for the identification o f specific virulence  factors,  indicating that genes induced inside mammalian cells often  Salmonella pathogenesis.  play a key role in  T h r e e o f the four genes (ssaR, sigD/sopB, and pipB) identified were  virulence factors, i n a s m u c h as insertional inactivation o f these genes decreased mouse mortality in an a n i m a l m o d e l . T h e fourth gene, iicA, was not found to reduce mouse mortality, h o w e v e r its role i n virulence has yet to be tested in other animal models, e.g.  calf ileal l o o p m o d e l .  The  four genes identified were tightly regulated by the bacteria, w i t h very little expression from extracellular bacteria.  T h e three genes identified as virulence factors (ssaR, sigD/sopB,  and  pipB) were contained w i t h i n c h r o m o s o m a l regions k n o w n as islands o f pathogenicity ( S P I ) , and at least t w o o f the gene products had p r e v i o u s l y been s h o w n to be i n v o l v e d w i t h a t y p e III secretion system ( S s a R and S i g D / S o p B ) . Pathogenicity islands have been f o u n d i n numerous bacterial pathogens, on their c h r o m o s o m e s and extrachromosomal elements.  These regions  often carry genes e n c o d i n g type III secretions systems and proteins for secretion, as w e l l as other products w h i c h a l l o w the different bacteria to s u r v i v e and cause disease w i t h i n defined niches. T h e further characterization o f these genes and their products w i l l lead to an enhanced understanding about h o w Salmonella functions as an intracellular pathogen. Future experiments w i l l define the regulation o f the various genes and the specific conditions required to induce their e x p r e s s i o n .  F o r example, both sigD/sopB  and pipB  were  contained w i t h i n S P I - 5 and both were induced w i t h i n cells; h o w e v e r their expression levels varied over time indicating that they are expressed under separate and unique  promoters.  Chapter 5 113 Specific deletion mutations w i l l help define w h i c h regions o f the genes are important for bacterial v i r u l e n c e , e.g. defined. homology  the regions necessary for secretion o f sigD/sopB  have not  been  F u r t h e r m o r e , the function o f both P i p B and S s a R have o n l y been deduced by to  other  b i o c h e m i c a l l y defined.  D N A sequences  and  the  corresponding  proteins  have  not  been  F i n a l l y , other Salmonella genes may be d i s c o v e r e d u s i n g this assay,  and the techniques m a y be applied to other pathogenic bacteria to isolate virulence factors.  References 114 References  1.  Abshire,  K . Z., and F.  Salmonella  typhimurium  1993. Analysis of proteins synthesized by  C. Neidhardt.  during growth within a host macrophage. J Bacteriol.  175(12):3734-43. 2.  Abshire,  K . Z., and F.  typhimurium 3.  1993. Growth rate paradox of  Neidhardt.  Salmonella  within host macrophages. J Bacteriol. 175(12):3744-8.  Alpuche Aranda,  1992.  C.  C. M . , J. A . Swanson,  Salmonella typhimurium  W . P. Loomis,  a n d S.  I.  Miller.  activates virulence gene transcription within acidified  macrophage phagosomes. Proc Natl Acad Sci USA. 89(21): 10079-83. 4.  Alpuche-Aranda, S.  I.  Miller.  correlates with  C. M . , E . P. Berthiaume,  B. Mock,  J. A . Swanson,  and  1995. 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T h e names o f the corresponding genes are g i v e n b e l o w the start o f the  T h e sequences w h i c h are underlined were actually sequenced i n this study;  n o n - u n d e r l i n e d sequences were obtained from p r e v i o u s l y p u b l i s h e d sequences.  A:  D11H5  ACCESSION  (ssaR) X 9 9 9 4 4 ( S P I - 2 S. typhimurium)  10 20 30 40 50 60 CCTGCAGTAA TCTACCACAT CAGCTAGCGT TGCATATTAA ATGGACAGTT GAAGAGCATG GGACGTCATT AGATGGTGTA GTCGATCGCA ACGTATAATT TACCTGTCAA CTTCTCGTAC ..C..S..N. .L..P..H.. Q..L..A..L ..H..I..K. .W..T..V.. E..E..H..E  (ssaQ) 70 80 90 100 110 120 A G T T C C A T A G C A T T A T T T T T ACATGGCCAA CGGGTTTTTT GCGCAATATA GTCGGAGAGC TCAAGGTATC GTAATAAAAA TGTACCGGTT GCCCAAAAAA CGCGTTATAT CAGCCTCTCG ..F..H..S. . I . . I . . F . . T..W..P..T . . G . . F . . L . .R..N..I.. V . . G . . E . . L 130 140 150 160 170 180 TTTCTGCTGA GCGACAACAG ATTTATCCTG CCCCTCCTGT GGTAGTCCCT GTATATTCAG AAAGACGACT CGCTGTTGTC TAAATAGGAC GGGGAGGACA CCATCAGGGA CATATAAGTC . . S . . A . . E . .R..Q..Q.. I . . Y . . P . . A ..P..P..V. .V..V..P.. V..Y..S..G 190 200 210 220 230 240 GCTGGTGCCA GCTTACATTA ATCGAACTTG AGTCTATCGA AATCGGCATG GGCGTTCGGA CGACCACGGT CGAATGTAAT TAGCTTGAAC TCAGATAGCT TTAGCCGTAC CCGCAAGCCT ..W..C..Q. . L . . T . . L . . I . . E . . L . . E . . S . . I . . E . .I..G..M.. G..V..R..I 250 260 270 280 290 300 T T C A T T G C T T CGGCGACATC AGACTCGGTT TTTTTGCTAT T C A A C T A C C T GGGGGAATCT AAGTAACGAA GCCGCTGTAG TCTGAGCCAA AAAAACGATA AGTTGATGGA CCCCCTTAGA ..H..C..F. .G..D..I.. R..L..G..F . . F . . A . . I . .Q..L..P.. G..G..I..Y 310 320 330 340 350 360 ACGCAAGGGT GTTGCTGACA GAGGATAACA CGATGAAATT TGACGAATTA GTCCAGGATA TGCGTTCCCA CAACGACTGT CTCCTATTGT GCTACTTTAA ACTGCTTAAT CAGGTCCTAT ..A..R..V. . L . . L . . T . . E..D..N..T ..M..K..F. .D..E..L.. V..Q..D..I  Appendix  153  370 380 390 400 410 420 TCGAAACGCT ACTTGCGTCA GGGAGCCCAA TGTCAAAGAG TGACGGAACG TCTTCAGTCG AGCTTTGCGA TGAACGCAGT CCCTCGGGTT ACAGTTTCTC ACTGCCTTGC AGAAGTCAGC ..E..T..L.  .L..A..S..  .D..G..T..  S..S..V..E  430 440 450 460 470 AACTTGAGCA GATACCACAA CAGGTGCTCT TTGAGGTCGG ACGTGCGAGT TTGAACTCGT CTATGGTGTT GTCCACGAGA AACTCCAGCC TGCACGCTCA  480 CTGGAAATTG GACCTTTAAC  ..L..E..Q.  L..E..I..G  .I..P..Q..  G..S..P..M  Q..V..L..F  ..S..K..S.  ..E..V..G.  .R..A..S..  490 500 510 520 530 540 GACAATTACG A C A A C T T A A A ACGGGGGACG TTTTGCCTGT AGGTGGATGT TTTGCGCCAG CTGTTAATGC TGTTGAATTT TGCCCCCTGC AAAACGGACA T C C A C C T A C A AAACGCGGTC ..Q..L..R.  .Q..L..K..  T..G..D..V  ..L..P..V.  .G..G..C.  F..A..P..E  550 560 570 580 590 600 AGGTGACGAT AAGAGTAAAT GACCGTATTA TTGGGCAAGG TGAGTTGATT GCCTGTGGCA TCCACTGCTA T T C T C A T T T A CTGGCATAAT AACCCGTTCC A C T C A A C T A A CGGACACCGT ..V..T..I.  .R..V..N..  D..R..I..I  ..G..Q..G.  .E..L..I..  A..C..G..N  610 620 630 640 650 660 A T G A A T T T A T GGTGCGTATT ACACGTTGGT ATCTTTGCAA AAATACAGCG TAAACCTGAT T A C T T A A A T A CCACGCATAA TGTGCAACCA TAGAAACGTT TTTATGTCGC ATTTGGACTA ..E..F..M.  . V . . R . . I . . T..R..W..Y  ..L..C..K.  .N..T..A..  *  710 720 670 680 690 700 AAGAAAAATA ATATGCGAAC A A T A T A A T A G CGTTCCAGGT CGTGTCATGA GAGATACAGT T T C T T T T T A T TATACGCTTG TTATATTATC GCAAGGTCCA GCACAGTACT CTCTATGTCA 730 740 750 760 770 780 ATGTCTTTAC CCGATTCGCC TTTGCAACTG ATTGGTATAT TGTTTCTGCT TTCAATACTG TACAGAAATG GGCTAAGCGG AAACGTTGAC TAACCATATA ACAAAGACGA AAGTTATGAC M..S..L..P  ..D..S..P.  .L..Q..L..  I..G..I..L  ..F..L..L.  .S..I..L..  {ssaR) C C T C T C A T T A TCGTCATGGG A A C T T C T T T C CTTAAACTGG CGGTGGTATT TTCGATTTTA GGAGAGTAAT AGCAGTACCC TTGAAGAAAG GAATTTGACC GCCACCATAA AAGCTAAAAT P..L..I..I  ..V..M..G.  .T..S..F..  L..K..L..A  ..V..V..F.  .S..I..L..  850 860 870 880 890 900 CGAAATGCTC TGGGTATTCA ACAAGTCCCC CCAAATATCG CACTGTATGG CCTTGCGCTT GCTTTACGAG ACCCATAAGT TGTTCAGGGG GGTTTATAGC GTGACATACC GGAACGCGAA R..N..A..L  ..G..I..Q.  .Q..V..P..  P..N..I..A  ..L..Y..G.  .L..A..L..  910 920 930 940 950 960 GTACTTTCCT T A T T C A T T A T GGGGCCGACG CTATTAGCTG TAAAAGAGCG CTGGCATCCG CATGAAAGGA A T A A G T A A T A CCCCGGCTGC GATAATCGAC ATTTTCTCGC GACCGTAGGC V..L..S..L  ..F..I..M.  .G..P..T..  L..L..A..V  ..K..E..R.  .W..H..P..  Appendix 970  980  990  1000  1010  154  1020  GTTCAGGTCG CTGGCGCTCC TTTCTGGACG T G A G T G G G ACAGTAAAGC ATTAGCGCCT CAAGTCCAGC GACCGCGAGG AAAGACCTGC A C T C A C C C TGTCATTTCG TAATCGCGGA V..Q..V..A ..G..A..P. .F..W..T.. S..E..W..D ..S..K..A. .L..A..P.. C T  G A  1030 1040 1050 1060 1070 1080 TATCGACAGT T T T T G C A A A A AAACTCTGAA GAGAAGGAAG C C A A T T A T T T TCGGAATTTG ATAGCTGTCA A A A A C G T T T T TTTGAGACTT CTCTTCCTTC GGTTAATAAA AGCCTTAAAC Y..R..Q..F ..L..Q..K. .N..S..E.. E..K..E..A ..N..Y..F. .R..N..L.. 1090 1100 1110 1120 1130 1140 ATAAAACGAA CCTGGCCTGA AGACATAAAA AGAAAGATAA AACCTGATTC TTTGCTCATA TATTTTGCTT GGACCGGACT T C T G T A T T T T T C T T T C T A T T TTGGACTAAG AAACGAGTAT I..K..R..T ..W..P..E. .D..I..K.. R..K..I..K ..P..D..S. . L . . L . . I . . 1150 1160 1170 1180 1190 1200 TTAATTCCGG CATTTACGGT GAGTCAGTTA ACGCAGGCAT TTCGGATTGG A T T A C T T A T T AATTAAGGCC GTAAATGCCA CTCAGTCAAT TGCGTCCGTA AAGCCTAACC TAATGAATAA L . . I . . P . . A ..F..T..V. .S..Q..L.. T..Q..A..F ..R..I..G. . L . . L . . I . . 1210 1220 1230 1240 1250 . 1260 T A T C T T C C C T TTCTGGCTAT TGACCTGCTT A T T T C A A A T A TACTGCTGGC TATGGGGATG ATAGAAGGGA AAGACCGATA ACTGGACGAA TAAAGTTTAT ATGACGACCG ATACCCCTAC Y..L..P..F . . L . . A . . I . .D..L..L.. I..S..N..I ..L..L..A. .M..G..M.. 1270 1280 1290 1300 1310 1320 ATGATGGTGT CGCCGATGAC C A T T T C A T T A CCGTTTAAGC TGCTAATATT TTTACTGGCA T A C T A C C A C A GCGGCTACTG GTAAAGTAAT GGCAAATTCG A C G A T T A T A A AAATGACCGT M..M..V..S ..P..M..T. . I . . S . . L . . P..F..K..L . . L . . I . . F . .L..L..A.. 1330 1340 1350 1360 1370 1380 GGCGGTTGGG A T C T G A C A C T GGCGCAATTG GTACAGAGCT TTTCATGAAT GATTCTGAAT CCGCCAACCC TAGACTGTGA CCGCGTTAAC CATGTCTCGA A A A G T A C T T A CTAAGACTTA G..G..W..D . . L . . T . . L . .A..Q..L.. V..Q..S..F ..S..* M..N.. D..S..E..L  (ssaS)  1390 1400 1410 1420 1430 1440 TGACGCAATT TGTAACGCAA CTTTTATGGA TCGTCCTTTT TACGTCTATG CCGGTAGTGT ACTGCGTTAA ACATTGCGTT GAAAATACCT AGCAGGAAAA ATGCAGATAC GGCCATCACA ..T..Q..F.  .V..T..Q.. L..L..W..I  ..V..L..F.  .T..S..M.. P..V..V..L  1450 1460 1470 1480 1490 1500 TGGTGGCATC GGTAGTTGGT GTCATCGTAA GCCTTGTTCA GGCCTTGACT CAAATACAGG ACCACCGTAG C C A T C A A C C A CAGTAGCATT CGGAACAAGT CCGGAACTGA GTTTATGTCC ..V..A..S.  .V..V..G.. V..I..V..S  ..L..V..Q.  .A..L..T.. Q..I..Q..D  Appendix  B.  A1A1 (sigD/sopB)  155  a n d E12A2 ipipB)  A C C E S S I O N A F 0 2 1 8 1 7 (sigDE  S.  typhimurium)  10 20 30 40 50 60 TATCTGTTCA AGCATGGAAT AGGAAAAACG A A T A T T C T T C GTCACGGTCT TACTTGTCCG ATAGACAAGT TCGTACCTTA TCCTTTTTGC TTATAAGAAG CAGTGCCAGA ATGAACAGGC 70 80 90 100 110 120 GGGCTTTGCT GGCATACACA CACCTGTATA ACATTTGATG TAACGCCGTT ACTTTACGCA CCCGAAACGA CCGTATGTGT GTGGACATAT TGTAAACTAC ATTGCGGCAA TGAAATGCGT 130 140 150 160 170 180 GGAGTAAATC GGTGAATTTG ATCTGAGTCA AGAAGGTGGG T T T T C A A T A A AAGTTGTGCC CCTCATTTAG CCACTTAAAC TAGACTCAGT TCTTCCACCC A A A A G T T A T T TTCAACACGG 190 200 210 220 230 240 ATAAATTGTG AAGTTTGTAG A T T T T A T G A A CATTTGATGT ACCGATCTCC CCCATGATCG TATTTAACAC TTCAAACATC T A A A A T A C T T GTAAACTACA TGGCTAGAGG GGGTACTAGC 250 260 270 280 290 300 CCACTACGCT ATGGACGTCA GGATGCCTCC CCGCCTGATC AGAAGCGTTT CCTCATTAAA GGTGATGCGA TACCTGCAGT CCTACGGAGG GGCGGACTAG TCTTCGCAAA GGAGTAATTT 310 320 330 340 350 360 AAGGACATTT TTTTAAAGTT CCTGGTGCAT AAAAGTCACA T C C T T T T A A A GGGTTGTTAA TTCCTGTAAA A A A A T T T C A A GGACCACGTA TTTTCAGTGT AGGAAAATTT CCCAACAATT 370 380 390 400 410 420 CCCTGTTGAA TGTTCCCACT CCCCTATTCA GGAATATTAA AAACGCTATG CAAATACAGA GGGACAACTT ACAAGGGTGA GGGGATAAGT CCTTATAATT TTTGCGATAC GTTTATGTCT M.. Q..1..Q..S (sigD/sopB) 430 440 450 460 470 480 GCTTCTATCA CTCAGCTTCA CTAAAAACCC AGGAGGCTTT TAAAAGCCTA CAAAAAACCT CGAAGATAGT GAGTCGAAGT GATTTTTGGG TCCTCCGAAA ATTTTCGGAT GTTTTTTGGA ..F..Y..H. .S..A..S.. L..K..T..Q ..E..A..F. .K..S..L.. Q..K..T..L 490 500 510 520 530 540 TATACAACGG AATGCAGATT CTCTCAGGCC AGGGCAAAGC GCCGGCTAAA GCGCCCGACG ATATGTTGCC TTACGTCTAA GAGAGTCCGG TCCCGTTTCG CGGCCGATTT CGCGGGCTGC ..Y..N..G. .M..Q..I.. L..S..G..Q ..G..K..A. .P..A..K.. A..P..D..A  580 590 600 550 560 570 CTCGCCCGGA AATTATTGTC CTGCGAGAAC CCGGCGCGAC ATGGGGGAAT TATCTACAGC GAGCGGGCCT TTAATAACAG GACGCTCTTG GGCCGCGCTG TACCCCCTTA ATAGATGTCG ..R..P..E. . I . . I . . V . . L..R..E..P ..G..A..T. .W..G..N.. Y..L..Q..H  Appendix  156  610 620 630 640 650 660 ATCAGAAGGC GTCTAACCAC TCGCTGCATA A C C T C T A T A A CTTACAGCGC GATCTTCTTA TAGTCTTCCG CAGATTGGTG AGCGACGTAT TGGAGATATT GAATGTCGCG CTAGAAGAAT ..Q..K..A.  .S..N..H..  S..L..H..N  ..L..Y..N.  .L..Q..R..  D..L..L..T  670 680 690 700 710 720 C T C G C G G C AACCGTTCTG GGTAAACAAG ACCCGGTTCT AACGTCAATG GCAAACCAAA G A G C G C C G TTGGCAAGAC CCATTTGTTC TGGGCCAAGA TTGCAGTTAC CGTTTGGTTT C G  G C  ..V..A..A...T..V..L...G..K..Q..D...P..V..L...T..S..M...A..N..Q..M  730 740 750 760 770 780 TGGAGTTAGC CAAAGTTAAA GCGGACCGGC CAGCAACAAA ACAAGAAGAA GCCGCGGCAA A C C T C A A T C G GTTTCAATTT CGCCTGGCCG GTCGTTGTTT TGTTCTTCTT CGGCGCCGTT ..E..L..A.  .K..V..K..  A..D..R..P  ..A..T..K.  .Q..E..E..  A..A..A..K  790 800 810 820 830 840 AAGCATTGAA GAAAAATCTT ATCGAACTTA TTGCAGCACG CACTCAGCAG CAGGATGGCT TTCGTAACTT CTTTTTAGAA TAGCTTGAAT AACGTCGTGC GTGAGTCGTC GTCCTACCGA ..A..L..K.  .K..N..L..  I..E..L..I  ..A..A..R.  .T..Q..Q..  Q..D..G..L  850 860 870 880 890 900 TACCTGCAAA AGAAGCTCAT CGCTTTGCGG CAGTAGCGTT TAGAGATGCT CAGGTCAAGC ATGGACGTTT TCTTCGAGTA GCGAAACGCC GTCATCGCAA A T C T C T A C G A GTCCAGTTCG ..P..A..K.  .E..A..H.. R..F..A..A  ..V..A..F.  .R..D..A..  Q..V..K..Q  910 920 930 940 950 960 A G C T T A A T A A CCAGCCCTGG CAAACCATAA A A A A T A C A C T CAC GCATAAC GGGCATCACT TCGAATTATT GGTCGGGACC GTTTGGTATT TTTTATGTGA GTGCGTATTG CCCGTAGTGA ..L..N..N.  .Q..P..W..  Q..T..I..K  ..N..T..L.  .T..H..N..  G..H..H..Y  970 980 990 1000 1010 1020 ATACCAACAC GCAGCTCCCT GCAGCAGAGA TGAAAATCGG CGCAAAAGAT A T C T T T C C C A TATGGTTGTG CGTCGAGGGA CGTCGTCTCT ACTTTTAGCC GCGTTTTCTA TAGAAAGGGT ..T..N..T.  .Q..L..P.. A..A..E..M  ..K..I..G.  .A..K..D..  I..F..P..S  1030 1040 1050 1060 1070 1080 GTGCTTATGA GGGAAAGGGC GTATGCAGTT GGGATACCAA GAATATTCAT CACGCCAATA CACGAATACT CCCTTTCCCG CATACGTCAA CCCTATGGTT CTTATAAGTA GTGCGGTTAT ..A..Y..E.  .G..K..G..  V..C..S..W  ..D..T..K.  .N..I..H..  H..A..N..N  1090 1100 1110 1120 1130 1140 ATTTGTGGAT GTCCACGGTG AGTGTGCATG AGGACGGTAA AGATAAAACG CTTTTTTTTG T A A A C A C C T A CAGGTGCCAC TCACACGTAC TCCTGCCATT TCTATTTTGC GAAAAAAAAC ..L..W..M.  .S..T..V..  S..V..H..E  ..D..G..K.  .D..K..T..  L..F..F..D  1150 1160 1170 1180 1190 1200 ACGGGATACG TCATGGCGTG CTTTCCCCCT ATCATGAAAA AGATCCGCTT CTGCGTCACG TGCCCTATGC AGTACCGCAC GAAAGGGGGA TAGTACTTTT TCTAGGCGAA GACGCAGTGC ..G..I..R.  .H..G..V..  L..S..P..Y  ..H..E..K.  .D..P..L.. L..R..H..V  A p p e n d i x 157  1210 1220 1230 1240 1250 1260 TCGGCGCTGA AAACAAAGCC AAAGAAGTAT TAACTGCGGC ACTTTTTAGT AAACCTGAGT AGCCGCGACT TTTGTTTCGG T T T C T T C A T A ATTGACGCCG TGAAAAATCA TTTGGACTCA ..G..A..E.  .N..K..A..  K..E..V..L  ..T..A..A.  .L..F..S..  K..P..E..L  1270 1280 1290 1300 1310 1320 TGCTTAACAA AGCCTTAGCG GGCGAGGCGG TAAGCCTGAA ACTGGTATCC GTCGGGTTAC ACGAATTGTT TCGGAATCGC CCGCTCCGCC ATTCGGACTT TGACCATAGG CAGCCCAATG ..L..N..K.  .A..L..A.. G..E..A..V  ..S..L..K.  .L..V..S..  V..G..L..L  1330 1340 1350 1360 1370 1380 TCACCGCGTC GAATATTTTC GGCAAAGAGG GAACGATGGT CGAGGACCAA ATGCGCGCAT AGTGGCGCAG CTTATAAAAG CCGTTTCTCC CTTGCTACCA GCTCCTGGTT TACGCGCGTA ..T..A..S.  .N..I..F..  G..K..E..G  ..T..M..V.  .E..D..Q..  M..R..A..W  1390 1400 1410 1420 1430 1440 GGCAATCGTT GACCCAGCCG GGAAAAATGA T T C A T T T A A A AATCCGCAAT AAAGATGGCG CCGTTAGCAA CTGGGTCGGC CCTTTTTACT AAGTAAATTT TTAGGCGTTA TTTCTACCGC ..Q..S..L.  .T..Q..P..  G..K..M..I  ..H..L..K.  .I..R..N..  K..D..G..D  1450 1460 1470 1480 1490 1500 ATCTACAGAC GGTAAAAATA AAACCGGACG TCGTCGCCGC ATTTAATGTG GGTGTTAATG TAGATGTCTG C C A T T T T T A T TTTGGCCTGC AGCAGCGGCG TAAATTACAC CCACAATTAC ..L..Q..T.  .V..K..I..  K..P..D..V  ..V..A..A.  .F..N..V..  G..V..N..E  1510 1520 1530 1540 1550 1560 AGCTGGCGCT CAAGCTCGGC TTTGGCCTTA AGGCATCGGA TAGCTATAAT GCCGAGGCGC TCGACCGCGA GTTCGAGCCG AAACCGGAAT TCCGTAGCCT A T C G A T A T T A CGGCTCCGCG ..L..A..L.  .K..L..G.. F..G..L..K  ..A..S..D.  .S..Y..N..  A..E..A..L  1570 1580 1590 1600 1610 1620 TACATCAGTT ATTAGGCAAT GATTTACGCC CTGAAGCCAG ACCAGGTGGC TGGGTTGGCG ATGTAGTCAA TAATCCGTTA CTAAATGCGG GACTTCGGTC TGGTCCACCG ACCCAACCGC ..H..Q..L.  .L..G..N..  D..L..R..P  ..E..A..R.  .P..G..G..  W..V..G..E  1630 1640 1650 1660 1670 1680 AATGGCTGGC GCAATACCCG GATAATTATG AGGTCGTCAA TACATTAGCG CGCCAGATTA TTACCGACCG CGTTATGGGC CTATTAATAC TCCAGCAGTT ATGTAATCGC GCGGTCTAAT ..W..L..A.  .Q..Y..P..  D..N..Y..E  ..V..V..N.  .T..L..A..  R..Q..I..K  1690 1700 1710 1720 1730 1740 AGGATATATG GAAAAATAAC CAACATCATA AAGATGGCGG CGAACCCTAT AAACTCGCAC TCCTATATAC C T T T T T A T T G GTTGTAGTAT TTCTACCGCC GCTTGGGATA TTTGAGCGTG ..D..I..W.  .K..N..N..  Q..H..H..K  ..D..G..G.  .E..P..Y.. K..L..A..Q  1750 1760 1770 1780 1790 1800 AACGCCTTGC CATGTTAGCC CATGAAATTG ACGCGGTACC CGCCTGGAAT TGTAAAAGCG TTGCGGAACG GTACAATCGG GTACTTTAAC TGCGCCATGG GCGGACCTTA ACATTTTCGC ..R..L..A.  .M..L..A..  H..E..I..D  ..A..V..P.  .A..W..N..  C..K..S..G  Appendix  158  1810 1820 1830 1840 1850 1860 GCAAAGATCG TACAGGGATG ATGGATTCAG AAATCAAGGG AGAGATCATT TCCTTACATC CGTTTCTAGC ATGTCCCTAC TACCTAAGTC TTTAGTTCCC TCTCTAGTAA AGGAATGTAG ..K..D..R.  .T..G..M..  M..D..S..E  ..I..K..G.  .E..I..I..  S..L..H..Q  1870 1880 1890 1900 1910 1920 AGACCCATAT GTTAAGTGCC CCTGGTAGTC TTCCGGATAG CGGTGGACAG AAAATTTTCC TCTGGGTATA CAATTCACGG GGACCATCAG AAGGCCTATC GCCACCTGTC TTTTAAAAGG ..T..H..M.  .L..S..A..  P..G..S..L  ..P..D..S.  .G..G..Q.. K..-I..F..Q  1930 1940 1950 1960 1970 1980 AAAAAGTATT ACTGAATAGC GGTAACCTGG AGATTCAGAA A C A A A A T A C G GGCGGGGCGG T T T T T C A T A A TGACTTATCG CCATTGGACC TCTAAGTCTT TGTTTTATGC CCGCCCCGCC ..K..V..L.  .L..N..S..  G..N..L..E  ..I..Q..K.  .Q..N..T..  G..G..A..G  1990 2000 2010 2020 2030 2040 GAAACAAAGT AATGAAAAAT TTATCGCCAG AGGTGCTCAA TCTTTCCTAT CAAAAACGAG CTTTGTTTCA T T A C T T T T T A AATAGCGGTC TCCACGAGTT AGAAAGGATA GTTTTTGCTC ..N..K..V.  .M..K..N..  L..S..P..E  ..V..L..N.  .L..S..Y..  Q..K..R..V  2050 2060 2070 2080 2090 2100 TTGGGGATGA AAATATTTGG CAGTCAGTAA AAGGCATTTC TTCATTAATC ACATCTTGAG A A C C C C T A C T TTTATAAACC GTCAGTCATT TTCCGTAAAG AAGTAATTAG TGTAGAACTC ..G..D..E.  .N..I..W..  Q..S..V..K  ..G..I..S.  .S..L..I..  2110 2120 2130 ' 2140 2150 TCTTGAGGTA ACTATATGGA AAGTCTATTA AATCGTTTAT ATGACGCGTT AGAACTCCAT TGATATACCT TTCAGATAAT TTAGCAAATA TACTGCGCAA M..E.  .S..L..L..  N..R..L..Y  ..D..A..L.  T..S..*  2160 AGGCCTGGAT TCCGGACCTA .G..L..D..  (sigE) 2170 2180 2190 2200 2210 2220 GCGCCAGAAG ATGAGCCACT GCTTATCATT GATGATGGGA TACAGGTTTA TTTTAATGAA CGCGGTCTTC TACTCGGTGA CGAATAGTAA CTACTACCCT ATGTCCAAAT A A A A T T A C T T A..P..E..D  ..Q..V..Y.  .F..N..E..  2230 2240 2250 2260 2270 TCCGATCATA CACTGGAAAT GTGCTGTCCC TTTATGCCAT TGCCTGACGA AGGCTAGTAT GTGACCTTTA CACGACAGGG AAATACGGTA ACGGACTGCT  2280 CATCCTGACT GTAGGACTGA  S..D..H..T  ..E..P..L.  ..L..E..M.  .L..I..I..  .C.C.P..  D..D..G..I  F..M..P..L  ..P..D..D.  .I..L..T..  2290 2300 2310 2320 2330 2340 TTGCAGCATT TTTTACGTCT GAACTACACC AGCGCCGTCA CTATCGGCGC TGACGCAGAC AACGTCGTAA AAAATGCAGA CTTGATGTGG TCGCGGCAGT GATAGCCGCG ACTGCGTCTG L..Q..H..F  ..L..R..L.  .N..Y..T..  S..A..V..T  ..I..G..A.  .D..A..D..  2350 2360 2370 2380 2390 2400 AATACTGCTT TAGTGGCGCT TTATCGCTTG CCGCAAACCA GTACCGAAGA AGAGGCGCTC TTATGACGAA ATCACCGCGA AATAGCGAAC GGCGTTTGGT CATGGCTTCT TCTCCGCGAG N..T..A..L  ..V..A..L.  .Y..R..L..  P..Q..T..S  ..T..E..E.  .E..A..L..  Appendix  159  2440 2450 2460 2410 2420 2430 ACTGGTTTTG A A T T A T T C A T TTCAAACGTG AAGCAATTGA AAGAGCATTA TGCATAATTT TGACCAAAAC TTAATAAGTA AAGTTTGCAC TTCGTTAACT TTCTCGTAAT ACGTATTAAA T..G..F..E  ..L..F..I.  .S..N..V..  K..Q..L..K  ..E..H..Y.  .A..*  2500 2510 2520 2470 2480 2490 AATACGTCAA C A T A C T T T C T TAATGAGATA AAACGCGATA CGTATGCCCT TTACAAGAGA TTATGCAGTT GTATGAAAGA A T T A C T C T A T TTTGCGCTAT GCATACGGGA AATGTTCTCT 2560 2570 2580 2530 2540 \ 2550 CAAGACCAGA ATCTTTGGTG GAAATGTAAG GGGCAAACGT TCATCTCTCT CATTTTGCTC GTTCTGGTCT TAGAAACCAC CTTTACATTC CCCGTTTGCA AGTAGAGAGA GTAAAACGAG 2620 2630 2640 2590 2600 2610 TGTTTGCGGG A G C A T T T T T A GTGTGTAAGT ATTCCTGCTC ATCAGGTTTT TACGCCATCA ACAAACGCCC TCGTAAAAAT CACACATTCA TAAGGACGAG TAGTCCAAAA ATGCGGTAGT 2680 2690 2700 2650 2660 2670 CGCGCATTTA TTCTGGTATA AGTTGAAATA CTGCAAAAAA TATTGGTGCT TATTATTTTT GCGCGTAAAT AAGACCATAT T C A A C T T T A T GACGTTTTTT A T A A C C A C G A A T A A T A A A A A 2740 2750 2760 2710 2720 2730 TCTTTAAGTA AATTTTCGCT GAACAAACTT AATTGTTTAT TCAATGATGA TGAAGCGTAA AGAAATTCAT TTAAAAGCGA CTTGTTTGAA TTAACAAATA AGTTACTACT ACTTCGCATT 2800 2810 2820 2770 2780 2790 GCTATGCTGG AAATGAAGGA ATCAATAGCA AGGATAATCT T A T T A T T C A C GGGTGATATT CGATACGACC TTTACTTCCT TAGTTATCGT TCCTATTAGA ATAATAAGTG CCCACTATAA 2830 2840 2850 2860 2870 2880 ACTTCTGCTT CACCGTTATG GCAGATATCA TCGCCTCTTG TCAGATGCCA GACACCTACT TGAAGACGAA GTGGCAATAC CGTCTATAGT AGCGGAGAAC AGTCTACGGT CTGTGGATGA 2890 2900 2910 2920 2930 2940 CATACTCAAC CAAAGCTCTA A A T A C A A A A A TCACCTTATA T C T T T T T T T A TTATTCCTTG GTATGAGTTG GTTTCGAGAT TTATGTTTTT AGTGGAATAT AGAAAAAAAT AATAAGGAAC 2950 2960 2970 2980 2990 3000 TATAAATGTG A C T T G A C T C A CACCTATAAG GAGTCGGCTC A C T T C C A T A A GAAGGAATCA A T A T T T A C A C TGAACTGAGT GTGGATATTC CTCAGCCGAG TGAAGGTATT CTTCCTTAGT 3010 3020 3030 3040 3050 3060 AAATGCCAAT AACTAACGCG TCCCCAGAAA A T A T A T T A A G ATATTTGCAT GCGGCCGGTA TTTACGGTTA TTGATTGCGC AGGGGTCTTT TATATAATTC TATAAACGTA CGCCGGCCAT M..P..I.  .T..N..A..  S..P..E..N  ..I..L..R.  .Y..L..H..  A..A..G..T  {pipB) 3070 3080 3090 3100 3110 3120 CCGGTACGAA AGAAGCAATG AAAAGTGCAA CTTCACCACG CGGTATACTG GAATGGTTTG GGCCATGCTT TCTTCGTTAC TTTTCACGTT GAAGTGGTGC GCCATATGAC CTTACCAAAC ..G..T..K.  .E..A..M..  K..S..A..T  ..S..P..R.  .G..I..L..  E..W..F..V  Appendix 3130  3140  TCAATTTTTT  TACCTGTGGT  AGTTAAAAAA  ATGGACACCA  ..N..F..F.  .T..C..G..  3190  3200  TTGGAAAACT  GACCACATCA  AACCTTTTGA  CTGGTGTAGT  ..G..K..L.  .T..T..S..  3250  3260  3150  3160  GGAGTAAGAA GAAGCAATGA CCTCATTCTT CTTCGTTACT  G..V..R..R  ..S..N..E.  3170  160  3180  AAGATGGTTT  CGGGAGGTAA  TTCTACCAAA  GCCCTCCATT  .R..W..F.. R..E..V..I  3220 3230 3240 T A A A T A A A A A T G C T T T C T T C G A T G G T A ATA TTATTATATG ACGAAAGAAG CTACCATTAT A A T A A T A T A C ATTTATTTTT L..L..Y..V . . N. .K. .N. . .A. . F . . F . . D..G..N..K 3210  3280  3290  3300  A A A T A T T T C T GGAGGATGTC TTTATAAAGA CCTCCTACAG . . I . . F . . L . .E..D..V..  AACGGGTGTT C T A T A T C T T T G C C C A C A A GATATAgjvGA N..G..C..S . . I . . C T 7 L .  GTCATGTGGA CAGTACACCT  GCAGCATCCG CGTCGTAGGC  .S..C..G..  A..A..S..E  3310 3320 AAAATACGGA TCCCATGGTC TTTTATGCCT AGGGTACCAG ..N..T..D. .P..M..V..  3340 3350 3360 3330 ATTATTGAAG TGAACAAAAA TGGAAAAACT GTAACGGATA CATTGCCTAT TAATAACTTC ACTTGTTTTT ACCTTTTTGA I . . I . . E . . V ..N..K..N. .G..K..T.. V..T..D..K  3270  G T  3400 3410 3420 3370 3380 3390 AAGTTGATAG TGAGAGATTT TGGAATGTAT GTCGAATGTT AAAACTGATG AGTAAACATA TTCAACTATC ACTCTCTAAA ACCTTACATA CAGCTTACAA TTTTGACTAC TCATTTGTAT ..V..D..S. .E..R..F.. W..N..V..C ..R..M..L. .K..L..M.. S..K..H..N 3430 3440 3450 3460 3470 A T A T A C A A C A GCCTGATTCA CTTATAACCG GAGGATGGTT TTCTGAACCT TATATGTTGT CGGACTAAGT GAATATTGGC CTCCTACCAA AAGACTTGGA ..I..Q..Q. .P..D..S.. L . . I . . T . . G ..G..W..F. .S..E..P..  3480 GCGCGGAGTA CGCGCCTCAT A..R..S..K  3490 3500 3510 3520 3530 AACCTGGCTC ATAAAGATTT CCAGGGGGAA GATTTGTCAA AAATAGATGC TTGGACCGAG TATTTCTAAA GGTCCCCCTT CTAAACAGTT TTTATCTACG ..P..G..S. .* ( c o r r e s p o n d i n g pipB gene is continuous i n S. dublin)  3540 TTCTAATGCA AAGATTACGT M..L.  3550 3560 3570 3580 3590 3600 GATTTCCGTG AAACAACTTC TATCTAATGT AAATTTAGTC GGTGCAAATT TGTGTTGTGC CTAAAGGCAC TTTGTTGAAG ATAGATTACA TTTAAATCAG CCACGTTTAA ACACAACACG .L..M..Q.. I..S..V..K ..Q..L..L. .S..N..V.. N..L..V..G ..A..N..L. 3610 AAATCTACAC TTTAGATGTG .C.C..A..  3620 3630 3640 3650 3660 GCTGTAAATC TAATGGGTTC AAACATGACT AAAGCAAACC TGACTCACGC CGACATTTAG ATTACCCAAG TTTGTACTGA TTTCGTTTGG ACTGAGTGCG N..L..H..A ..V..N..L. . M . . G . . S . . N..M..T..K ..A..N..L.  Appendix  161  3710 3720 3690 3700 3670 3680 GCTGCAATTC TATTCGGCTC TGTCCGGTGT A A A C T T A A C C AGACCTGACT TGCGCTAACA TCTGGACTGA ACGCGATTGT ACAGGCCACA TTTGAATTGG CGACGTTAAG ATAAGCCGAG • T . . H . . A . .  D . . L . . T . . C  . . A . . N . . M .  . S . . G . . V . .  N. . L . . T . . A  . .A.  . I . . L .  3760 3770 3780 3730 3740 .' 3 7 5 0 AGACTTAACT GACACCAAAC TAAATGGTGC GAAATTAGAT AAGATAGCTC TAACTTTAGC TCTGAATTGA CTGTGGTTTG ATTTACCACG CTTTAATCTA TTCTATCGAG ATTGAAATCG • F . . G . . S . .  D . . L . . T . . D  . . T . . K . . L .  . N . . G . . A . .  K . . L . . D . . K  . . I . . A . . L .  3790 3800 3810 3820 3830 3840 GAAAGCATTA ACAGGAGCCG ATCTGACAGG TAGTCAACAT ACCCCTACTC CACTCCCGGA CTTTCGTAAT TGTCCTCGGC TAGACTGTCC ATCAGTTGTA TGGGGATGAG GTGAGGGCCT . T . . L . . A . .  K . . A . . L . . T  . . G . . A . . D .  . L . . T . . G . .  S..Q..H..T  . . P . . T . . P .  3850 3860 3870 3880 3890 3900 TTACAATGAT AGAACTCTTT TCCCCCATCC GATATTTTAG TCGAGATAAA GGGATTTTAT A A T G T T A C T A TCTTGAGAAA AGGGGGTAGG CTATAAAATC AGCTCTATTT CCCTAAAATA . L . . P . . D . .  Y . . N . . D . . R  . . T . . L . . F .  3910 3920 AAACAAGAAG T A T T C A A A C A GA TTTGTTCTTC ATAAGTTTGT CT  . P . . H . . P . .  I . . F . . *  Appendix  C.  G7H1  162  (iicA)  A C C E S S I O N # A F 1 6 4 4 3 5 (iicA S. typhimurium) 10 20 30 40 50 60 GAATGCAAAA GAGAAGTTAC TGGATTTTGT GGAGTTAGAA GAAAACGAAT CGCTGATTTT CTTACGTTTT CTCTTCAATG A C C T A A A A C A CCTCAATCTT CTTTTGCTTA GCGACTAAAA E..C..K..R ..E..V..T. .G..F..C. G..V..R..R ..K..R..I. .A..D..F.. ( p o t e n t i a l ORF) 70 80 90 100 110 120 GGTAAAGATA TTCGCATCGT TCTGGCATCA GCGGATTTCA GTAAAGAATT AAC GACAAC C CCATTTCTAT AAGCGTAGCA AGACCGTAGT CGCCTAAAGT C A T T T C T T A A TTGCTGTTGG G..K..D..I ..R..I..V. .L..A..S.. A..D..F..S ..K..E..L. .T..T..T.. 160 170 180 130 140 150 GCAATATGGC TAAGAGATAA AGGTGTCGAT ATTCGCTGTG TTCGCTTAAC GCCTTACAAC CGTTATACCG A T T C T C T A T T TCCACAGCTA TAAGCGACAC AAGCGAATTG CGGAATGTTG A..I..W..L ..R..D..K. .G..V..D.. I..R..C..V ..R..L..T. .P..Y..N.. 190 200 TTTAAGGGTG AAGTGCTGAT AAATTCCCAC TTCACGACTA F..K..G..E ..V..L..I.  220 230 240 210 TAATGCTGAA CAAATAATAC CAGTCCCTGA AC TGGAAGAA ATTACGACTT GTTTATTATG GTCAGGGACT TGACCTTCTT .N..A..E.. Q..I..I..P ..V..P..E. .L..E..E..  250 260 270 280 290 300 TATCAGGTCA GATTCAGAGA GAAACGCACG GAACAAATTA TTAGCAGTCA AAAGTCGGAG ATAGTCCAGT CTAAGTCTCT CTTTGCGTGC CTTGTTTAAT AATCGTCAGT TTTCAGCCTC Y..Q..V..R ..F..R..E. .K..R..T.. E..Q..I..I ..S..S..Q. .K..S..E.. 310 320 330 340 350 360 AGGGATTATT C C T T A T A T A A ATATAAAGGA AAAACCTTCA ATAAACGGAA GCTTGCACTT T C C C T A A T A A GGAATATATT TATATTTCCT TTTTGGAAGT TATTTGCCTT CGAACGTGAA R..D..Y..S ..L..Y..K. .Y..K..G.. K..T..F..N ..K..R..K. .L..A..L.. 370 380 390 . 400 410 420 GAACTTTTCA CTGACTGGAT TAATAAACAT AATCCTGCGA ATATAGATGA TCTCAAGAAT CTTGAAAAGT GACTGACCTA ATTATTTGTA TTAGGACGCT TATATCTACT AGAGTTCTTA E..L..F..T ..D..W..1. •N..K..H.. N..P..A..N ..I..D..D. .L..K..N.. 430 440 450 460 470 480 AAATTGAGTG A A G A C T T A C A GAAAAGAACA GTAGCACTGG TTGAGCAGAT CCCTGAAAAA TTTAACTCAC TTCTGAATGT CTTTTCTTGT CATCGTGACC AACTCGTCTA GGGACTTTTT K..L..S..E ..D..L..Q. .K..R..T.. V..A..L..V ..E..Q..I. .P..E..K.. 490 500 510 520 530 540 AGGAAAAACA GATATCATAT GCAGGAAGAT GCGTTGATTG AGTTGCCGTC CGGTGAGCGT TCCTTTTTGT CTATAGTATA CGTCCTTCTA CGCAACTAAC TCAACGGCAG GCCACTCGCA R..K..N..R ..Y..H..M. .Q..E..D.. A..L..I..E ..L..P..S. .G..E..R..  Appendix 590  163 600  550  560  570  580  ATTGCTATAT  CGATAAATGG  GGGTTAGGGA  CTATAGAACT  GCTTATATGA  TTTTGTTCGG  TAACGATATA  GCTATTTACC  CCCAATCCCT  GATATCTTGA  CGAATATACT  AAAACAAGCC  I . . A . . I . . S  . . I . . N . . G .  640  610  .G.  .* 630  620  TCAGGATAAT  TTTGTAGTTG AAAAAGTAGG  AGTCCTATTA  AAACATCAAC  TTTTTCATCC 690  650  660  TTGACAGGAA GTAATAATAA  AATAGATCCC  CATTATTATT  TTATCTAGGG  AACTGTCCTT  670  680  700  710  720  ATTCATTAAT  GGGATCTCAC  GTTTCATCCG  ATACGAAGAC  CATGGTCTCT  TTGTCAGTAG  TAAGTAATTA  CCCTAGAGTG  CAAAGTAGGC  TATGCTTCTG  GTACCAGAGA  AACAGTCATC  730  740  760  770  780  CGTCATAATT  ACGCAAGCCT  CTTTACTTTG  CTTATCATTT  ATATTTAATG  TAAATATTCA  GCAGTATTAA  TGCGTTCGGA  GAAATGAAAC  GAATAGTAAA  TATAAATTAC  ATTTATAAGT  790  750  800  810  820  830  840  CGCAACACCA  TTAAAAAATA  AGAAAAAATG  GCTCACTGTT  GAACTGATAT  TAATACCTGA  GCGTTGTGGT  AATTTTTTAT  TCTTTTTTAC  CGAGTGACAA  CTTGACTATA  ATTATGGACT  850  860  870  880  ACCACTGAAT  TAGAGTAATG  TGGCGCTATT  CATAGCGTAA  TTTTTTCTGT  TGCGGTTACA  TGGTGACTTA  ATCTCATTAC  ACCGCGATAA  GTATCGCATT  AAAAAAGACA  ACGCCAATGT  910  920  930  940  950  960  900  GGGGGAGGAA  TGCACACCTT  TAGA  TCACTAAGGC  ATAGCGATCT  GTTATATGAA  CCCCCTCCTT  ACGTGTGGAA  ATCTQQTATG  AGTGATTCCG  TATCGCTAGA  CAATATACTT  . . H . . T . . F .  . R . . P . . Y . .  S . . L . . R . . H  . . S . . D . . L .  . L . . Y . . E . .  M  C  C  ATAC  890  (iicA) 970  980  990  GATATTCCGT  TAGAAATACG  CGAGCAAATA  ATCTTATTGA  TTATCAATAC  GCTAGGAAAC  CTATAAGGCA  ATCTTTATGC  GCTCGTTTAT  TAGAATAACT  AATAGTTATG  CGATCCTTTG  . E . . Q . . I . .  I . . L . . L . . I  . . I . . N . . T .  . L . . G . . N . .  D . . I . . P . . L 1030  . . E . . I . . R . 1040  1050  1000  1060  1010  1070  1020  1080  TGCTCCTCTT  TTTATGATAT  GACATTATAC  TGCTATCATA  ATAGTCATTC  TGACGAAGTT  ACGAGGAGAA  AAATACTATA  CTGTAATATG ACGATAGTAT  TATCAGTAAG  ACTGCTTCAA  C . . S . . S . . F  . . Y . . D . . M .  . . S . . H . . S .  . D . . E . . V . .  1090  1100  . T . . L . . Y . . 1110  C . . Y . . H . . N 1120  1130  1140  TATCGAAGAA  TATGTAAAAC  GTTGCGCAAA  GAGTATGGCT  TATTCACCTT  ATAGGCGCAT  ATAGCTTCTT Y . . R . . R . . I  ATACATTTTG . . C . . K . . T .  CAACGCGTTT . L . . R . . K . .  CTCATACCGA E . . Y . . G . . L  ATAAGTGGAA . . F . . T . . L  TATCCGCGTA  Appendix  164  1150 1160 1170 1180 1190 1200 TCAACGTCAT ATCTGGATGA AATGAGTAAT CTGTTATTAA AAACAGATGA TAAAAGAAAG AGTTGCAGTA TAGACCTACT TTACTCATTA GACAATAATT TTTGTCTACT ATTTTCTTTC M..S..N..  (potential  L . . L . . L . . K  . . T . . D . . D .  . K . . R . . K . .  ORF2)  1210 1220 1230 1240 1250 1260 CATATTGATA CCATTGAGCT TGCTTTTAAC TATATAGATA CCTACCTTCG GACCTATGAA GTATAACTAT GGTAACTCGA ACGAAAATTG A T A T A T C T A T GGATGGAAGC CTGGATACTT H . . I . . D . . T  . . I . . E . . L .  . A . . F . . N . .  Y . . I . . D . . T  . . Y . . L . . R .  . T . . Y . . E . .  1270 1280 1290 1300 1310 1320 GTTACGCTTG GGTTAGAACC GGATAAGGCG ATTAGTGAAT T A A A T A A T A T ATTTCATGAG CAATGCGAAC CCAATCTTGG CCTATTCCGC TAATCACTTA A T T T A T T A T A TAAAGTACTC V . . T . . L . . G  . . L . . E . . P .  . D . . K . . A . .  I . . S . . E . . L  . . N . . N . . I .  . F . . H . . E . .  1330 1340 1350 1360 1370 CATAGTTTAA A A T A T C G A T A TGAAAAATGG TTAGGATTGT TAAGGTTGCG C GTATCAAATT TTATAGCTAT ACTTTTTACC AATCCTAACA ATTCCAACGC G H . . S . . L . . K  . . Y . . R . . Y .  . E . . K . . W . .  L . . G . . L . . L  . . R . . L . . R .  .  

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