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The impact of cell wall alterations in Mycobacterium tuberculosis on macrophage interactions and virulence Lynett, Jennifer Theresa 2006

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THE IMPACT OF C E L L WALL ALTERATIONS IN MYCOBACTERIUM TUBERCULOSIS ON MACROPHAGE INTERACTIONS AND VIRULENCE By Jennifer Theresa Lynett A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA August 2006 © Jennifer Theresa Lynett, 2006 11 Abstract While Mycobacterium tuberculosis is known to infect and persist within macrophages, the characterization of the mycobacterial gene products that are involved during host cell interactions remains incomplete. For the present study, we screened a transposon library of M. tuberculosis using a positive selection strategy to identify mutants with an enhanced binding affinity for macrophages. Our expectation was that this approach would identify genes that are involved in the maintenance of the cell wal l architecture and could provide novel insights into the virulence of the organism. The initial library screen identified five mutants with transposon disruptions within genes that encode enzymes associated with l ipid synthesis, a putative membrane protein and proteins with unknown functions. One mutant selected for further study contained a transposon insertion within the fadD23 gene. The mutant was found to be devoid of sulfolipid production, impaired for survival within macrophages and unable to cause disease in aerosol-infected mice. A second mutant was also characterized where the transposon insertion mapped to the Rv1505c gene which codes for a conserved hypothetical protein. This mutant also exhibited a significantly reduced rate of intracellular replication within macrophages and failed to actively replicate in vivo. However, the interpretation o f the in vivo findings with the Rv1505c mutant were complicated by the fact that genetic complementation of the mutant with the wi ld type gene failed to demonstrate a restoration of virulence within the mouse model of infection. A second screen of the transposon library for alterations in colonial morphology resulted in the identification of a mutant with a disruption in the mtrB gene which encodes the sensor kinase of a two-component regulatory system. Studies in other Ill mycobacterial species have also found that the inactivation of this locus leads to alterations in cell wall structure. We found that the absence of mtrB affected the distribution of the phosphatidylinositol mannosides and the phthiocerol dimyococerosates lipids of the cell envelope. In addition, the mutant was also compromised for virulence within the murine model of infection. IV Table of Contents Page Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xiii Chapter 1 Introduction 1 1.1 The global burden of Tuberculosis 1 1.2 Therapeutics and drug resistance 1 1.3 The B C G vaccine and future prospects 3 1.4 Investment into T B control 4 1.5 Understanding the Pathogen 5 1.5.1 General Biology 5 1.5.2 Highlights of the M. tuberculosis genome sequence . . . 6 1.5.3 Evolution of the M. tuberculosis complex 7 1.5.4 The M. tuberculosis cell envelope 8 1.6 Pathogenesis of Tuberculosis 15 1.6.1 Recognition of M. tuberculosis by the innate immune system 15 1.6.2 Intramacrophage survival of M. tuberculosis 17 1.6.3 The onset of the adaptive immune response 20 1.6.4 Kill ing mechanisms of IFN-y-activated macrophages 21 1.6.5 Chronic persistence of M. tuberculosis 23 1.7 Introduction to the thesis 25 1.8 Literature cited 27 Chapter 2 Identification of transposon mutants of Mycobacterium tuberculosis with increased macrophage infectivity 51 V 2.1 Preface 51 2.2 Introduction 51 2.3 Materials and Methods . . . : 54 2.4 Development of a library screening methodology 59 2.5 Transposon mutagenesis and selection 61 2.6 Molecular characterization of mutants that display enhanced macrophage binding 66 2.7 Persistence of the mutant strains in vivo 66 2.8 Discussion 70 2.9 Literature cited 75 Chapter 3 Disruption offadD23 in Mycobacterium tuberculosis affects sulfolipid production and virulence 80 3.1 Preface 80 3.2 Introduction 80 3.3 Materials and Methods 83 3.4 Identification of the fadD2 3mutmt 87 3.5 Survival of the fadD23 mutant within THP-1 cells 88 3.6 Lipid analysis of the fadD 2 3m\itant 92 3.7 Growth and Persistence of the fadD23 mutant in vivo 94 3.8 Discussion 97 3.9 Literature cited 103 Chapter 4 Disruption of the Rvl505c gene reduces the intracellular survival of M. tuberculosis and impacts virulence 109 4.1 Preface 109 4.2 Introduction 109 4.3 Material and Methods 110 4.4 The macrophage binding and intracellular survival of the Rv1505c mutant 114 4.5 The Rv1505c mutant is not hypersensitive to acidic conditions or to the presence of nitrite 117 4.6 Lipid analysis of the Rv1505c mutant 117 vi 4.7 Disruption of Rvl505c adversely affects bacterial growth and persistence within mice 120 4.8 Discussion 120 4.9 Literature cited 126 Chapter 5 Inactivation of the Mycobacterium tuberculosis mtrB gene alters colony morphology, lipid biosynthesis and reduces bacterial virulence 130 5.1 Preface 130 5.2 Introduction 130 5.3 Materials and Methods 133 5.4 Identification of the mtrB mutant 135 5.5 Lipid analysis of the mtrB mutant 136 5.6 Growth of the mtrB mutant in vivo 138 5.7 Discussion 141 5.8 Literature cited 146 Chapter 6 Discussion and Future Directions 153 6.1 Introduction 153 6.2 Advances in the genetic tools for mycobacteria 154 6.3 Further characterization of the fadD23 mutant 157 6.4 Further characterization of the Rvl505c mutant 159 6.5 Issues with Genetic Complementation 161 6.6 Further characterization of the mtrB mutant 162 6.7 Literature cited 164 Vll Lis t of Tables Table Tit le p a g e 2.1 Mutants isolated from the transposon library that exhibit an increased binding affinity to THP-1 cells 67 viii List of Figures Figure Title Page 1.1 Schematic model of the M. tuberculosis envelope 9 1.2 Structure of mycobacterial lipoglycans 11 1.3 Schematic representation of selected surface lipids and glycolipids o f M tuberculosis 13 2.1 Library screening to identify mutants with an increased macrophage binding affinity 60 2.2 Competitive binding of syringed and sonicated mycobacteria 62 2.3 Southern hybridization of randomly selected mutants 63 2.4 Pools of mutants that exhibit increasing levels of macrophage association 64 2.5 Macrophage binding of the mutants identified from the library selection 68 2.6 Murine survival studies with the selected library mutants 69 2.7 Virulence of randomly selected transposon mutants as compared to wild type and the fadD23 mutant 71 3.1 Disruption of the fadD23 gene results in an increased association between M. tuberculosis and macrophages 89 3.2 Disruption offadD23 impairs the intracellular growth of M. tuberculosis 91 3.3 Disruption offadD23 in M. tuberculosis leads to a loss of SL-1 production 93 3.4 The fadD23 mutant and the complemented strain produce lower levels of P D I M as compared to wild type 95 3.5 fadD23 inactivation affects replication and persistence within the mouse model of infection 96 4.1 Disruption of the Rvl505c gene affects the binding and intracellular survival Of M. tuberculosis 115 4.2 Disruption of the Rvl505c gene does not affect bacterial resistance ix to conditions of low p H or acidified nitrite 118 4.3 Two-dimensional T L C analysis of the apolar lipid fractions 119 4.4 Growth and persistence of the M. tuberculosis strains in mice following aerosol infection 121 5.1 The disruption of mtrB affects colonial morphology and microscopic cording 137 5.2 Disruption of the mtrB gene affects the synthesis of cell wall lipids... 139 5.3 The mtrB mutant fails to thrive within the mouse model of infection. 140 X List of Abbreviations AIDS acquired immunodeficiency syndrome B C G Bacille Calmette-Guerin B L A S T P basic local alignment search tool protein bp base pair C D cluster of differentiation C R complement receptor D A T diacylated trehalose D N A deoxyribonucleic acid D O T S directly observed treatment short course E E A 1 early endosomal antigen 1 F A A L fatty acyl -AMP ligases G+C guanosine + cytosine G A S glycerol alanine salts G F P green fluorescent protein H I V human immunodeficiency virus IFN Interferon IL Interleukin kDa Kilodalton L A M Lipoarabinomannan L B Luria-Bertani broth L M i Left-Handed Parallel beta-Helix L M Lipomannan LPS Lipopolysaccharide M B L mannose binding lectin Mbp megabase pairs MD88 myeloid differentiation factor 88 M D G millenium development goals M D R - T B multidrug resistant tuberculosis M O I multiplicity of infection M T B Mycobacterium tuberculosis NOS2 inducible nitric oxide synthase nt. Nucleotide O A D C oleic acid-albumin-dextrose complex P A T polyacylated trehalose PBS phosphate buffered saline P D I M phthiocerol dimycocerosate P E proline-glutamine P G R S polymorphic G C rich repetitive sequence P I L A M phosphatidylinositol capped lipoarabinomannan P I M phosphatidylinositol mannoside P M A phorbol 12-myristate 13-acetate PPE proline-proline glutamine R N A ribonucleic acid RNI reactive nitrogen intermediates ROI reactive oxygen intermediates SL-1 sulfolipid 1 S N A R E soluble N S F attachment receptor S O D superoxide dismutase SP surfactant protein T A C O trytophan aspartate containing coat T B tuberculosis T h l T helper type 1 T L C thin layer chromatography T L R toll-like receptor T N F tumour necrosis factor W H O World Health Organization Xlll Acknowledgments I would like to thank my thesis advisor, Rick Stokes, for his guidance and advice over the course of this project. In addition his technical help and expertise were crucial to completing the in vivo studies of this project. I would also like to thank the members of my advisory committee, Brett Finlay, Jim Kronstad and Wan Lam who have always been supportive during my graduate work. The past and present members of the Stokes laboratory have also aided in this project with their ideas and technical support. I would like to acknowledge Lucy Brooks who greatly helped my studies by sharing her knowledge of mycobacterial genetics and cell wall chemistry. I would also like to extend my thanks to Shelley Small who kept me in the loop with all the departmental requirements and always had a moment to chat when I visited the campus office and the members of the B C R I third floor for the extended coffee breaks. This work would not have been possible without the collaborations of W.R. Jacobs who provided the transposon constructs for mycobacteria, Yossef Av-Gay for the G F P -expressing strain of B C G and K . G . Papavinasasundaram for constructs and advice on mutant complementation. I would also like to acknowledge the Child & Family Research Institute and the University of British Columbia who provided Graduate Fellowships over the course of my studies. Finally, I must also extend my thanks to Peter, my family and my closest friends who have been unwavering in their support over the years. 1 Chapter 1 Introduction 1.1 The global burden of Tuberculosis With each passing second, it is estimated that another person has become infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). The bacterium is highly transmissible with 10-15 new infections arising annually from a single T B case (1, 2) and an infectious dose that can be as low as 3 bacilli (3). It is thought that 32% of the global population is already infected, making M. tuberculosis one of the most successful pathogens of man (4, 5). However, only 10% of those infected wi l l develop active T B in their lifetime while the balancing 90% remain as latently infected carriers (4, 6). Thus, in most cases, the immune response of the host successfully contains the bacilli and prevents the onset of clinical disease (7, 8). Nevertheless, the numbers of T B cases and TB-related deaths continue to climb with 9 mill ion new cases and 2 mill ion deaths reported in 2004 (9). The worldwide resurgence of T B is compounded by the pandemic spread of the human immunodeficiency virus (HIV) that has generated an expanding population o f immunocompromised individuals who are highly susceptible to infection (10). The coinfection of H I V and M. tuberculosis leads to a 50% chance of reactivating a latent M. tuberculosis infection (10). In addition, HIV-patients that are newly-infected with M. tuberculosis are more likely to progress to active disease and have a higher incidence of extrapulmonary T B (11, 12). 1.2 Therapeutics and drug resistance There are five first-line drugs available for T B treatment; rifampin, isoniazid, pyrazinamide, ethambutol and streptomycin. Isoniazid and ethambutol target the M. 2 tuberculosis cell wall by inhibiting the synthesis of mycolic acids and arabinogalactan, respectively (13-15). Pyrazinamide causes acidification of the M. tuberculosis cytoplasm and affects membrane function although its precise mechanism of action is unclear (16, 17). Rifampin inhibits transcription by binding the M. tuberculosis R N A polymerase (18) and streptomycin blocks protein synthesis by targeting the ribosome (19). Both isoniazid and pyrazinamide are prodrugs that require mycobacterial enzymes for activation; the toG-encoded catalase peroxidase activates isoniazid (20) while the nicotinamidase encoded by the pncA gene converts pyrazinamide to its active state as pyrazinoic acid (21). The short-course chemotherapy that is employed in countries with a high T B prevalence is a combined prescription of isoniazid, rifampin, pyrazinamide and either ethambutol or streptomycin for a span of two months known as the intensive phase. This is followed by a continuation phase of rifampin and isoniazid that lasts for a further four months (22). The treatment is typically successful and boasts a cure rate > 85% among drug sensitive strains (23, 24) whereas, if left untreated, T B mortality is approximately 50-60% (25). In 1993, the World Health Organization (WHO) set out to implement a system, known as directly observed treatment short course (DOTS), to address the rising incidence of T B in developing nations (26, 27). D O T S combines the use of standardized diagnostic methods and drug regimens along with strict case management to improve T B detection, reduce the frequency of transmission and prevent the emergence of resistance to first-line drugs (28-31). Problems arise in remote and impoverished regions where effective D O T S programs are hindered by an inadequate access to health care services. The resulting 3 non-compliance to chemotherapy increases the risk of disease relapse and contributes to bacterial drug resistance (10, 32, 33). Multi-drug resistant M. tuberculosis ( M D R - T B ) is defined as resistance to at least isoniazid and rifampin. M D R - T B requires longer treatment protocols that involve second-line therapies which are less effective, more expensive and not as well tolerated as first-line agents (24). At present, the global incidence of M D R - T B appears relatively stable at 3% of all T B cases (34). Regional hot spots in areas of Eastern Europe, Russia and China are a growing concern where M D R -T B now accounts for > 10% of all new T B cases and exceeds 25% among cases that have previously been treated (23, 24, 35). 1.3 The B C G vaccine and future prospects The only T B vaccine that is currently available is the live attenuated strain of mycobacteria known as Bacille Calmette-Guerin (BCG). The vaccine originated from a virulent isolate of M. bovis that underwent extended passage in the laboratory over a span of 13 years. Over time, the bacteria lost the ability to cause disease in animal models (36) and, in 1921, the vaccine was first administered to humans (37). B C G is still widely used for protection against childhood forms of T B meningitis and miliary T B with an estimated 100 million newborns inoculated each year (38). However, B C G has failed to consistently demonstrate protection against adult pulmonary T B with clinical trial results varying from 80% to 0% protection (39, 40). The reasons behind the lack of efficacy are not completely understood but may relate to prior exposure to environmental mycobacteria, a waning immune response after vaccination or antigenic differences that exist between B C G and M. tuberculosis (41). 4 Clearly, a more effective vaccine is necessary that, ideally, would prevent disease in the absence of prior M. tuberculosis exposure and block the onset of active T B in latently infected individuals (8). The current strategies for vaccine development attempt to boost the T-helper type 1 (Thl) immune response beyond that which is generated by conventional B C G vaccination or by natural M. tuberculosis infection. One approach is to vaccinate using immunodominant antigens of M. tuberculosis to drive the expansion of specific T-cell clones (42). The current candidate antigens, including Antigen 85A and E S A T - 6 , are under evaluation as protein subunit and D N A vaccines (43-45). The main drawback to this approach is that it relies on a limited T-cell repertoire to provide protection which could be problematic because the antigens that prevent disease reactivation will likely be different from those that protect nai've individuals (8, 46). A n alternate approach is to improve the B C G vaccine by having the bacteria express M. tuberculosis antigens (47). Live attenuated mutants of M. tuberculosis that are auxotrophic (48, 49) or deficient in virulence factors (50) are also under consideration, although this approach is met with concerns over patient safety (51). 1.4 Investment into T B control Historically, both industry and government have overlooked the so-called 'neglected diseases' that predominantly afflict developing nations but have little impact on industrialized countries (52). In 2000, the United Nations announced the Millennium Development Goals (MDG) in an attempt to redirect global attention toward the rising incidence of T B . The M D G targets are to attain a 50% reduction in T B prevalence and death rates by 2015 (53). In addition, a new anti-TB drug and vaccine are to be available 5 by 2010 and 2015, respectively (54). This has sparked new alliances between public and private partners such as the Global Alliance for T B Drug Development and the Global Stop T B Plan which are mandated to expedite the process of target validation and progression through clinical trials (55-57). There is still much that remains unknown about the biology of M. tuberculosis including a complete elucidation of the intracellular survival tactics of the bacillus, the factors that are required to maintain chronic persistence as well as the host triggers and bacterial factors that lead to disease reactivation. Achievement of the M D G targets will require coordinated efforts from the public health sector to improve the infrastructure needed for treatment and, importantly, the research community will also need to combine its resources to identify optimal targets and strategies for novel treatments. 1.5 Understanding the Pathogen 1.5.1 General Biology A l l mycobacteria are aerobic, non-motile, rod-shaped bacilli that synthesize a thick cell envelope with approximately 60% of its weight as lipid. Mycobacteria are classified as acid-fast bacilli, which refers to the ability of the cell wall to retain dyes such as carbol fuschin or auramine O even in the presence of acid-alcohol. The genus Mycobacterium is classified into over 60 distinct species most of which are nonpathogenic inhabitants of soil and water environments. The most notorious members of the genus are the few species that are causative agents of disease in humans, including M. tuberculosis (TB), M. leprae (leprosy) and M. ulcerans (buruli ulcers). Other species such as the members of the M avium-intracellulare complex are recognized 6 opportunistic pathogens that frequently cause disseminated infections in children, the elderly, A I D S patients and other immunocompromised populations. There are also species that are prominent pathogens in the agricultural industry including M. bovis and M. avium ssp. paratuberculosis which are the causative agents of T B and Johne's disease, respectively. 1.5.2 Highlights of the M. tuberculosis genome sequence The release of the complete genome sequence of M. tuberculosis in 1998 ushered in a new era for mycobacterial research (58). Prior to that point, significant advances had been hampered by the slow growth of the organism, the intractability of the bacteria to genetic manipulation and the lack of available tools for mutagenesis (59). The circular genome is 4.4 Mbp in size with a G+C content of 65.6% (58). There is a total o f 4043 annotated genes for which 52% have a predicted function (60). One of the striking features o f the genome is the high number o f genes that are thought to be involved in lipid synthesis and fatty acid metabolism (58). In addition, approximately 9% of the genome encodes members of two related gene families; the P E and P P E families (58). Each family shares conserved N-terminal domains that contain consensus proline-glutamate (PE family) or proline-proline-glutamate (PPE family) residues. The P E family also comprises a further subgroup known as the P E _ P G R S proteins that encodes a conserved C-terminal region. The precise functions of these families are unknown, although they have been proposed to be involved in antigenic variation (61) and at least some members have been localized to the bacterial surface (62, 63). 7 The genome sequence of the laboratory strain H37Rv was soon followed by that of an M. tuberculosis clinical isolate known as C D C 1551 which allowed for a direct assessment of intra-species variability (64). Interestingly, nearly 50% of the insertions and deletions between the two strains occur within the P E and P P E families (64). Overall the level of nucleotide variation between the two strains is quite limited with single nucleotide polymorphisms occurring, on average, once every four kilobases (60, 64). A t present, there are three other mycobacterial genomes that have been fully sequenced including M. bovis (65), M. leprae (66) and M. avium ssp. paratuberculosis (67). The availability of these genomes wi l l allow for further comparative analyses to help elucidate the bacterial factors that dictate pathogenic differences (60). 1.5.3 Evolution of the M. tuberculosis complex Mycobacterium tuberculosis along with M. bovis, M. africanum, M. microti and M. canettii, all combine to form the M. tuberculosis complex. The members of the complex are genetically similar based on 16s r R N A sequence and D N A - D N A hybridization (68). However, the complex members exhibit vast differences in pathogenicity and host tropism. M. tuberculosis, M. canettii and M. africanum cause T B in humans, M. microti is pathogenic to voles while M. bovis infects a much broader host range including bovines and humans (68). From a phylogenetic perspective, it was traditionally thought that M. bovis represented the ancestral member of the complex and that M. tuberculosis arose during the domestication of cattle and subsequently lost the ability to infect species other than humans (69). Later comparative genomic studies refuted this hypothesis by the novel finding that M. bovis contains 16 genomic deletions 8 relative to M. tuberculosis, suggesting that the common ancestor for the complex was likely already pathogenic to humans (70). 1.5.4 The M. tuberculosis cell envelope Mycobacteria synthesize a complex cell envelope that is remarkable for its abundance of lipids and the diversity in lipid structure. The M. tuberculosis envelope exhibits a reduced permeability that is thought to confer protection against the toxic environments within the host and effectively limit the influx of antibiotics (71). Recent evidence also suggests that various cell wall factors have the ability to directly interact with and modulate the innate and adaptive arms of the immune response (72, 73). The cell envelope of M. tuberculosis can be divided into 3 main components shown in Figure 1.1; the plasma membrane, the mycolic acid-arabinogalactan-peptidoglycan complex and the surface capsule (74). The cytoplasmic contents of the bacteria are contained within a phospholipid plasma membrane that is typical of other eubacteria and includes phosphatidylglycerol, cardiolipin and phosphatidylethanolamine (74) . A distinctive feature of the M. tuberculosis plasma membrane is the presence of large lipoglycans including lipoarabinomannan ( L A M ) , lipomannan (LM) and the L A M precursors known collectively as the phosphatidylinositol mannosides (PIMs). The lipoglycans are anchored in the plasma membrane by their phosphatidylinositol moiety (75) . L A M and L M have been proposed to extend through the cell wall allowing exposure at the bacterial surface, although this has not been directly confirmed (74). Interestingly, the PIMs have been localized to the outer surface of the bacillus within the capsular layer (76). 9 Capsule-like material Figure 1.1 Schematic model of the M. tuberculosis envelope. Adapted from Crick et al. (77). 10 The phosphatidylinositol anchor of the PIMs is further modified with up to six mannose residues, with the dimannoside and hexamannoside versions being the most prevalent within the cell wall (78). The PIMs can also be acylated with up to four fatty acyl chains that can be either palmitic, octadecenoic or tuberculostearic acids (74, 79). Several of the PIMs have been suggested as ligands that are recognized by the macrophage via the mannose receptor (80) or complement receptor 3 (CR3) (81, 82). In addition, the PIMs have been shown to function as T cell antigens when complexed with CD1 (83) and contribute to the survival of M. tuberculosis within the macrophage (84). L M is a multi-mannosylated version of PIM and is thought to be a precursor of L A M , however the two lipoglycans differ in their immunomodulatory effects (85). In general, L M is proinflammatory while L A M from M. tuberculosis is immunosuppressive (85). However, the immunomodulatory effects of L A M strongly depend upon the mycobacterial species as shown in Figure 1.2. In some rapidly growing species such as M. smegmatis, the terminal mannoses on L A M are capped with inositol phosphate, referred as P I L A M , which is a potent inducer of proinflammatory cytokines by macrophages (85). This is in contrast to M. tuberculosis L A M where only mannose residues cap the arabinan termini and the lipoglycan is an immunosuppressive factor (85). L A M from M. tuberculosis has been shown to inhibit macrophage activation by interferon-gamma (IFN-y) (86), block the expression of interleukin 12 (IL-12) (87, 88), inhibit macrophage apoptosis (88) and halt phagosomal maturation (89). L M , regardless of the mycobacterial species, is proinflammatory and induces IL-12, tumour necrosis 11 ManLAM PILAM (Mannose-cap) (Phospho-/wyo-inositol-cap) M. tuberculosis M. smegmatis, M. species M. kansasii O P - A r a / # a -Man / 7 Phospho-wyo- inos i to l /s/w fatty ac id Figure 1.2 Structure of mycobacterial lipoglycans L A M , L M and dimannoside PIM (PIM 2) from M. tuberculosis and P I L A M from M.smegmatis. Modified from Dao et al. (88) 12 alpha (TNF-cc), nitric oxide production as well as apoptosis in unactivated macrophages (88, 90). The core skeleton of the cell envelope is a covalently linked complex o f peptidoglycan, arabinogalactan and a layer of mycolic acids. The peptidoglycan is typical of most Gram-positive bacteria (91) with a couple of added features; the N-acetyl functional groups on the muramic acid residues can be further oxidized to glycolic acid (92, 93) and a high degree of crosslinking exists between the peptidoglycan peptide chains (94). Arabinogalactan constitutes the major polysaccharide component of the cell wall and is linked via a phosphodiester bridge to the peptidoglycan layer (77, 95). A subset of the non-reducing termini of the arabinan component is linked to clusters of four mycolic acids (96). The mycolic acids are a-alkyl , P-hydroxy fatty acids that fall into three categories; the a-, methoxy- and keto-mycolic acids (97). The mycolate layer is thought to be a very hydrophobic region of the cell wall with the long alkyl chains packing tightly together (98). A collection of surface lipids and glycolipids covers the mycolic acid layer through non-covalent interactions and also likely intercalates with the outer capsule layer, shown in Figure 1.3 (74, 99). The surface lipids include the acylated trehaloses, the phthiocerol/phenolphthiocerol dimycocerosates, and the PIMs. The mycocerosate lipids have been the focus of intense research since the results of several studies have implicated this group of cell wall factors in the virulence of M. tuberculosis (100-102). Phthiocerol dimycocerosate (PDIM) is important in maintaining the low permeability of the cell envelope (103) and is also suggested to play a role in countering the toxic effects of nitric oxide (104). The phenolphthiocerol dimycocerosate (also known as phenolic 13 E Figure 1.3 Schematic representation of selected surface lipids and glycolipids of M. tuberculosis (A) P D I M (B) Phenolphthiocerol dimycocerosate (C) Trehalose dimycolate (D) Diacylated trehalose and (E) Sulfolipid-1. Adapted from Reed et al. (102) and Woodruff etal. (105). 14 glycolipid) is expressed by hypervirulent strains of M. tuberculosis from the W-Beijing family that are highly transmissible and associated with epidemic outbreaks of T B (106). The phenolic glycolipid is thought to confer an advantage to the bacilli by inhibiting proinflammatory responses by effectors of the innate immune system (102, 107). The acylated trehaloses include the family of sulfolipids, trehalose dimycolate and the di- and poly-acylated trehaloses ( D A T / P A T ) . The sulfolipids can have pro- or anti-inflammatory effects on monocytes in vitro depending on their concentration (108, 109). More recently, diacylated sulfolipids have been shown to be recognized by specific T-cell subsets (110) and their accumulation in the M. tuberculosis cell wall has a negative impact on virulence in mice (111, 112). Trehalose dimycolate (also known as cord factor) acts as a strong proinflammatory agent that can activate macrophages (113) and contributes to the granulomatous response (114, 115). The D A T and P A T glycolipids have been suggested to anchor the capsular polysaccharides to the bacillus surface (116) and the inactivation of D A T / P A T synthesis increases the surface hydrophobicity leading to bacterial aggregation (117). Suggestions of a mycobacterial capsule originated from electron micrographs that showed a diffuse outer layer that extended from the bacillus surface (118, 119). Subsequent chemical analysis of the mycobacterial surface components identified an extensive amount of surface polysaccharide and protein with a paucity of lipid (99). The main capsular polysaccharides are the a-1,4 glucan (70%), arabinomannan (28%) and mannan (2%) (120-122). The presence of capsule at the outermost surface suggests that it resides at the interface between the bacteria and the extracellular environment and is likely to play a role in host cell interactions (99). 15 1.6 Pathogenesis of Tuberculosis 1.6.1 Recognition of M. tuberculosis by the innate immune system Transmission of M. tuberculosis between human hosts occurs by the inhalation of infectious aerosols that are generated by coughing or sneezing from an active T B patient. The aerosolized droplets range from 5-10 microns in size and have an extremely slow settling rate that can promote a wide dispersal via air currents before gaining entry to the lung alveoli (123). The resident alveolar macrophages and dendritic cells are the primary effectors for the innate immune system and serve as the first line of defense against invading pathogens. These cells express germline-encoded pattern-recognition receptors that detect conserved microbial components known collectively as pathogen-associated molecular patterns (124). The M. tuberculosis-infected macrophages and dendritic cells aid in the development of adaptive immunity through antigen presentation and cytokine production (125). A variety of macrophage receptors can participate in the internalization of M. tuberculosis under both opsonic and nonopsonic conditions (126, 127). In the absence o f infection, the lung environment is thought to have a very low level o f serum opsonins (128) and as such, the initial uptake of M. tuberculosis is likely to proceed nonopsonically. The direct recognition of surface moieties on the bacilli can involve the mannose receptor (129, 130), CD14 (131), CD43 (132, 133), CD44 (134), C R 4 (135), scavenger receptors (136, 137) and C R 3 (81, 138). The specific ligands are still unknown for several of the receptors, although L A M and its precursor PIMs are thought to bind the mannose receptor via their terminal mannose caps (80). The capsular PIMs are involved in CR3 binding at a site that is distinct from the C3bi binding site (81, 82). 16 The collectin system, which includes the mannose binding lectin ( M B L ) and the surfactant proteins A and D (SP-A and SP-D), also contributes to the innate defenses of the lung (139, 140). Each collectin member contains a carbohydrate recognition domain and a collagenous domain with three monomers combining to form a complex that binds microbial carbohydrates in the presence of calcium (139, 140). M B L has been reported to opsonize M. tuberculosis (141, 142) leading to complement activation and phagocytosis (143). Genetic polymorphisms within the M B L gene that lead to increased serum M B L concentrations have been correlated with heightened T B susceptibility in some ethnic groups (144, 145). The results with SP-A are more equivocal with some studies reporting its role in an increased macrophage uptake of M. tuberculosis (146-148) while others find that SP-A has no impact on bacterial binding (149). The presence of SP-D causes agglutination of mycobacteria but does not appear to promote macrophage internalization (150, 151). At later stages of disease, the bacilli may also be opsonized with IgG to allow entry via the Fc receptors (152). In the presence of other serum opsonins, the bacilli can be coated with C3b and C3bi particles that are recognized by the complement receptors ( C R 1 , C R 3 , CR4)(136, 153). Recently, various members of the Toll-like receptor (TLR) family have also been implicated in the recognition of M. tuberculosis or mycobacterial products (125, 154, 155). T L R 2 , either alone or in combination with T L R 1 / T L R 6 (156), is thought to recognize L M (157), PIMs (158) as well as a 19kDa lipoprotein (159-161) from M. tuberculosis. T L R 4 dependent recognition of M. tuberculosis has also been reported (162) with a recent study suggesting that mycobacterial heat shock proteins are the 17 correlating ligands (163). There are conflicting reports following M. tuberculosis infection of T L R knockout mice. Mice deficient in T L R 2 (164, 165), T L R 4 (164) or T L R 6 (165) are similar to wi ld type mice in their ability to control M. tuberculosis following a low dose aerogenic infection. However, two groups found that T L R 4 is required to control M. tuberculosis infection following either intranasal (166) or low dose aerosol challenge (167). Furthermore, T L R 2 knockout mice are reportedly susceptible to a high dose aerosol infection of M. tuberculosis (164, 168). Additional evidence which lends support to a role for T L R signaling in protective immunity is that mice deficient in the T L R adaptor molecule, myeloid differentiation factor 88 (MyD88) , are unable to control M. tuberculosis (169, 170). 1.6.2 Intramacrophage survival of M. tuberculosis The receptor-mediated phagocytosis of invading microbes typically results in the destruction of the pathogen. The bacteria are internalized into a phagosomal vacuole that undergoes sequential fusion with other endosomes by interactions between specific Rab GTPases and S N A R E proteins (171-173). Eventually, the phagosome accumulates lysosomal markers and acidifies which leads to the activation of lysosomal hydrolases and the degradation of the phagosomal contents (171-173). A t the early stages of infection, the lung alveolar macrophages are largely ill-equipped to control the infection as M. tuberculosis has evolved several survival strategies to resist elimination. The phagocytosis of M. tuberculosis is an actin-dependent process that requires the presence o f cholesterol within the host plasma membrane (174). Following macrophage uptake, M. tuberculosis immediately begins the process o f remodeling the 18 phagosomal environment to promote replication (175). The phagosome retains membrane markers that are typical of early endosomes, such as Rab5, while restricting the accumulation of Rab GTPases that are associated with late endosomal/lysosomal compartments, such as Rab7 (176, 177). The mycobacterial phagosome fails to acidify due to a paucity of the vacuolar-type proton ATPases (178-180). It is thought that an on-going interaction with other Rab5-containing early endosomes allows the intracellular mycobacteria to maintain access to extracellular nutrients; studies have shown that iron conjugated to the transferrin receptor cycles from the plasma membrane to the M. tuberculosis-contzimmg vacuole (181, 182). Alterations in calcium levels also contribute to the aberrant trafficking of the M. tuberculosis phagosome (183-185). Live M. tuberculosis inhibits the rise of cytosolic 2+ Ca ions that is observed following uptake of dead bacteria or other control particles (184). Maturation of the M. tuberculosis phagosome can be induced with a calcium ionophore to release the suppression of C a 2 + ions and this coincides with a reduction in bacterial survival (183). Subsequent studies demonstrated that M. tuberculosis inhibits the macrophage sphingosine kinase that prevents Ca 2 +-mediated signaling and leads to the failed recruitment of calmodulin to the phagosomal membrane (184, 185). The block * 2+ in Ca signaling also limits the phagosomal accumulation of hVPS34, a phosphatidylinositol-3 kinase, which in turn blocks the recruitment of the early endosomal antigen 1 (EEA1) (186). The exclusion of E E A1 inhibits the fusion of Rab5-positive early endosomes with late endosomes (186-188). A comparison of the phagosomal membrane proteins from live and dead mycobacteria indicated that a protein termed T A C O (trytophan aspartate containing coat) 19 was retained within the phagosomes of live B C G , suggesting that T A C O retention was linked to phagosomal maturation (189). T A C O is identical to the previously described actin-binding protein, known as coronin, that is involved in phagocytosis (190, 191). A role for TACO/coronin in the intracellular survival of M. tuberculosis is unclear as others have been unable to repeat the initial observations, instead noting that membrane coronin is only transiently associated with the mycobacterial phagosome (192). Early studies reported that mycobacterial proteins and lipids were actively released from the M. tuberculosis phagosome within small vesicles or could intercalate within the phagosomal membrane (176, 193-195). This suggested that cell wall components could act as important modulators that determine the fate of intracellular M. tuberculosis. Indrigo et al. found that cord factor was necessary for the block in phagosomal fusion since the removal of apolar lipids from the surface of M. tuberculosis resulted in a reduced intracellular replication and led to complete maturation of the mycobacterial phagosome (196, 197). Others have found that L A M and PIMs are linked to the intracellular survival of M. tuberculosis. Polystyrene beads that are coated with L A M can reproduce the block in phagosomal maturation by restricting syntaxin 6-dependent vesicular traffic from the trans Golg i network (198). This observation links L A M to the reduced accumulation of E E A 1 which also requires syntaxin 6 for delivery (198, 199). Surprisingly, the precursors of L A M exert a completely different effect on the phagosome. The presence of PIMs promotes the fusion between early endosomal compartments and the M. tuberculosis phagosome (84). While several mycobacterial genes have been implicated by mutagenesis studies in the intracellular survival of M. tuberculosis (200-202), very few proteins have been 20 thoroughly characterized for their biological role during macrophage infection. The exceptions include the SapM protein which is a lipid phosphatase that hydrolyzes phosphotidylinositol-3 phosphate to interfere with hVPS34 signaling (203). In addition, mycobacterial mutants inactivated in the pknG gene are unable to block phagosomal maturation and are avirulent in mice (204, 205). PknG is a eukaryotic-like serine/threonine kinase that is actively secreted from phagosomal mycobacteria and can be localized within the host cytosol, leading to speculation that PknG may interfere with host cell signaling (204, 205). 1.6.3 The onset of the adaptive immune response The M. tuberculosis-infected macrophages secrete proinflammatory cytokines and chemokines that serve to attract additional immune cells to the site of infection. Since M. tuberculosis is able to subvert the defense mechanisms of these unactivated phagocytes, the bacteria replicate logarithmically until the host cell is killed. The newly released bacteria are phagocytosed by the infiltrating macrophages, dendritic cells and neutrophils but these cells are also incapable of eradicating the bacteria. The infected dendritic cells and macrophages will also migrate to the draining lymph nodes where they present mycobacterial antigens to circulating T lymphocytes (206). Antigen presentation to T cells can occur in the context of both major histocompatibility complex ( M H C ) class I and II giving rise to antigen-specific CD8+ and CD4+ T cells, respectively (206, 207). Protective immunity against M. tuberculosis relies on the development of a robust T-cell response that is primarily composed of CD4+ and CD8+ T lymphocytes (207-210), 21 although evidence also suggests a role for T cells that recognize nonpeptide antigens such as the CD1 restricted T cells and the y5T cells (211-213). After 2-4 weeks, the site of infection is surrounded by a myriad of cell types that culminates in the formation of a granulomatous lesion. The granuloma consists of activated lymphocytes (both T and B cells), monocyte-derived macrophages and fibroblasts (214-216). From the host's perspective, the goal of granuloma formation is to sequester the infection from the rest of the lung and prevent bacterial dissemination to other sites in the body. The granuloma is also necessary from the point of view of the pathogen. For bacterial transmission to be successful, M. tuberculosis must induce a granulomatous lesion that, ideally, will erupt and release the bacteria into the airways (217,218). 1.6.4 K i l l i n g mechanisms of IFN-y-activated macrophages Within the granuloma environment, the infected macrophages secrete cytokines, such as IL-12, IL-23 and IL-18, that will direct T-cell activation toward a Th-1 phenotype (219-223). In response, the activated lymphocytes produce IFN-y which is a key cytokine in the development of immunity against M. tuberculosis (214). Studies in mice (224, 225) and humans (226-228) demonstrate that a lack of IFN-y or the IFN-y receptor results in an increased susceptibility to mycobacterial infections. IFN-y, along with T N F -a, activate the antimycobacterial defenses of host macrophage to inhibit the intracellular replication of M. tuberculosis (125,214). Activated macrophages are able to bypass the M. tuberculosis-mediated block in phagosomal maturation leading to acidification of the phagosome and increased killing of 22 M. tuberculosis (229). These events are dependent upon the expression of the IFN-y-inducible L R G - 4 7 GTPase which is recruited to the M. tuberculosis phagosome in activated macrophages (230, 231). Mice deficient in L R G - 4 7 are susceptible to M. tuberculosis and similarly LRG-47" A macrophages fail to control the bacterial replication even following activation with IFN-y (230). The localization of the GTPase within the phagosomal membrane promotes fusion with late endosomal/lysosomal compartments and is necessary for the recruitment of the vacuolar-type proton ATPases that mediate the acidification of the M. tuberculosis phagosome (230). A recent study also suggests that the IFN-y/LRG-47 pathway is required for the acquisition of Beclin-1 by the phagosome that promotes an autophagic cell death to reduce M. tuberculosis survival (232). A separate antimicrobial defense that is triggered by IFN-y is the production of reactive nitrogen (RNI) and oxygen intermediates (ROI) by infected macrophages (233, 234). The increased expression of the inducible nitric oxide synthase (NOS2) results in the production of toxic nitric oxide from L-arginine which can accumulate as nitrite within the acidic phagosome or can complex with ROI to form peroxynitrite which is also antimycobacterial (233). Studies in mice demonstrate that RNI are important during both acute (235, 236) and chronic phases (237) of M. tuberculosis infection. Reactive oxygen species within macrophages are produced by the N A D P H oxidase (238) but appear to have a dispensable role in the killing of M. tuberculosis (239). The resistance of M. tuberculosis to R O S is due in part to the KatG catalase peroxidase that counters the effects of hydrogen peroxide in vitro (240, 241) and is required for infection in vivo (240). Interestingly, the virulence of the M. tuberculosis KatG mutant is identical to wild 23 type in mice deficient in the N A D P H oxidase, thus confirming its involvement in R O S defenses of M. tuberculosis (240). Infected macrophages also k i l l intracellular mycobacteria through the induction of apoptosis and evidence suggests that M. tuberculosis actively interferes with host apoptotic signaling pathways in order to maintain its intracellular niche (242). Macrophage infection with the virulent H37Rv strain of M. tuberculosis results in a lower level of apoptotic cell death as compared to the avirulent H37Ra strain (243, 244). This disparity seems to depend on the ability of virulent strains to limit the apoptosis-inducing effects of T N F - a by the upregulated expression of the soluble T N F - a receptor 2 to limit the bioactivity of the cytokine on macrophages (245). Others studies find that the limited apoptosis in M. tuberculosis-infected macrophages involves members of the Bcl -2 family of pro- and anti-apoptotic proteins (246). Macrophage infection with H37Rv upregulates the anti-apoptotic Mcl-1 protein (247) and L A M induces the phosphorylation of the Bad protein which leads to an increased presence of the prosurvival Bcl -2 protein in the host cytosol (248). The increased presence of the anti-apoptotic members of the Bcl -2 family prevents the onset of apoptosis by blocking the release of cytochrome c from the mitochondrial membrane and preventing caspase activation (246, 249). 1.6.5 Chron ic persistence of M. tuberculosis While the IFN-y-activated macrophages can limit replication o f M. tuberculosis, there are often bacilli that continue to persist within the confines of the granuloma. Several studies have utilized in vitro models that mimic aspects of the granuloma environment in order to gain further insight into the mycobacterial factors that are 24 required for persistence (250-252). In addition, a number of M. tuberculosis mutants have also added to our understanding of the pathogenesis of latency. The transition from acute to chronic infection depends on the ability of M. tuberculosis to shift toward the catabolism of lipids as a carbon source. This is reflected in mutants that are disrupted in the id gene that are unable to maintain persistence in mice or survive within activated macrophages (253). The id gene codes for the isocitrate lyase enzyme which is necessary for the metabolism of fatty acids via the glyoxylate cycle (254). Cel l wall factors are also involved as demonstrated by the pcaA knockout in M. tuberculosis that lacks cyclopropane-modified mycolic acids and is unable to maintain a chronic infection (255). The long-term survival of M. tuberculosis also relies heavily upon transcriptional regulation to induce the appropriate transcriptional response when faced with changing environmental conditions. The dormant-like state of M. tuberculosis that is induced during latency is the result of a coordinated regulon of 48 genes that is under the control of the DevR-DevS two-component regulatory system (251). The regulon is induced by conditions of hypoxia and the presence of nitric oxide as well as other stresses (256-258). A separate two-component system, encoded by the mprA and mprB genes, is also necessary for persistence in vivo (259) and has recently been shown to control the transcription of the SigE and SigB sigma factors (260). Previous studies have linked particular sigma factors, including SigE and SigH, to the recruitment of lymphocytes to the site of infection. M. tuberculosis mutants that are deficient in these sigma factors induce less severe lung pathology during chronic infection in vivo. In the presence of a robust and prolonged immune response, the necrotic center of the granuloma becomes toxic to M. tuberculosis presumably due to the reduced oxygen 25 tension and an overly high content of fatty acids. Over time, the granuloma may decrease in size and calcify which correlates with low levels of viable bacilli . Alternatively, a perturbation in the host immune response wi l l cause the liquefaction of the caseous granuloma which enables the extracellular replication of M. tuberculosis and promotes dissemination. T N F - a plays an important role in the successful maintenance of the granuloma but also contributes to the tissue damage and pathology in the lung (214). The requirement for T N F - a during latency is clearly demonstrated by cases of reactivated T B in patients with Crohn's disease or rheumatoid arthritis that undergo treatment with anti-T N F - a antibodies (261). However, since M. tuberculosis is also a potent inducer of T N F - a , it implies the host granuloma is equally important for the pathogen. The transmission of M. tuberculosis between hosts would be ablated without the induction of a strong immune response thus demonstrating the fine balance that exists between the immune response of the host and the virulence strategies of M. tuberculosis (214). 1.7 Introduction to the thesis Research into the molecular events that govern the interaction between M. tuberculosis and the host macrophage is necessary to further our understanding of this human pathogen. The presence of the M. tuberculosis capsular carbohydrates at the bacillus surface (262, 263) implies an involvement in mediating host cell interactions (81, 262-265). To date, there have been no reports that detail the M. tuberculosis genes involved in capsule synthesis, regulation or transport to the cell surface. Previous findings from our lab indicated that disruption of the M. tuberculosis'surface capsule by mechanical treatment (brief pulses of sonication) resulted in an increased association with 26 macrophages (264). This suggests that the cell wall capsular components may somehow act to limit the interaction between the bacteria and the host macrophage (264). Thus, the initial aim for this thesis work was to use a genetic approach to explore the role of the capsule layer during macrophage infection. As outlined in Chapter 2, a transposon library of M. tuberculosis was constructed and screened for mutants that exhibited an increased propensity to bind macrophages. We reasoned that mutants displaying an enhanced ability to bind macrophages would have incurred alterations to the cell envelope that resulted in the exposure of bacterial ligands which, under in vitro culture conditions, would be normally masked by the surface capsule. A total of five mutants were isolated and two of the mutants were selected for further characterization. As described in Chapters 3 and 4, the virulence of the fadD23 and Rvl505c mutants was assessed using macrophage monolayers and mice. 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March 2006. 2.2 Introduction A central feature of TB pathogenesis is the ability of M. tuberculosis to infect and persist within the host macrophage. However the characterization of M. tuberculosis gene products that are involved during host cell interactions remains incomplete. Studies on the initial M. tuberculosis-host cell binding interactions have primarily focused on the identification of macrophage (1, 2) and dendritic cell (3) receptors that can mediate phagocytosis while much less is known about the identity of bacterial surface components that participate in the ligand-receptor interactions. Thus, a thorough understanding of the molecular mechanisms employed by M. tuberculosis when faced with host cells could potentially identify targets for antimicrobial design and lead to the development of novel therapies. The M. tuberculosis cell envelope is a complex structure comprised of polysaccharides, proteins and an array of lipids of which many are unique to mycobacteria. The major component of the cell wall is a complex of peptidoglycan and arabinogalactan that connects to a perpendicular layer of mycolic acids which forms the 52 inner leaflet of an asymmetric lipid bilayer (4, 5). The outer leaflet of the bilayer comprises a collection of surface lipids and glycolipids that non-covalently associates with the mycolic acids (4, 6). The outermost surface of the bacillus, which is referred to as the capsule layer, is a mixture of polysaccharides and proteins that intercalate with the surface lipids and glycolipids (7, 8). The initial interaction between the host cell and the bacterial surface is a critical event that likely influences both microbial recognition and the subsequent immune response to infection. Several studies have utilized purified cell wall components or coated polystyrene beads to investigate putative M. tuberculosis ligands for their ability to induce host signaling pathways or bind host cell receptors (9-11). The main drawback to these in vitro model systems is that they do not take into consideration the architecture of the cell envelope. Thus, there remains the distinct possibility that the cell wall factors under study are not exposed on the bacterial surface and are not available for receptor binding. An alternate strategy has been to employ genetic tools in order to facilitate the identification of cell wall moieties that are necessary for optimal binding to macrophages or epithelial cells. In non-tuberculous mycobacteria, genetic complementation has identified genes whose expression confers an increased host cell association (12-14). Similarly, several gene-disrupted mutants have also been characterized that display cell wall alterations which coincide with a reduced ability to bind host cells (15-18). There are also examples in the literature where gene inactivation actually results in an improved association between the bacteria and the host cell. M. smegmatis mutants lacking the cell wall glycopeptidolipids are more rapidly internalized by monocyte-derived macrophages 53 than wild type (19). In M. tuberculosis, the inactivation of the pks3-pks4 locus also coincides with an increase in macrophage binding due to the loss of certain cell wall lipids or the weakened retention of the surface capsule (20, 21). We have studied the role of the M. tuberculosis surface capsule during macrophage infection and demonstrated that that disruption of the capsule by brief sonication markedly improves the association of M. tuberculosis with cultured macrophages (22). Altogether, these findings suggest that cell wall modifications may expose additional cell wall components that are readily recognized by the macrophage. Furthermore, our results imply that the function of the surface capsule may be to support the presentation of some ligands while masking others, perhaps as a means to direct bacterial entry through a specific receptor (22-25). For the present study, we were interested in identifying M. tuberculosis genes that are involved during the initial stages of macrophage infection. A transposon library of M. tuberculosis was screened for mutants that showed an increased level of macrophage association, similar to that which we observed following mechanical disruption of the capsule using sonication (22). Our expectation was that this screening strategy would identify M. tuberculosis genes that contribute, in some manner, to the biogenesis of the cell wall. A transposon library containing 10,000 mutants was screened and a total of five mutants were selected for further analysis. The mutants displayed increased levels of macrophage association as compared to wild type and four of the mutants also showed reduced virulence in vivo. Two of the disrupted genes are likely involved in lipid biosynthesis, one encodes a hypothetical membrane protein and the two remaining genes encode hypothetical proteins with little functional information. 54 2.3 Materials and Methods Bacterial strains M. tuberculosis Erdman (ATCC 35801) and M. bovis B C G were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose complex (OADC) and 0.05% Tween-80 or on Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol and 10% OADC. OADC was prepared as described previously (26). M. bovis B C G transformed with a plasmid harbouring the gene coding for the green-fluorescent protein (GFP) was cultured as above but with the addition of 50ug/ml of hygromycin (27). Single cell suspensions of mycobacteria for use during infection assays were prepared using two separate techniques, essentially as described previously (22). Briefly, the bacteria were pelleted by centrifugation (16,000x g for 5 min) and resuspended in 500pl of binding medium (138 m M NaCl, 8.1 m M Na 2 HP0 4 , 1.5 m M K H 2 P 0 4 , 2.7 m M K C l , 0.6 m M CaCl 2 , 1 m M M g C l 2 , and 5.5 m M D-glucose) (28). The bacterial clumps were then dispersed with 10 repeated passages through a 25-gauge needle attached to a lml syringe or with three 30 second pulses of bath sonication using a VC50T 50-W microcup horn (Sonics & Materials) as indicated for each experiment (22). The volume of the cell suspension was then adjusted using binding medium to achieve the desired bacterial concentration. Transposon mutagenesis A transposon library of M. tuberculosis containing approximately 10000 mutants was generated using the phAE87 vector and Tn5370 as described previously (29). 55 Briefly, a 100ml culture of M. tuberculosis was grown to an OD600 of 0.8-1.0. The cells were washed twice and resuspended in a final volume of 10ml of phage adsorption buffer (10 m M Tris/HCl, pH 7.6, 100 mM NaCl, 10 m M M g S 0 4 , 2 mM CaCl 2). The bacteria were infected with the phage lysate at a multiplicity of infection (MOI) of 10:1 (phage particle:bacteria) for 4 hrs at 39°C. The infected cell suspension was centrifuged at 16000xg for 5 min to recover the bacteria before plating onto selective media. After 4-6 weeks of growth, hygromycin-resistant colonies were scraped from the agar plates, cultured in 7H9 and then stored at -80°C in pools each containing approximately 500 mutants. A total of 20 library pools were used in the screening experiments. Infection of THP-1 cells The human macrophage-like cell line, THP-1 (ATCC TIB202), was cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Gibco), lOmM L -glutamine (Gibco) and lOmM sodium pyruvate (Gibco) and maintained in an atmosphere of 5% CO2 at 37°C as described previously (30). For infection assays, THP-1 cells were seeded onto acid-washed glass coverslips in a 24 well tissue culture plate (2x105 cells/well) or in T-75cm flasks (5x10 cells) and differentiated in the presence of lOOnM of phorbol 12-myristate 13-acetate (Sigma-Aldrich) over 3 days. Prior to infection, the monolayers were washed with binding medium to remove any residual P M A . The differentiated THP-1 cells were infected with mycobacteria for three purposes; i) to conduct competitive binding assays as proof-of-principle experiments to show the efficacy of the screening strategy, ii) to screen the transposon library and iii) to test the macrophage binding affinities for the mutant clones selected for further study. 56 For i) and iii), syringed and sonicated suspensions of mycobacteria were prepared as described above and used to infect THP-1 monolayers on coverslips. To maximize the level of bacterial association, we selected a high MOI of 100:1 (bacteria: macrophage) for a relatively short infection period of 3 hrs. At the end of the infection period, the monolayers were washed, fixed (10 min in 10% formaldehyde in ethanol) and stained using Kinyoun's Carbol Fuschin and malachite green. For the competitive binding assays using BCG-GFP and wild type B C G , the association of sonicated BCG-GFP was done using fluorescence microscopy. The slides were then stained as described above and the association of acid-fast bacteria (syringed B C G and sonicated BCG-GFP) was counted using light microscopy. The percentage of infected macrophages was determined by counting 100 macrophage cells for each coverslip. For procedure ii), THP-1 cells cultured in T-75cm2 flasks and on coverslips were infected with the library pools (syringed suspensions) at an MOI of 100:1 (bacteria:macrophage) for 4hrs. Non-adherent mycobacteria were removed by repeated washing of the monolayer. A high MOI and a longer period of infection was employed for the library screening in order to increase the probability of macrophage association for the desired mutants within the input pool from the transposon library. The infected coverslips in the 24 well plate were used to monitor the level of bacterial binding for each mutant pool by microscopic count as described above. The infected THP-1 cells in the T-75cm flasks were used to enrich for mutants with enhanced host cell binding capabilities; the THP-1 cells were dislodged using a cell scraper, resuspended in phosphate-buffered-saline containing 0.1 % Tween-80 (PBS-Tween) and briefly sonicated to lyse the macrophages. The recovered bacteria that were macrophage-associated were then 57 cultured for 7-10 days in 7H9 and used in a subsequent THP-1 binding selection experiment. After six to eight rounds of enrichment, mutant pools that displayed an increased level of macrophage association (as determined by microscopic count) were plated onto selective agar plates. Individual mutant clones were then assessed for macrophage infectivity as described for procedure iii). Identification of the transposon insertion site Genomic D N A from the selected transposon mutants was extracted (31) and analyzed by Southern hybridization to verify that a single copy of the transposon had incorporated into the chromosome. Briefly, the genomic D N A was digested with Pvu-II, which does not cut within the transposon, and transferred onto nylon membrane as described elsewhere (32). The blots were hybridized with a DIG-labeled D N A fragment derived from the hydrogmycin-resistance gene according to the directions from the manufacturer (Roche Applied Science). The precise location of the transposon insertion was determined by sequencing the amplicons generated by ligation-mediated inverse PCR (33). Briefly, 2.5 pg of D N A was digested with Rsa-I. The restriction digest was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. A n aliquot of the digested D N A (~200ng) was ligated overnight at 16°C, followed by ethanol precipitation. The D N A sequences adjacent to the transposon were amplified using primers corresponding to the left (084L-F 5'-G T C A T C C G G C T C A T C A C C A G - 3 ' and 084L-R 5 ' -AACTGGCGCAGTTCCTCTGG-3') and right (084R-F 5 ' -ATACACGCGCACCGGTTCTAGC-3 ' and 084R-R 5'-58 CACGGCGAACCGCTGGTG-3 ' ) ends of the transposon. Sequence comparisons were done using B L A S T 2.0 at www.ncbi.nlm.nih.gov/. Mouse experiments Broth cultures of the bacterial strains were normalized by O.D.6oonm and diluted to an estimated concentration of 4xl0 7cfu/ml. The bacterial suspensions were used to deposit 500-1000 bacilli into the lungs of CD-I mice (4-6 weeks of age, Charles River) via aerosol inhalation using a Glas-Col inhalation chamber. For morbidity studies, the health status of the infected mice (8 mice per strain) was monitored by visual inspection and by determining weight loss on a weekly basis. Animals were euthanized upon evidence of morbidity in accordance with the Canadian Council On Animal Care guidelines. Mice were also infected with mutants that were chosen at random from the transposon library in order to confirm that the transposon construct itself was not adversely affecting bacterial virulence. For these studies, CD-I mice (4 mice per group) were infected as described above and the bacterial load in the lungs and spleens was measured at 28 days post infection. Briefly, the infected mice were euthanized and the organs were aseptically removed and homogenized. Serial dilutions of the organ homogenates were plated onto 7H10 supplemented with hygromycin for the transposon mutants and 7H10 for wild type M. tuberculosis. Statistical Analysis The results shown are the means + standard error of the mean. Statistical significance between groups was determined with the two-tailed, unpaired Student's t test 59 or analysis of variance. The survival curve data was analyzed using the log-rank test. P values < 0.05 were considered significant. 2.4 Development of a l ib ra ry screening methodology We devised a biological screen that relied on iterative rounds of macrophage infection to identify M. tuberculosis mutants that possessed an increased affinity to bind macrophages. As outlined in Figure 2.1, large pools of mutants from the transposon library were used to infect TFfP-1 cell monolayers for a period of three hours. Mutants that associated with the host cells were recovered, cultured in broth and used as the inoculum in a subsequent TFfP-1 infection experiment. We hypothesized that enrichment of the desired phenotype would occur within the output mutant population after successive rounds of selection. Nevertheless, before we embarked on the library screening, we first needed to establish whether this approach would be sufficiently sensitive to select for a mutant phenotype that was likely a minor representative of the entire library. To address this issue, we conducted competitive binding assays using differentially labeled suspensions of M. bovis B C G that had undergone mechanical treatments to alter the surface properties of the bacilli. Previous findings from our group have shown that short bursts of sonication disrupt the capsule layer of the cell envelope and result in significantly higher rates of macrophage association (22). Milder treatments, such as repeated passages through a syringe, do not affect capsule integrity and have no impact on host cell binding (22). Therefore a syringed suspension of M. bovis B C G and a sonicated suspension of M, bovis B C G transformed with a GFP-plasmid 60 A. Infect THP-1 cells D. Microscopic count of bacterial binding B. Detergent lysis C. Culture the macrophage-associated bacteria Figure 2.1 Library screening to identify mutants with an increased macrophage binding affinity. (A) Pools of transposon mutants from the library were serially passaged using binding to THP-1 cells as the selection criteria. (B) Cell-associated bacteria were recovered from the monolayer. (C) These bacteria were cultured in broth and used as the infecting inoculum in the next round of selection. (D) The level of bacterial binding was assessed at each passage by microscopic count. 61 (BCG-GFP) were combined at various ratios and used to infect THP-1 cell monolayers. As shown in Figure 2.2, when the two B C G strains were equally represented in the initial inoculum (1:1 ratio), the sonicated bacteria were the dominant organisms associated with the monolayer. Even when the sonicated bacteria were a small percentage of the initial inoculum, they continued to represent a significant proportion of the bacteria that were cell-associated. Thus by analogy, we reasoned that i f the desired mutant was present within a pool of library mutants, the screening strategy should enable the mutant to increase its representation within the output mutant population over sequential infection assays. 2.5 Transposon mutagenesis and selection A transposon library of M. tuberculosis was generated using the Tn5370 transposon in conjunction with a temperature-sensitive mycobacteriophage delivery vector (29). Previous studies have shown that the integration of this transposon may not be completely random as there is evidence of some hot-spots for insertion (34). We conducted Southern blot analysis from a collection of mutants that were chosen at random from the library. As shown in Figure 2.3, the hybridization revealed that significant diversity appears to exist within the library. For the library screening, a total of 20 pools, each containing ~500 mutants, were used to infect differentiated THP-1 monolayers as outlined in Figure 2.1. Between the sixth and eighth round of serial macrophage infections, three library pools emerged that consistently bound the cell monolayers more readily than the syringed M. tuberculosis control (Figure 2.4). The three pools were plated onto 7H10 agar and Southern blot analysis was used to identify 62 70 i a i so -SYR SON 1:1 10:1 20:1 50:1 100:1 syringed BCG: sonicated BCG-GFP Figure 2.2 Competitive binding of syringed and sonicated mycobacteria. Bacterial suspensions of B C G and BCG-GFP were syringed and sonicated, respectively to generate single cell suspensions. The two strains were combined at ratios of 1:1, 10:1, 20:1, 50:1 and 100:1 (syringed B C G : sonicated BCG-GFP) and used to infect THP-1 monolayers for 3 hrs. The percentage of the macrophage population binding > 10 B C G -GFP was determined by fluorescence microscopy (white bars). The slides were then stained and the percentage of macrophages binding >10 acid fast bacilli (both B C G and BCG-GFP) was determined by light microscopy (black bars). The mean + S E M is shown for three independent experiments each conducted in triplicate (N=9). 63 12 3 4 5 0 7 8 91011 ! } ' 5 8 7 8 9 10 11 kb 23.1-9.4 -6.8 -4.3 -2.3 2.0 23.1 _ 7 8 9 10 11 12 13 14 IS 16 Figure 2.3 Southern hybridization of randomly selected mutants Genomic D N A from individual mutants chosen at random from the library was digested with Pvu-II and subjected to Southern blot analysis using the hygromycin resistance gene from the transposon as a probe. The pattern of hybridization indicates mutant diversity within the library. 64 Figure 2.4 Pools of mutants that exhibit increasing levels of macrophage association During the library screening, three mutant pools (A-C) were identified that showed increasing levels of macrophage association with each round of selection. The level of macrophage binding was determined by microscopic count of fixed monolayers. The graphs represent the % of macrophages binding >10 syringed-treated M. tuberculosis (white bars) or > 10 syringed-treated bacteria from the library pool (black bars). The data shown in the mean + S E M of three infected coverslips (* denotes P < 0.05 when compared to syringed M. tuberculosis). 65 100 80 A o A „ U> ~ £ 5 60 c « e g. 40 20 Pool A o *™ A a; CD ™ C « c w e £ 100 80 £ 2 60 40 A 20 H Poo! B • • i i J 6 7 8 Round of Select ion Figure 2.4 Pools of mutants that exhibit increasing levels of macrophage association 66 the dominant siblings within each pool (data not shown). 2.6 Molecular characterization of mutants that display enhanced macrophage binding The site of transposon insertion was identified for the selected mutants using inverse-PCR and D N A sequencing which yielded a total of five mutants shown in Table 2.1. The level of macrophage association was tested using a pure culture of each mutant that was syringed prior to infection. As shown in Figure 2.5, all of the mutants identified from the selection procedure exhibited a binding phenotype that more closely mimicked the sonicated preparation of wild type M. tuberculosis rather than syringed M. tuberculosis. The increases in macrophage infectivity suggested that the transposon insertions lead to an altered bacterial surface and an increased recognition by the host macrophage. 2.7 Persistence of the mutant strains in vivo A subset of the identified mutants was examined for their ability to cause disease within the mouse model of infection. The parental wild type and four of the library mutants (Apks6, ARvl505c, AfadD23, ARv3335c) were used to infect mice via aerosol and mouse survival was monitored over time. As shown in Figure 2.6, all of the mutants analyzed displayed an impaired ability to cause morbidity in mice as compared to M. tuberculosis. In the first experiment (Figure 2.6A), mice were infected with M. tuberculosis, ARv1505c or AfadD23. The median survival for the wild type-infected mice was 132 days, while mice infected with the mutants did not reveal any signs of 67 M. tuberculosis H37Rv Gene ID Gene Name Predicted function Rv3826 fadD23 Fatty-acyl A M P ligase Rvl505c Rvl505c Conserved hypothetical protein Rv0405 pks6 Polyketide synthase Rv3335c Rv3335c Probable conserved integral membrane protein R v l l l 5 RvlllS Possible exported protein Table 2.1 Mutants isolated from the transposon library that exhibit an increased binding affinity to THP-1 cells. 68 100 Figure 2.5 Macrophage binding of the mutants identified from the l ib rary selection. The individual mutant clones were cultured to logarithmic phase and syringed prior to infecting THP-1 macrophages. Wild type M. tuberculosis, either syringed (SYR) or sonicated (SON), was included as controls. Black bars represent the % of macrophages associated with at least one bacterium, the gray bars represent the % of macrophages associated with greater than 10 bacteria. The mean + SEM is shown for two independent experiments each with triplicate coverslips (N=6). * P< 0.05, ** PO.OOl when compared to syringed M. tuberculosis. 69 A. 120-- 100-co | 80H 3 8 40H MTB •Tn::Rv1505c -&-Tn::fadD23 -T— 20 40 60 80 100 120 140 160 180 200 Time in Days B. 120 100 co I 80H I 60H c o 40H °" 20H MTB Tn::pks6 Tn::Rv3335c 50 100 150 200 250 Time in Days 300 Figure 2.6 Murine survival studies with the selected library mutants Survival studies for four of the mutants strains in comparison to wild type M. tuberculosis (MTB) were conducted over two experiments. (A) Mice were infected via aerosol with M T B , Tn::Rvl505c or Tn::fadD23. (B) Mice were infected with M T B , Tn::pks6 or Tn::R3335c. Time till morbidity (loss of 15% body weight) was followed over time. The Kaplan-Meier survival curves were analyzed using the log-rank test (GraphPad Prism). 70 disease throughout the 180 day experiment. In the second experiment, the median survival for the M. tuberculosis-infected mice was 210 days (Figure 2.6B). When this experiment was terminated at Day 256, 5/8 mice for the pks6 group and 6/8 mice for the Rv3335c did not show any signs of disease. Analysis of the survival curves using the log-rank test indicated significant differences between mice infected with M. tuberculosis and the Rvl505c (P=0.0001) and fadD23 mutants (P=0.0001) and between M. tuberculosis and the pks6 (P= 0.0429) and Rv3335c (P= 0.0102) mutant strain. To confirm that the reductions in virulence were not merely a byproduct of the mutagenesis, we compared the in vivo replication of the fadD23 mutant with three mutants that were selected from the library at random. As shown in Figure 2.7, the bacterial loads within the lungs and spleens of mice infected with the fadD23 mutant were significantly reduced compared to either the parental wild type (P< 0.001) or the random transposon mutants (P< 0.001). It should be noted that bacterial loads in the lungs of wild type-infected mice was also significantly higher than the randomly selected mutants (P< 0.001). However, no significant difference existed in the splenic counts from mice infected with the randomly selected mutants and wild type M. tuberculosis. Thus while transposon mutagenesis may have some detrimental effect on the virulence of M. tuberculosis in the lungs, the striking loss of virulence seen with the fadD23 mutant could not be attributed to any non-specific effects from the transposon insertion into the genome. 2.8 Discussion 71 Figure 2.7 Virulence of randomly selected transposon mutants as compared to wild type and the fadD23 mutant. M. tuberculosis (MTB), Tn::fadD23 and three mutants selected from the library at random (Tnl, Tn2, Tn3) were assessed for bacterial growth within CD-I mice (four to five mice per group). At four weeks post infection, the number of cfu in the lungs (Panel A) and spleens (Panel B) was determined by plate count. (** P< 0.001 when compared to Tn: :fadD23, f P < 0.001 when compared to MTB) . 72 It is well known that some mycobacterial species have a strong tendency to aggregate during culture, even in the presence of dispersal agents such as Tween-80. As a result, mechanical treatments such as repeated syringing, pulses of sonication or shaking with glass beads are commonly used to break up bacterial clumps that would otherwise interfere during in vitro infection studies. Previously, we reported that these methods vary in their impact on the surface properties of the bacteria; sonication of an M. tuberculosis suspension disrupted the outermost capsule layer of the cell envelope which surprisingly, also translated into an increased infectivity of cultured macrophages (22). We hypothesized that this observation could be used as a library screening tool to identify mutants that had incurred cell wall alterations as a result of mutagenesis. In this investigation, we screened a transposon library of M. tuberculosis using successive rounds of infection assays with THP-1 cell monolayers. Five mutants were identified from the screen that exhibited an increased binding affinity for host cells as compared to the parental wild type. Two of the mutants identified have been previously linked to M. tuberculosis cell wall processes. The pks6 gene codes for a predicted polyketide synthase that was initially identified by signature-tagged mutagenesis (35). In that study, the pks6 mutant in M. tuberculosis strain Mtl03 was impaired for growth in the lungs of Balb/c mice (35). A pks6 mutant in M. tuberculosis was also isolated by differential screening of a signature-tagged library (36). The replication rates of a pks6 mutant in different transgenic mice suggested that pks6 is required for countering host defense mechanisms that involve IFN-y but separate from pathways that include the inducible nitric oxide synthase (36). In addition, studies in M. bovis B C G have shown that pks6 expression is 73 induced following macrophage infection (37). Although the precise biological function of the pks6 in M. tuberculosis will require further study, a recent report suggests the involvement of the pks6 in the synthesis of polar lipids within the cell envelope (38). The fadD23 gene encodes a fatty-acyl Co A ligase (39) that likely participates in sulfolipid biosynthesis. The fadD23 gene is adjacent to the pks2 gene on the M. tuberculosis genome which encodes the polyketide synthase that is required for the synthesis of the acyl components of the cell wall sulfolipids (40). Early studies demonstrated strong immunomodulatory properties of the sulfolipid family however, a potential contribution to bacterial virulence has been recently subject to debate (41-43). The Rv3335c gene codes for a predicted membrane protein that is conserved in both M. bovis and M. leprae. B L A S T analysis of the amino acid sequence reveals the presence of two overlapping conserved domains; pFAM03631 and COG1295. Proteins within these families consist primarily of hypothetical membrane proteins from various bacterial species as well as the Ribonuclease B N tRNA-processing enzymes. A transposon insertion within the Rv3335c gene has been previously shown to be defective for growth within the spleens of C57/BL6 mice using the T R A S H system (44). Rvlll5 and Rvl505c both encode hypothetical proteins with little functional information. The disruption of the Rvlll5 gene has been linked to an inability to control phagosomal maturation in M. bovis B C G , leading to localization of the bacteria within acidified phagosomes (45). In a separate study, an Rvlll5 mutant was isolated from a transposon library of M. tuberculosis H37Rv and was unaltered for virulence within the SCID mouse model of infection (34). The predicted protein of Rv1505c harbours a conserved domain that is associated with enzymes belonging to the hexapeptide repeat 74 superfamily of acetyltransferases (46). This collection of proteins includes enzymes that function in a wide variety of roles including amino acid and cell wall bisosynthesis (46-48). To date much of the research surrounding the interaction between M. tuberculosis and the host macrophage has centered on the host cell receptors that can mediate bacterial internalization. Furthermore, while great strides have been made in elucidating the chemical composition of the mycobacterial cell envelope, much less is known about the biological function of the different cell wall moieties during infection. Several studies have reported that differences within the cell envelopes of various M. tuberculosis strains can contribute to the progression of disease. Recently, Reed et al. demonstrated that M. tuberculosis isolates belonging to the W-Beijing family express a phenolic glycolipid that is linked to the hypervirulence of these strains in animal models of infection (49, 50). Cywes et al. reported differences in the levels of capsule production amongst different laboratory strains and clinical isolates of M. tuberculosis that correlated with the bacterial binding affinity to complement receptor 3 on host cells (23). 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In vivo phenotypic dominance in mouse mixed infections with Mycobacterium tuberculosis clinical isolates. J Infect Dis 192:600. Chapter 3 80 Disruption of fadD23 in Mycobacterium tuberculosis affects sulfolipid production and virulence 3.1 Preface This chapter has been submitted for publication as: Lynett, J. and R.W. Stokes. Disruption offadD23 in Mycobacterium tuberculosis affects bacterial sulfolipid production, interaction with macrophages and virulence. Submitted to Cellular Microbiology. March 2006 . 3.2 Introduction Infection with M. tuberculosis, the causative agent of tuberculosis continues to be an enormous burden to public health worldwide. It is estimated that up to one third of the world's population is infected with the bacillus resulting in nearly 2 million deaths each year (1). The cell wall of M. tuberculosis has been proposed as a key mediator for protection against the antimicrobial defenses of the host and is also thought to play a role in modulating the immune response to infection (2, 3). At the onset of infection, the initial recognition and binding of the bacteria by the macrophage can proceed through a number of host receptors (3, 4) and accordingly, there are likely numerous M. tuberculosis ligands that are exposed at the bacterial surface. Recent studies have detailed the involvement of specific mycobacterial lipids during binding and uptake of M. tuberculosis by host cells (5-9). Mycobacterial lipids have also been implicated in the intracellular survival of M. tuberculosis by contributing to the aberrant maturation of the mycobacterial phagosome (10-12). Furthermore, lipid effectors affect cytokine production from macrophages either by lipid release from the phagosome (13-15) or by 81 shedding from extracellular organisms (16-19). Thus there is growing interest with regard to the lipid constituents of the cell that can influence the interactions with the host. The backbone of the M. tuberculosis cell wall is a covalently linked complex of peptidoglycan, arabinogalactan and mycolic acids that interacts with a collection of surface lipids to form an asymmetric lipid bilayer (20). The surface lipids are also thought to intercalate with the overlying surface capsule (21). Within this group of lipids are a number of glycolipids that share a common structural theme; namely a trehalose core that is modified with various acyl chains. This subgroup includes the strongly immunogenic mycobacterial cord factor, the diacylated and polyacylated trehaloses (DAT, PAT) and the sulfolipids (22). The sulfolipid contingent of the M. tuberculosis cell wall includes five structurally related lipids that differ in the number and composition of acyl chain residues (23, 24). The principal sulfolipid is known as sulfolipid-1 (SL-1) and its structure has been elucidated as 2,3,6,6'-tetra-acyl-trehalose-2'-sulfate. The four modifying acyl chains include two hydroxyphthioceranic residues, one phthioceranic acid residue and either a palmitic or a stearic acid residue (23, 25). SL-1 has been historically linked, albeit indirectly, to the virulence of M. tuberculosis. Early reports utilizing a panel of clinical isolates described a significant correlation between sulfolipid abundance and strain virulence in guinea pigs (26). In addition, the administration of SL-1 to mice in combination with cord factor resulted in a synergistic increase in the level of cord factor-induced toxicity (27). Studies conducted using cells cultured in vitro have also attributed strong immunomodulatory activity to SL-1. Human monocytes pretreated with SL-1 are subsequently resistant to the activating effects of lipopolysaccharide (LPS) or IFN-y, leading to a block in the release of 82 superoxide (28). The opposite effect was observed with human neutrophils, where high doses of SL-1 actually induced the release of superoxide (29). In contrast to these compelling findings, recent studies using isogenic mutants have failed to clearly identify a direct requirement for SL-1 in disease pathogenesis. Disruption of the polyketide synthase (pks2) responsible for generating the phthioceranic and hydroxyphthioceranic acyl groups of SL-1 (25) had no effect on mycobacterial replication within macrophages (30). Furthermore, the disruption of pks2 in M. tuberculosis Erdman strain or H37Rv did not affect bacterial growth within the lungs of mice (30, 31). The mmpL8 gene, located downstream of pks2, encodes a membrane transporter responsible for shuttling sulfatide precursors across the cytoplasmic membrane (31, 32). Interestingly, the disruption of mmpL8 leads to disparate in vivo phenotypes depending on the initial route of infection (31, 32). Converse et al. reported that mice infected via intravenous injection with a mmpL8 deletion mutant constructed in M. tuberculosis Erdman strain had significantly reduced bacterial loads in the lung, liver and spleen as compared to wild type-infected mice by as early as 6 weeks post infection (31) . A more subtle in vivo phenotype was reported in mice infected via aerosol with an mmpL8 mutant constructed in M. tuberculosis H37Rv. Under these conditions, a difference was only noted during long term mouse survival studies, whereas the in vivo replication rates were indistinguishable between the mutant and the parental wild type (32) . Taken together, these studies do support the conclusion that, although mature SL-1 is not directly required for M. tuberculosis replication during in vivo infection, (as evidenced by the pks2 knockouts), the interruption of SL-1 biosynthesis by inactivation of the putative MmpL8 transporter can impact bacterial virulence (31, 32). The MmpL8 83 transporter may also have additional functions in M. tuberculosis separate from SL-1 synthesis that impact replication and persistence in vivo (31). The genomic region surrounding the pks2 and mmpL8 genes likely includes other loci that are also involved in SL-1 production. Immediately upstream of the pks2 gene lies a putative fatty acyl-CoA ligase (fadD23) (33). Recently, the functional classification of the 36 fadD genes within the M. tuberculosis genome has revealed that a subset of these genes do not code for fatty acyl-CoA synthetases as originally annotated (34); rather this group of 12 genes, which includes fadD23, actually encode fatty acyl-A M P ligases (FAAL) and function in lipid biosynthesis rather than in lipid degradation (33, 35). In this report, we characterize the effects of a transposon disruption within the fadD23 gene of M. tuberculosis. The mutant is devoid of mature sulfolipid in the cell envelope as assessed by thin-layer chromatography (TLC). In addition, the fadD23 mutant associates with macrophages more readily than wild type, however its intracellular survival within these cells is reduced as compared to wild type. Bacterial replication of the fadD23 mutant was attenuated in vivo and this phenotype was partially complemented in the presence of the wild type gene. 3.3 Materials and Methods Bacterial strains and cells M. tuberculosis Erdman strain (ATCC 35801) was grown in Middlebrook 7H9 broth (Difco) or Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol, 10%o oleic acid-albumin-dextrose complex (OADC) (Becton-Dickinson) and 0.05% Tween-80. For the preparation of lipid extracts, mycobacteria were cultured in glycerol-alanine-salts 84 (GAS) media (36). Escherichia coli strains were grown in Luria-Bertani (LB) broth.or on LB-agar plates. When required, hygromycin and kanamycin were used at 50pg/ml and 25 ug/ml respectively. Identification of the fadD23 mutant and genetic complementation The fadD23 mutant was isolated from a transposon library constructed in M. tuberculosis Erdman strain by screening for mutants that preferentially associated with THP-1 cell monolayers (37). The transposon insertion within the fadD23 gene was confirmed by inverse-PCR as described previously (37, 38) and by PCR using primers that flank the site of transposon integration. The FADD-L 5'-T C T G A C A A C A T C C G C G A T A A - 3 ' primer corresponds to nucleotides (nt.) 348-368 and the FADD-R 5 ' - G T C A C T T C G A A G G C G A A G T T - 3 ' primer matches nt. 807-827 of the fadD23 gene. The complementation construct was generated by amplifying the fadD23 gene along with 455 bp of upstream sequence from chromosomal D N A of M. tuberculosis (39) using Pfu D N A polymerase (Stratagene) using the following primers (FADJLL 5'-C G T A G A A C T C G T C G C A A T C C -3' and FADJLR 5 ' - G T G G A T C C G A C C G T A A G A C C -3'). The amplicon was blunt-end ligated into the mycobacterial integration vector, pMV306, that had been digested with EcoRV (40). The resulting plasmid construct was sequenced and transformed into the mutant by electroporation as described elsewhere (41). Transformed bacteria were recovered using kanamycin selection. The presence of the wild type fadD23 gene was confirmed by PCR in the complemented mutant using the F A D D - L and FADD-R primers. 85 Infection of THP-1 cells The human macrophage-like cell line, THP-1 (ATCC TIB202), was maintained at 37°C and 5% C 0 2 in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Gibco), lOmM L-glutamine (Gibco) and lOmM sodium pyruvate (Gibco). For infection assays, THP-1 cells were seeded onto acid-washed glass coverslips (2x105 cells/coverslip) and differentiated over 3 days in the presence of 1 OOnM of P M A (Sigma-Aldrich). The monolayers were washed twice with binding medium (42) prior to infection in order to remove any residual P M A . Single-cell suspensions of mycobacteria were prepared as described previously (43). Briefly, frozen stock vials for each strain were thawed, centrifuged and resuspended in binding medium by syringing ten times using a 25-gauge syringe. The cell monolayers were infected for a period of 3 hrs using an MOI of 50:1 (bacteria:macrophage) to monitor intracellular survival as described previously (44). At the end of the infection period, the monolayers were washed three times with binding medium to remove any non-associated bacteria. The infected coverslips were transferred to new wells containing 1ml of supplemented RPMI. Fresh media was added to the wells on every second day and the infected monolayers were processed for viable colony forming units (cfu) on days 0, 4 and 7. On day 0, the infected coverslip was added to 2ml of PBS supplemented with 0.1% Tween-80 (PBS-Tween). The suspension was briefly sonicated to lyse the monolayer and disperse bacterial clumps. Serial dilutions of the suspension were plated onto 7H10 agar. On days 4 and 7, the overlying media as well as two washes of PBS-Tween were pooled together with the infected coverslip before sonication and plating. After 3-4 weeks of growth, colonies were enumerated and used to 86 calculate the number of bacteria for each infected cell monolayer. The intracellular doubling times for the bacterial strains were calculated with the equation N t = N 0 e k t where N t = number of bacteria at day 7, N 0 = number of bacteria at day 0, k = growth rate constant and t = time. Lipid analysis Apolar and polar lipids were extracted from heat killed, lyophilized bacterial pellets as described previously (45). Lipids were separated by two-dimensional thin layer chromatography (2D-TLC) on Silica Gel 60-plates (EM Science) using five different solvent systems (A-E) that have been described for mycobacterial lipid analysis (45, 46). Sulfolipids were localized using solvent system D which uses chloroform:methanol:water (100:14:0.8) for the first dimension solvent system and chloroform:acetone:methanol:water (50:60:2.5:3) for the second dimension. Lipids were visualized by spraying TLC plates with either 5% ethanolic phosphomolybdic acid or 2.5% tt-naphthol-10% sulfuric acid followed by charring. SL-1 was also detected by staining TLC plates with cresyl violet as described elsewhere (47). Briefly, the lipids were resolved on T L C plates using solvent system D and the plates were immersed in a solution of 0.02%o cresyl violet in 1% aqueous acetic acid for 10 min followed by briefly destaining with 1%> acetic acid. To detect the cell wall mycocerosates, the apolar lipid fractions were analyzed by TLC using solvent system A. Briefly, plates were developed three times in the first dimension solvent system [petroleum ethenethyl acetate (98:2)] and once in the second dimension solvent system [petroleum ethenacetone (98:2)]. The 87 plates were sprayed with 5% ethanolic phosphomolybdic acid and charred for lipid detection. Growth and persistence in mice Actively growing broth cultures of the M. tuberculosis strains were used to infect female CD-I mice (4-6 weeks old) via aerosol inhalation. The Glas-Col inhalation chamber was set up to deposit approximately 500-1000 bacteria into the lung. At each experimental time point, four mice per group were euthanized and the lungs and spleens were aseptically removed and homogenized in PBS-Tween. Serial dilutions of the organ homogenates were plated onto 7H10, 7H10-hygromycin or 7H10-hygromycin/kanamycin. Statistical Analysis Data are expressed as the mean + standard error of the means. The two-tailed Student t test or two-way A N O V A followed by the Tukey-Kramer Multiple Comparisons Test were performed as appropriate, using GraphPad Prism Software. Differences were considered significant with a P < 0.05. 3.4 Identification of the /a</D23mutant As outlined in Chapter 2, previous findings from our group have reported that brief pulses of sonication of an M. tuberculosis suspension leads to a marked increased in the level bacterial binding to macrophages (43). We subsequently screened a transposon library of M. tuberculosis to select for mutants that also displayed increases in 88 macrophage infectivity. Our hypothesis was that the selected mutants would exhibit an altered cell wall composition that allowed the exposure of additional ligands at the bacillus surface (37). One particular mutant was repeatedly isolated from the library screening and the transposon insertion mapped to nucleotide (nt.) 630 of the fadD23 gene, shown in Figure 3.IB. The fadD23 mutant displayed an increased binding affinity to THP-1 cells as compared to wild type (P < 0.05). Complementation of the mutation with the fadD23 gene resulted in binding levels that were intermediate between the wild type and the mutant (Figure 3.1C). 3.5 Survival of the fadD23 mutant within THP -1 cells One of the hallmarks of mycobacterial virulence is the ability to survive within the phagosomal environment of the macrophage. As evidenced by the library screening, the disruption of the fadD23 gene leads to changes that affect the bacterial interaction with the host macrophage (37). To determine whether the disruption of fadD23 would also impact the intracellular replication of M. tuberculosis, we infected THP-1 monolayers with the wild type, the mutant and the complemented strain and monitored bacterial growth over 7 days. As shown in Figure 3.2, the fadD23 mutant exhibited a significant growth defect within macrophage monolayers as compared to M. tuberculosis (P< 0.001). Complementation of the mutant with the fadD23 gene in trans reversed this phenotype resulting in significantly higher bacterial numbers at day 7 as compared to the mutant (P< 0.05). Importantly, all three bacterial strains exhibited similar growth in broth culture. The calculated bacterial doubling times also reflect the impaired 89 Figure 3.1 Disruption of the fadD23 gene results in an increased association between M. tuberculosis and macrophages. (A) Structural schematic of sulfolipid-1. Adapted from Mongous et al. (48). (B) Diagram of the genomic region that encompasses the fadD23 gene showing the site of transposon insertion at nt.630. (C) Bacterial association of M. tuberculosis (MTB), AfadD23 and the complemented strain (complement) to THP-1 monolayers. The binding level of the fadD23 mutant is significantly increased relative to wild type (* P < 0.05,), data shown is the mean + S E M of three independent experiments, each conduced in triplicate (N=9) 90 Hydroxyphthioceranic o OH -O3SO Hydroxyphthioceranic O O OH C16-20 VVi -H-o Phthioceranic mmpL8 papA1 pks2 fadD23 Figure 3.1 Disruption of the fadD23 gene results in an increased association between M. tuberculosis and macrophages. 91 100 0 2 4 6 8 DAYS Figure 3.2 Disruption of fadD23 impairs the intracellular growth of M. tuberculosis Differentiated THP-1 cells were infected with M. tuberculosis (<>),fadD23 (•) and the complemented strain (A) and bacterial growth was monitored by colony count. At day 7 post infection, macrophages infected with the fadD23 mutant harbored significantly fewer bacteria than macrophages infected with the wild type or the complemented strain (* P< 0.05 , ** P< 0.001 when compared to the fadD23 mutant). Results shown are the mean + S E M from three independent experiments each conducted in triplicate (N=9). 92 growth of the fadD23 mutant within the macrophage. The doubling time of the parental wild type (21.6 + 0.5 h) was significantly faster than that of the fadD23 mutant (31.3 + 1.7 h) (P< 0.001 ). The doubling time of the complemented mutant (24.0 + 0.3 h) was also significantly faster relative to the mutant (P< 0.001). The doubling times of wild type and the complemented mutant were not significantly different. 3.6 L i p i d analysis of the/<w/Z)2Jmutant As outlined in the Introduction, several groups have reported on the biochemical characterization of the Pks2 and MmpL8 proteins and have confirmed their involvement in SL-1 production (25, 30-32). To investigate whether the fadD2 3-encoded protein also participated in SL-1 biosynthesis, we extracted the apolar and polar lipid fractions from whole cell pellets of the parental wild type, the mutant and the complemented strain for analysis using TLC. As shown in Figure 3.3A, comparison of the apolar lipid fractions revealed a lack of SL-1 production in the absence of the fadD23 gene. Importantly, SL-1 production could be restored following complementation of the mutant with the wild type fadD23 gene. The loss of SL-1 production in the fadD23 mutant was also confirmed with cresyl violet staining of the TLC plates (Figure 3.3B) which specifically stains the sulfatides a blue colour, while cord factor is stained violet (47). Interestingly, both the mutant strain and the complemented strain also seemed to produce a greater abundance of cord factor as compared to wild type. This could signal alterations in the utilization of trehalose which is the carbohydrate component in both cord factor and SL-1. In addition, the TLC plates from the mutant and the complemented strains also reveal the presence of a novel lipid. While this unidentified lipid has a 93 Figure 3 . 3 Disrupt ion of fadD23 in M. tuberculosis leads to a loss of S L - 1 production. (A) Apolar lipids from M. tuberculosis (MTB), AfadD23 and the complemented strain were separated by 2D-TLC to resolve SL-1. Plates were stained with a-naphthol and charred. (B) TLC plates stained with cresyl violet to specifically identify SL-1. DAT: diacyltrehalose, CF: cord factor, SL-1: sulfolipid-1, ?: unknown lipid. 94 similar retention to SL-1 during the first dimension of chromatographic separation, its migration is retarded during the second solvent separation. This may represent a precursor of SL-1 that accumulates within these strains, however more detailed analysis will be required to assign a chemical structure to this lipid. Domenech et al. reported that the accumulation of an SL-1 precursor within the polar lipid fraction of the mmpL8 mutant (32) however, we did not observe any polar lipid differences in the fadD23 mutant relative to wild type(data not shown). Further analysis of the apolar lipids, as shown in Figure 3.4, indicates that the fadD23 mutant and the complemented strain both produce a lower abundance of the PDIM lipid family as compared to the parental wild type. The reduced production of the PDIM lipids in the mutant and the complemented strains was consistently observed across different phases of growth and with different types of growth medium (7H9 broth and GAS broth) (data not shown). 3.7 Growth and Persistence of the fadD23 mutant in vivo The reduced replication of the mutant within the macrophage model of infection prompted us to investigate whether virulence as assessed in vivo would also be compromised by the disruption of the fadD23 gene. As shown in Figure 3.5, when compared to M. tuberculosis, both the mutant and the complemented strain demonstrated very poor growth within the lungs and spleens of mice. A potential caveat is that the initial deposition of the mutant and complemented mutant into the lungs of mice was nearly one log lower than the parental wild type even though the viable count of all the inocula used to infect the mice were comparable. At 16 weeks post infection, the bacterial loads were reduced by ~5 logs in the lungs and ~3 logs in the spleens of mice 95 Figure 3.4 The fadD23 mutant and the complemented strain produce lower levels of P D I M as compared to wild type. Apolar lipids from M. tuberculosis (MTB), AfadD23 and the complemented strain were separated by 2D-TLC to resolve the PDIM family. PDIM: phthiocerol dimycocerosates, T A G : triacylglycerol, M K : menaquinone. 96 A. B. WEEKS Figure 3.5 fadD23 inactivation affects replication and persistence within the mouse model of infection. Mice were infected via aerosol and the bacterial loads in the lungs (A) and spleen (B) were monitored for M. tuberculosis (MTB) (0), the fadD23 mutant (•) and the complemented strain (A). At 8 weeks post infection, the number of organisms recovered from the lungs of AfadD23-infected mice were significantly reduced as compared to mice infected with the complemented strain. Each point is the mean + SEM from 4 mice (* P.< 0.05, # P< 0.01, ** P< 0.001, as compared to the fadD23 mutant). 97 infected with the mutant strains as compared to wild type-infected mice. At 8 weeks post infection, there was an indication of restored virulence following complementation. The bacterial growth in the lung was significantly increased for the complemented mutant as compared to the fadD23 mutant (P< 0.05). This observation was restricted to the lung and was not sustained during the chronic phase of infection. 3.8 Discussion The pioneering work of Mayer Goren nearly 30 years ago first described the chemical structures of the M. tuberculosis sulfatides (23). Recent studies using mass spectrometry techniques have identified the sulfated metabolites produced by M. tuberculosis which has resulted in a proposed model for SL-1 biosynthesis (49). This is supported by the findings of several groups that have identified the gene products that contribute to various steps within the pathway. One of the earliest defined reactions in SL-1 synthesis is the activity of the StfO sulfotransferase to yield trehalose 2'rsulfate which is the core carbohydrate of SL-1 (48). A separate sulfotransferase encoded by the Rvl373 gene has also been identified in M. tuberculosis, although it is unclear whether this enzyme participates in SL-1 production (50). The Pks2 polyketide synthase generates the methyl branched acyl chains (hydroxylphthioceranic and phthioceranic acids) that are eventually transferred to trehalose 2'-sulfate (25). At this time, the identity of the acetyltransferase(s) that initiate the esterification reactions in order to transfer the four acyl chains are unknown, although the PapAl acetyltransferase is a likely candidate as the papAl gene lies between pks2 and mmpL8 on the genome (51). Lipid transport from the cytosol to the cell envelope is mediated by the MmpL8 protein. Both Converse 98 et al. and Domenech et al. reported the accumulation of a diacylated precursor of SL-1 in the absence of mmpL8, leading to speculation that the two remaining acyl chains are added during or after transport (31, 32). From the data presented in this report, we propose that the FadD23 protein should now also be included in the list of SL-1 biosynthetic enzymes. The fadD23, pks2, mmpL8 and papAl genes are all clustered within a 13kb region of the M. tuberculosis genome (34). This genetic organization, whereby a polyketide synthase gene is located in close proximity to a member of the mmpL gene family and genes with putative lipid biosynthetic functions (fadD and papA), is a theme that is repeated throughout the M. tuberculosis genome. The most well characterized example of this class of gene clusters is the ~ 50 kb locus responsible for the synthesis of the PDIM (35, 52). In this pathway, the synthesis of the phthiocerol moiety of PDIM involves the FadD26 enzyme which provides acyl-adenylated fatty acids to the polyketide synthase, PpsA, for further chain extension (33, 35). Similarly, it is thought that the FadD28 enzyme performs an analogous function by working in concert with the polyketide synthase, Mas, to generate the mycocerosic acid component of PDIM (33, 35). Using the synthesis of PDIM as a model, the FadD23 protein likely provides activated fatty acyl precursors which are utilized by the Pks2 enzyme to generate the hydroxyphthioceranic and phthioceranic acids of SL-1. Under this model, it seems likely that the disruption of fadD23 would lead to the loss of SL-1 and perhaps to an accumulation of trehalose 2'-sulfate precursors that may or may not be already modified with the palmitic/stearic acid side chain. Comparative TLC analysis of the apolar lipid fractions from the parental wild type and the fadD23 mutant confirmed the absence of 99 mature SL-1 on the cell surface of the mutant strain. In addition, SL-1 production could be restored by complementation of the mutant with the fadD23 gene. TLC analysis also indicated a relative increase in cord factor production and identified a novel lipid within the apolar fraction of the mutant. It is tempting to speculate that these findings could also be the result of the interruption in SL-1 biosynthesis. The additional lipid could represent a sulfatide precursor while the increase in cord factor could indicate that excess trehalose is being redirected into the synthesis of other trehalose-containing glycolipids. Further chemical analysis will be needed to explore these possibilities. We found that disruption of the fadD23 gene resulted in an increased level of bacterial association with macrophages (37). While the studies with the pks2 and mmpL8 knockouts did not specifically investigate the macrophage binding capabilities of these mutants, Domenech et al. did note changes to the surface properties of the mmpL8 mutant as assessed by neutral red staining (32). The neutral red assay relies on the cell surface binding of the cationic dye, neutral red, which yields a red coloration of the cells in a reducing environment (53, 54). Positive neutral red staining of mycobacteria has been used as a correlative marker of virulence (53, 54). While the cell wall moieties that are involved in binding the dye are not fully known (54, 55), the lack of staining in the mmpL8 mutant does suggest a change in the distribution of charged moieties at the cell surface (32). Further research with the fadD23 mutant will also examine the neutral red staining of the bacteria and focus on the identity of the mycobacterial surface ligands and the macrophage receptors that mediate the increase in bacterial binding. The findings presented in this study are in contrast with others reports that investigate the contribution of SL-1 during macrophage infection. Rousseau et al. 100 reported that the pks2 mutant in H37Rv was not compromised for growth in either THP-1 cells or bone-marrow derived murine macrophages (30). Our findings with the fadD23 mutant in M. tuberculosis Erdman strain demonstrate a significant reduction in intracellular replication. Macrophages infected with the fadD23 mutant contained significantly lower numbers of bacteria by day 7 post infection as compared to macrophages infected with either M. tuberculosis or the complemented mutant. In addition, the calculated doubling time of the fadD23 mutant was significantly higher as compared to M. tuberculosis or the complemented mutant. The difference in the intramacrophage survival for the pks2 and fadD23 mutants is puzzling since these two enzymes likely work together to produce the acyl chains of SL-1. One possibility is that the FadD23 protein performs additional functions that are unrelated to SL-1 production or perhaps the FadD23 protein participates in other stages of SL-1 synthesis that do not involve the Pks2 enzyme. There are also differing results in the literature with regard to the involvement of SL-1 during infection in vivo. The presence of SL-1 during the early acute phase of infection does not appear to be critical i f the mice are infected via aerosol inhalation, this is confirmed for mutations in both pks2 and mmpL8 (30-32). In contrast, bacterial replication is reduced in an mmpL8 mutant in M. tuberculosis Erdman strain when the mice are infected by i.v. injection (31). Aerosol infection of an mmpL8 mutant in H37Rv only impacts mouse survival at the later stages of disease without any observed defects in bacterial replication (32). The differences in methodology and bacterial strains make it difficult to draw any clear conclusions about the involvement of sulfatides in mycobacterial pathogenesis. 101 In the present study, the fadD23 mutant in M. tuberculosis Erdman strain showed a dramatic loss of virulence during both the acute and chronic phases of infection following aerosol infection. Complementation of the mutant led to a partial restoration of virulence in the murine lung at 8 weeks post infection although it is important to note that the initial seeding of bacteria into the lung was significantly different among the strains which could have influenced the replication kinetics. However, this is unlikely as we know that deposition of 50-100 wild type M. tuberculosis into the lungs of CD-I mice still results in 4-5 logs of growth (data not shown), demonstrating that deposition of low numbers of bacteria into the lung is not, in itself, an explanation for poor growth. Since the complementation of the fadD23 mutant did not fully reverse the in vivo phenotype, it is possible that additional factors are also contributing to the loss of virulence. One possibility is that the presence of a second antibiotic resistance marker in the complemented mutant has an adverse effect on the in vivo replication. Previous studies have also reported only partial complementation of mutant phenotypes in mycobacteria (52) or found that integration vector alone can impact bacterial fitness (56). An alternate possibility is that differences exist in the levels of fadD23 transcription between the parental wild type and the complemented mutant. Promoter studies with the fadD28 gene in M. bovis B C G found that promoter elements located over 300bp upstream of the translation start site were needed for maximal transcription (57). While we did try to include ample upstream sequence within the complementation construct, there remains the possibility that other regulatory regions were not included which could negatively impact the virulence of the complemented mutant in vivo. An additional possibility comes from our observation that expression of the PDIM class of lipids was 102 reduced in the mutant and complemented mutant as compared to wild type. The necessity of PDIM for full virulence has been confirmed in a number of studies (38, 58, 59) and so it is also possible that the reduced production of this cell wall component could have contributed to the loss of virulence of the fadD23 mutant and the complemented mutant as assessed in vivo. It should be noted, however, that the loss of virulence seen with the aforementioned PDIM mutants did not approach the dramatic loss of virulence observed with the fadD23 mutant. Thus, loss of PDIM can, at most, only partially explain the attenuation of the fadD23 mutant in vivo. In conclusion, we propose that the fadD23 gene of M. tuberculosis contributes to the biosynthesis of SL-1. The loss of SL-1 production in M. tuberculosis Erdman strain correlated with an increased infectivity of differentiated THP-1 cells while also compromising the subsequent intracellular survival of M. tuberculosis in these macrophages. Importantly, these phenotypes could be complemented by re-introducing the fadD23 gene into the mutant strain. 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Ensergueix, E. Perez, B. Gicquel, and C. Guilhot. 1999. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Molecular Microbiology 34:257. 59. Rousseau, C., N . Winter, E. Pivert, Y . Bordat, O. Neyrolles, P. Ave, M . Huerre, B . Gicquel, and M . Jackson. 2004. Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection. Cellular Microbiology 6:277. 109 Chapter 4 Disruption of the Rvl505c gene reduces the intracellular survival of M. tuberculosis and impacts virulence 4.1 Preface This chapter forms part of a manuscript in preparation. Lynett, J., and R.W. Stokes. The Mycobacterium tuberculosis Rvl505c gene is necessary for optimal replication in macrophages. 4.2 Introduction Over a decade has passed since the World Health Organization first declared that the resurgence of tuberculosis (TB) had reached the level of a global health crisis (1), yet the global incidence rate of TB has continued to climb by 1% every year (2). Drug resistance, combined with the expanding HIV epidemic, has further impeded progress towards reaching TB control in endemic regions. Clearly, the development of new therapeutic and preventative agents will be needed to halt the spread of disease. As outlined in Chapter 2, we reasoned that the characterization of M. tuberculosis genes that are involved in macrophage interactions would provide new insight into the architecture of the cell envelope and bacterial virulence. The complex waxy cell wall of M. tuberculosis has long been recognized as an effective permeability barrier (3) and recent studies now suggest that certain cell wall constituents are required for virulence both for their role in the physical integrity of the cell wall and for their ability to subvert the defenses of the host macrophage (4-8). The disruption within the Rvl505c gene resulted in an improved association with macrophages (Figure 2.5). The Rvl505c predicted protein is 221 amino acids and contains a conserved domain known as the Left-handed parallel beta-helix (L0H) motif that is typically found within acyltransferase 110 enzymes (9-11). Recently, two separate studies in M. marinum have characterized gene homologues that are in close proximity to Rv1505c on the M. tuberculosis genome. Mehta et al. found that disruption of Rvl502 reduced macrophage binding and intracellular survival of M. marinum following infection (12). In addition, the LosA protein that is encoded by the Rvl500 homologue is a glycosyltransferase involved in the synthesis of cell wall lipooligosaccharides (13) and is also involved in the bacterial association with macrophages (14). For the present study, we were interested in determining whether the Rvl505c mutant in M. tuberculosis would yield a similar defect in intracellular survival. The results presented herein show that disruption of the Rvl505c gene does indeed compromise the intracellular replication M. tuberculosis. Interestingly, the Rvl505c mutant is no more sensitive to nitrite or mild acid stress than wild type. When tested for virulence in vivo, the Rv1505c mutant displayed an attenuated phenotype within the murine lung and spleen yet this phenotype was unchanged following complementation with the wild type gene. 4.3 M a t e r i a l a n d M e t h o d s Bacterial strains M. tuberculosis Erdman (ATCC 35801) was grown in Middlebrook 7H9 broth (Difco) or on Middlebrook 7H10 agar (Difco) supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose c omplex (OADC) (15). 7H9 broth was also supplemented with 0.05%o Tween-80. Escherichia coli strains were grown in Luria-Bertani (LB) broth I l l or on LB-agar plates. When required, hygromycin and kanamycin were used at 50ug/ml and 25pg/ml respectively. Identification of the Rvl505c mutant and genetic complementation The transposon insertion was localized to between nt. 335-338 of the Rvl505c by sequencing the amplicons generated by ligation-mediated inverse PCR (16). The transposon insertion in Rv1505c was confirmed by PCR using primers which flanked the insertion site (1505L 5 ' -CTTTAGTGGCCTTCGACGAG-3 ' corresponds to nt. 143-163 and 1505R 5 ' - T A T A G A C A C C A T C C G C G T C A - 3 ' matches nt. 590-610 of Rvl505c). The complementation construct for the mutation was generated by amplifying the Rvl505c gene from M. tuberculosis genomic D N A using Pfu D N A polymerase (Stratagene) with primers (1505JL 5 ' - G A A G G A T C C A T G A C C A A A C C - 3 ' and 1505JR 5 ' -GGCGAATTCCTAGCCTTTTC-3 ' ) . The amplicon was digested with BamHI and ligated to 2tara//7-digested pSODIT-2 vector, to generate an in-frame transcriptional fusion with the mycobacterial superoxide dismutase (SOD) promoter (17). The recombinant plasmid was then digested with Xbal and Hindlll to release the pSOD-Rvl505c fragment which was subcloned into the mycobacterial integration vector pMV306. The resulting plasmid construct was transformed into the Rvl505c mutant by electroporation as described elsewhere (18) and successful complementation was confirmed by PCR using primers 1505L and 1505R. Macrophage infection experiments 112 The human macrophage-like cell line, THP-1 (ATCC TIB202), was maintained at 37°C and 5% C 0 2 in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Gibco), lOmM L-glutamine (Gibco) and lOmM sodium pyruvate (Gibco). For infection assays, THP-1 cells were seeded onto acid-washed glass coverslips differentiated over 3 days in the presence of lOOnM P M A (Sigma) as described previously (19). Prior to use, differentiated THP-1 cells were washed with binding medium (20) to remove any residual P M A . For the infection assays, the bacteria were prepared from frozen stock vials that were thawed, centrifuged and resuspended in binding medium as described in Chapter 2. THP-1 cells were infected at an MOI of 50:1 (bacteria:macrophage) for 3 hrs followed by washing (three times) to remove non-associated bacteria. The level of bacterial binding was determined by comparing the number of bacteria associated with the monolayer after three hours. Briefly, the infected coverslip was added to 2ml of PBS-Tween, the suspension was briefly sonicated to lyse the monolayer and serial dilutions were plated onto 7H10 agar. To compare intracellular survival, the infected monolayers were washed and transferred to new wells containing 1ml of supplemented RPMI. Fresh media was added to the wells on every second day and the infected monolayers were processed for viable colony forming units (cfu) on days 0, 4 and 7. The overlying media and two washes of PBS-Tween were pooled together with the infected coverslip before sonication and plating. The doubling time for intracellular bacteria was calculated from the colony counts using the equation N t = N 0 e k t where N t = number of bacteria at day 7, N 0 = number of bacteria at day 0, k = growth rate constant and t = time. 113 Lipid analysis Apolar and polar lipids were extracted from heat killed, lyophilized bacterial pellets as described previously (21). Lipids were separated by 2D-TLC on Silica Gel 60-plates (EM Science) using the five different solvent systems described for mycobacterial lipid analysis (21, 22). Lipids were visualized by spraying T L C plates with either 5% ethanolic molybdophosphoric acid or 2.5% a-naphthol-10% sulfuric acid following by charring. Bacterial viability following exposure to acidified sodium nitrite The level of resistance to sodium nitrite was determined for all mycobacterial strains essentially as described elsewhere (23). Briefly, bacteria were cultured to late log phase in 7H9 broth, pelleted by centrifugation and resuspended in 7H9 (pH 5.4) with or without the addition of 6mM sodium nitrite. The cultures were incubated for 24 hrs at 37°C and the number of surviving bacteria was determined by plating onto 7H10 agar. Growth and persistence in mice Actively growing broth cultures of the M. tuberculosis strains were normalized by OD600 and resuspended to an estimated final concentration of 4xl0 7cfu/ml. A 5ml aliquot of the bacterial suspension was used for infection of female CD1 mice (6-8 weeks old) via aerosol inhalation (Glas-Col) set up to deposit approximately 500-1000 bacteria into the lung. At each experimental time point, the lungs and spleens of the infected mice (four mice per strain) were aseptically removed, homogenized in PBS-Tween and serial dilutions were plated onto 7H10, 7H10-hygromycin or 7H10-hygromycin/kanamycin. 114 Statistical Analysis Data are expressed as the mean + standard error of the means. The two-tailed Student t test or two-way A N O V A followed by the Tukey-Kramer Multiple Comparisons Test was performed as appropriate, using GraphPad Prism Software. Differences were considered significant with aP< 0.05. 4.4 The macrophage binding and intracellular survival of the Rvl505c mutant Macrophage monolayers were infected with the wild type, the mutant and the complemented mutant to measure bacterial association and intracellular survival. Surprisingly, the complemented strain did not reverse the binding phenotype that was originally observed in the Rvl505c mutant, rather the constitutive expression of the Rvl505c gene resulted in binding levels that were higher than either the wild type or the mutant (Figure 4.1 A). The bacterial strains were then tested for the ability to replicate intracellularly within macrophages over a period of 7 days. As shown in Figure 4.IB, macrophages infected with the Rvl505c mutant harbored significantly fewer organisms by day 7 as compared to the parental wild type (P < 0.001). Complementation of the mutant strain with the wild type gene in trans effectively reversed the defect in intracellular growth. The number of cfu recovered at day 7 for the complemented strain was significantly increased relative to the mutant (P < 0.001) but were not significantly different from the wild type. These findings are also supported by comparison of the calculated bacterial doubling times within THP-1 cells. The doubling time for the Rvl505c mutant (38.5+4.4 hrs) was significantly increased as compared to wild type (21.7±0.2 hrs) or the complemented strain (28.2+1.5 hrs) (P < 0.001). Importantly, the 115 Figure 4.1 Disruption of the Rvl505c gene affects the binding and intracellular survival of M. tuberculosis. (A) Binding of the wild type (MTB), the Rvl505c mutant (Tn::Rvl505c) and the complemented mutant (complement) to THP-1 monolayers. The mean + S E M is shown from two experiments each done in triplicate (N=6). * P< 0.05, ** P< 0.001 when compared to M T B , f P< 0.01 when compared to the Rvl505c mutant. (B) The replication of the wild type (0), the mutant (•) and the complemented strain (A) was assessed within THP-1 cell monolayers over 7 days. (** /?<0.001 when compared to the Rvl505c mutant). Data shown is the mean + S E M of two experiments each done in triplicate (N=6). (C) Growth of the wild type (0), the mutant (•) and the complemented strain (A) was monitored in 7H9 broth by measuring absorbance at 600nm. 116 Figure 4.1 Disruption of the Rvl505c gene affects the binding and intracellular survival of M. tuberculosis. 117 three strains exhibited identical growth rates when cultured in broth (Figure 4.1C). These results confirm that deletion of the Rv1505c gene confers a marked disadvantage to the survival and replication of M. tuberculosis within macrophages in vitro. 4.5 The Rvl505c mutant is not hypersensitive to acidic conditions or the presence of nitrite In an attempt to understand why the Rv1505c mutant replicates at a reduced rate intracellularly, we tested the sensitivity of the wild type, mutant and complemented strains to in vitro conditions that could simulate the environmental pressures that are encountered within the phagosome. Bacteria were cultured to late log phase and exposed to a slightly acidic environment (pH 5.4) with or without the addition of sodium nitrite for 24 hrs. As shown in Figure 5.2, the Rv1505c mutant was as susceptible to reduced pH and the presence of nitrite as the parental wild type strain. 4.6 Lipid analysis of the Rvl505c mutant A diverse collection of lipids and glycolipids can be easily removed from cell wall using solvent extraction and therefore are thought to be at or near the bacillus surface. To investigate whether the disruption of Rvl505c would affect the distribution of cell wall lipids, the apolar and polar lipid fractions were extracted from the parental, mutant and complemented strains and separated using TLC. As shown in Figure 5.3, TLC analysis of the apolar lipid fraction indicated that the Rvl505c mutant and its complement synthesized significantly lower levels of the PDIM lipid family as compared to M. tuberculosis. The loss of PDIM in the Rv1505c mutant and the complemented 118 100 i 80 MTB Tn::Rv 1505c Complement • pH 5.4 • pH 5.4+nitrite Figure 4.2 Disruption of the Rvl505c gene does not affect bacterial resistance to conditions of low pH or acidified nitrite. The percentage survival of all strains was determined following incubation in acidified media (pH 5.4) with or without the addition of 6mM NaN02 for 24hrs as compared to untreated control cultures. Data represents the mean + S E M of three independent experiments each done in duplicate (N=6). 119 A r \ pomf/ * MK TAG J mmwr-B ^ ) IP c Figure 4.3 Two-dimensional T L C analysis of the apolar l ip id fractions Lipids from the wild type (A), the Rv1505c mutant (B) and the complemented strain (C). Plates were developed with solvent system A as described elsewhere (22). Lipids were visualized by spraying with 5% ethanolic molybdophosphoric acid followed by charring. Triacylglycerol (TAG), menaquinone (MK), phthiocerol dimycocerosates (PDIM). 120 strain was not affected by the bacterial growth phase or growth medium. Further analysis of the apolar and polar lipid fractions did not reveal any other differences between the parental wild type and the mutant strain (data not shown). 4.7 Disruption of Rvl505c adversely affects bacterial growth and persistence within mice To determine i f the disruption of Rvl505c would affect the disease outcome in vivo, mice were infected with aerosolized M. tuberculosis, the Rvl505c mutant or the complemented mutant strain. The bacterial loads in the lungs and spleens were monitored over 16 weeks (Figure 4.5). During both the acute and chronic phases of infection the mutant and complemented strain failed to replicate to the same degree as M. tuberculosis in the lungs and spleens of infected mice. The bacillary load within these organs remained largely constant throughout the experiment suggesting a defect in bacterial replication under in vivo conditions. Unfortunately, complementation of the Rvl505c had no restorative effect on attenuation of the Rvl505c mutant suggesting that other factors must also be contributing to the avirulent phenotype within the mouse model of infection. 4.8 Discussion Under most circumstances, the interaction between the host macrophage and a bacterial invader represents the endpoint of infection. M. tuberculosis is a notable exception, having evolved strategies to survive and persist intracellularly despite the onslaught of antimicrobial mechanisms by the host macrophage. Recently, several 121 Figure4.4 Growth and persistence of the M. tuberculosis strains in mice following aerosol infection. The bacterial loads from mice infected with the wild type (0), the Rvl505c mutant (•) and the complemented strain (A) were determined for the lung (A) and spleen (B). Each point is the mean + S E M from 4 mice. 122 landmark papers have described the transcriptional response of M. tuberculosis following macrophage infection (24) and outlined the mycobacterial gene sets required for intracellular survival (25) and for the aberrant maturation of the M. tuberculosis phagosome (26, 27). The results presented herein show that disruption of the Rvl505c gene leads to a reduction in the ability to thrive intracellularly. Using THP-1 cell monolayers as a model of infection, we found that intracellular replication of the Rvl505c mutant was markedly reduced compared to wild type or the complemented strain. Several scenarios could explain the observed defect in intracellular replication. One possibility is that Rvl505c mutant is more sensitive to the restrictive conditions of the phagosomal environment. For example, the mutant may be highly sensitive to the lack of available nutrients within the phagosome, similar to that observed with amino acid auxotrophs (28-30). Alternatively, the Rvl505c mutant may be unable to fully arrest the process of phagosomal maturation. Conceivably such a defect would lead to the fusion of the mycobacterial phagosome with late endosomal and lysosomal vesicles that would reduce the phagosomal pH and lead to the acquisition of lysosomal proteases. While our findings indicate that the Rvl505c mutation does not result in an overall increased sensitivity to environmental stresses (including reduced pH and nitrite stress), further studies will be required to fully delineate the intracellular events that lead to reduced intracellular survival of this mutant. Using T L C we could not detect changes to the cell wall lipid profile that could be solely attributed to the disruption of the Rvl505c gene. Although the mutant produced lower amounts of the PDIM lipid family, this effect was also observed in the 123 complemented strain. The PDIM lipid class has received particular attention due to its demonstrated role in disease pathogenesis (16, 31). Mutations that map within the ~50kbp cluster of genes required for PDIM biosynthesis (32) lead to growth defects specifically in the murine lung (16) or in both the lung and spleen depending on the route of infection (6). There have been other reports that have described loci that are unlinked to this region yet still generate a loss in PDIM synthesis (33, 34). Recently, there has been some debate as to whether these loci are in fact truly involved in PDIM biosynthesis given the lack of mutant complementation and conflicting data from other publications (35, 36). Given that the Rvl505c gene does not map near mycocerosic acid synthase region of the genome and that the complemented strain fails to produce PDIM levels similar to the parental wild type, this leads us to conclude that this observed difference could not be unequivocally due to the transposon disruption. A possible explanation can be found in previous reports in the literature which suggest that long term culture of M. tuberculosis (37, 38) or the act of phage transfection (39) can contribute to a spontaneous decline in PDIM production. In the case of the Rvl505c mutant, it is possible that either the act of mutagenesis, the repeated culturing in broth during the library screening or both events could have contributed to the altered profile of PDIM. The virulence of the Rvl505c mutant was assessed in mice following aerosol challenge. The Rvl505c mutant grew poorly in the lungs and spleens throughout both the acute and chronic stages of infection but this phenotype was unaffected by complementation with the wild type Rvl505c gene. In addition, the level of macrophage association did not return to wild type levels following complementation. One possible explanation is that the transposon insertion into the Rvl505c gene may be exerting polar 124 effects on the expression of downstream genes. The genomic region surrounding the Rvl505c gene encodes a number of tightly clustered genes which could be affected as a result of the disruption in Rvl505c. Alternatively, the binding interactions and virulence of the complemented mutant could have also been impacted by the constitutive expression of the Rv1505c by the SOD promoter or the presence of an additional antibiotic resistance cassette. There also remains the possibility that the reduced levels of PDIM in the cell envelope of the Rvl505c mutant could have contributed to the loss of the virulence within the mouse model of infection. Previous studies have reported that inhibition of PDIM synthesis by disruption of ithe mycocerosic acid synthase (mas), the proposed membrane transporter (mmpLT) or the fatty-acyl A M P ligase (fadD28) result in a ~2 log decrease in the cfu counts in the mouse lung relative to wild type at 3 weeks post infection (16, 31). The results presented with the Rv1505c mutant show a much more profound phenotype in the mouse, with a 3.5 log reduction in bacilliary load of the lung compared to wild type infected mice. Thus, it is unlikely that the avirulence could be solely attributed to reduced PDIM expression and there are likely other factors that are contributing to the in vivo phenotype of the Rv1505c mutant. In conclusion, this work identifies the protein product of Rvl505c as having a role during the interplay between M. tuberculosis and the macrophage. The predicted Rv 1505c protein encodes motifs that are associated with acetyltransfereases from other bacteria although a precise biological role is not clearly evident from its location on the genome. We propose that inactivation of Rv1505c leads to alterations within the cell envelope that comprises the ability of M. tuberculosis to survive within the phagosome. 125 Future studies will be aimed at isolating the components of the cell wall that are affected as a result of the Rvl505c mutation and elucidating the mechanisms that underlie the reduced intracellular replication of the mutant. 126 4.9 Literature cited 1. WHO. 1994. Global Tuberculosis Programme - Framework for effective tuberculosis control. World Health Organization, Geneva, Switzerland. 2. WHO. 2005. 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J Biol Chem 279:21257. 130 Chapter 5 Inactivation of the Mycobacterium tuberculosis mtrB gene alters colony morphology, lipid biosynthesis and reduces bacterial virulence 5.1 Preface This chapter has been submitted for publication as: Lynett, J. and R.W. Stokes. Inactivation of the Mycobacterium tuberculosis mtrB gene alters colony morphology, lipid biosynthesis and reduces bacterial virulence. FEMS Microbiology Letters. Submitted April 2006 5.2 Introduction In 1882, M. tuberculosis was identified as the causative agent of tuberculosis, becoming the first microbe to fulfill what are now known as Koch's postulates (1). Today, tuberculosis still remains as the leading cause of death from a single infectious agent despite significant advances in mycobacterial research and the delivery of monitored antibiotic therapy (2). The success of M. tuberculosis as a human pathogen can be attributed in part to its resilience within adverse surroundings. The ability to sense and adapt to environmental cues is thought to be critical for mycobacterial survival within the lung and contribute to the timely transitions both into and out of bacterial latency. The capacity of both pathogenic and nonpathogenic bacteria to detect environmental changes and rapidly initiate the required response is largely governed by two-component regulatory systems that consist of a membrane-associated sensor kinase and a response regulator (3, 4). At the functional level, the sensor kinase is autophosphorylated in response to a specific stimulus that leads to the activation of a cognate response regulator via a phosphotransfer reaction. Upon activation, the regulator 131 functions as a DNA-binding protein that recognizes regulatory sequences within the upstream region of its target genes to control transcriptional expression. The M. tuberculosis genome encodes 11 paired two-component systems as well as a number of orphaned kinases and regulators (5). While the majority of these systems have been characterized in some detail, in most instances the activating signals and gene regulons are still largely undefined. One approach has been to compare the sequences of two-component systems in other bacteria (6). This method has proven useful in the initial characterization of the KdpD-KdpE which responds to limiting concentrations of K+ ions (7). Similarly, bioinformatic analysis and structural modeling of the SenX3 sensor suggests that the SenX3-RegX3 system may respond to changes in redox potential and oxygen tension (8). Other studies have employed D N A microarrays to compare the expression profiles between wild type and two-component M. tuberculosis mutants (9-12). Yet, without prior knowledge of the activating signal, there can be difficulties in highlighting the genes that are under the direct control of the regulatory system (13). The task of functional characterization has also been approached by surveying the two-component systems that are upregulated following macrophage infection, with the expectation that these systems would coordinate the transcriptional response of M. tuberculosis required for intracellular survival. Using transcriptional reporters and SCOTS analysis, it is now evident that in fact several two-component systems are upregulated following phagocytosis (14-17). These findings have been extended using constructed mutants which have confirmed the involvement of several systems during intracellular replication as well as during growth and persistence in vivo (9, 17-19). Interestingly, Parish et al. noted that the disruption of some systems actually yielded a 132 hypervirulent phenotype in the SCID mouse model (20). To date, the most well characterized two-component system in M. tuberculosis is the DevR-DevS system. This system was originally identified by analyzing the transcriptional response of M. tuberculosis following exposure to hypoxia (21). Subsequent work has defined the D N A sequence that is recognized by the DevR regulator (10) and identified the gene regulon that is linked with the onset of dormancy (11, 22-25). Collectively, these findings imply that strict control over gene expression is a critical component of mycobacterial virulence and also suggest that these signal transduction systems, which are unique among bacteria, could serve as future therapeutic targets (26). The first two-component system to be cloned in mycobacteria was the mtrA-mtrB locus where the initial studies in M. bovis B C G reported an increase in the expression of the /wfrvl-encoded response regulator following macrophage infection (27, 28). However, later work with virulent M. tuberculosis found that mtrA-mtrB was in fact constitutively expressed during macrophage infection and was also expressed during in vitro broth culture (16, 17). Attempts to construct targeted deletions of either mtrA (29) or mtrB (20) in M. tuberculosis have thus far been unsuccessful. Studies using saturated mutagenesis have found that while the mtrB-encoded sensor is necessary for optimal growth in vitro (30), the mtrA gene is required for survival in mice (31). During the construction of the transposon library as outlined in Chapter 2, we screened for mutants that exhibited differences in colony morphology. One mutant was isolated for further study and the transposon insertion was mapped to the mtrB gene. The mutant displays extensive clumping during in vitro culture and does riot exhibit the characteristic cording phenotype of virulent M. tuberculosis. The lipid profile of the cell 133 envelope revealed an increased production of the cell wall mycocerosates but reduced elaboration of some forms of phosphatidylinositol mannoside. Following aerosol infection of mice, the mtrB mutant failed to persist within the lung with no indication of bacterial dissemination to the spleen. 5.3 Materials and Methods Bacterial strains and mutagenesis M. tuberculosis Erdman strain (ATCC 35801) and the mtrB mutant were cultured in Middlebrook 7H9 broth (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose complex (OADC) and 0.05% Tween-80 or on Middlebrook 7H10 agar (Difco) supplemented with 0.5%) glycerol and 10% OADC. OADC was prepared as described previously (32). Hygromycin was used, when required, at a concentration of 50ug/ml. A Tn5370 transposon library was generated using the phAE87 mycobacteriophage as described previously (33). Briefly, bacteria were grown to mid-logarithmic phase, concentrated 10-fold in phage adsorption buffer (10 mM Tris/HCl, pH 7.6, 100 m M NaCl, 10 mM M g S 0 4 , 2 m M CaCl 2) and infected with phage lysate at a multiplicity of infection (MOI) of 10:1 (phage:bacteria). Following the 4 hrs infection period at 39°C, the bacteria were pelleted by centrifugation (16000xg for 5 min) and plated onto selective agar plates. Identification of the transposon insertion site The site of transposon insertion was mapped by comparing the amplicon sequence from ligation-mediated inverse PCR with the published M. tuberculosis genome sequence 134 (http://genolist.pasteur.fr/TubercuList/). Briefly, genomic D N A was purified (34), digested with Rsa-I and ligated at 16°C. The genomic D N A adjacent to the transposon was amplified using primers corresponding to the left (084L-F 5'-G T C A T C C G G C T C A T C A C C A G - 3 ' and 084L-R 5 ' -AACTGGCGCAGTTCCTCTGG-3') and right (084R-F 5 ' -ATACACGCGCACCGGTTCTAGC-3 ' and 084R-R 5'-CACGGCGAACCGCTGGTG-3 ' ) ends of the transposon as described previously (35). The disruption of the mtrB gene was also confirmed by PCR using primers designed to amplify an internal region of the mtrB gene (MtrBl 5' -ATCGAGTTGCTGGTGGATCT-3' and MtrB2 5 ' -GCGCTGACGTTCCTTGTATT-3 ' ) . Southern hybridization was performed using the hygromycin resistance marker as a probe to verify that a single copy of the transposon had incorporated into the chromosome. Lipid analysis Apolar and polar lipids were extracted from heat killed, lyophilized bacterial pellets as described previously (36). Lipids were separated by 2D-TLC on Silica Gel 60-plates (EM Science) using five different solvent systems previously described for mycobacterial lipid analysis (36, 37). To specifically detect the cell wall mycocerosates, T L C plates were loaded with ~200ug of the apolar lipid fraction and developed three times in the first dimension solvent system [petroleum ether:ethyl acetate (98:2)] and once in the second dimension solvent system [petroleum ethenacetone (98:2)]. Lipids were visualized by spraying TLC plates with 5% ethanolic phosphomolybdic acid following by charring. To analyze the distribution of the phosphatidylinositol mannoside lipids, T L C plates were loaded with ~250ug of the polar lipid fraction and were 135 developed once in the first dimension solvent system [chloroform:methanol:water (60:30:6)] and once in the second dimension solvent system [chloroform: acetic acid: methanol: water (40:25:3:6) ]. The plates were sprayed with 2.5% a-naphthol-10%> sulfuric acid following by charring to specifically detect glycolipids. Bacterial Growth and persistence in mice Logarithmic phase cultures of the wild type and mutant were normalized by OD600 and resuspended to an estimated final concentration of 4xl0 7cfu/ml in sterile water. A 5ml aliquot of the bacterial suspension was loaded into the nebuliser of a Glas-Col inhalation chamber which was operated so as to deposit approximately 500-1000 bacteria into the lungs of 6-8 week old female CD-I mice (Charles River). At each experimental time point mice were euthanized (four mice per strain) and the lungs and spleens were aseptically removed, homogenized in PBS supplemented with 0.05% Tween-80 and serial dilutions were plated onto 7H10 or 7H10-hygromycin. 5.4 Identification of the mtrB mutant Recent studies have described a number of mycobacterial cell wall moieties that function as important virulence determinants during infection (38-41). Given this association between cell wall composition and pathogenesis, several groups have utilized visual markers such as colonial morphology or bacterial aggregation as library screening tools to identify surface components that may play a role during infection (42-46). We employed this strategy using a transposon library of M. tuberculosis and screened for mutants with alterations in colony morphology. One mutant was identified and sequence 136 analysis of the transposon insertion found that the disruption occurred within the mtrB gene (Figure 5.1 A). As shown in Figure 5.IB, the colonies of the mtrB mutant strain have an irregular bulging shape and are elevated from the agar surface. This is in contrast to the parental wild type colonies which are flat and exhibit the dry, rough morphology that is typical of M. tuberculosis. M. tuberculosis aligns into long braids, known as the cording phenotype, when cultured in liquid broth in the absence of dispersal agents such as the detergent Tween-80. The loss of bacterial cording has been causally linked to a decline in virulence as reported in studies using targeted gene disruptions (46) or as a result of spontaneous mutation, as is the case with the avirulent H37Ra strain (47). Due to the differences that were observed in the colonial morphology of the mtrB mutant, we were also interested in evaluating the cell-cell interactions and cording ability of this strain. As shown in Figure 5.1C, acid-fast staining of the parental wild type reveals the presence of long cords when cultured in the absence of Tween-80 while the mtrB mutant aggregated into large clumps. The tendency of the mutant to clump in vitro occurred even in the presence of Tween-80 with small clumps being clearly visible to the naked eye when cultured on a roller bottle apparatus. 5.5 L i p i d analysis of the mtrB mutant Since the disruption of the mtrB gene led to such dramatic changes to the surface properties of the bacteria, we next addressed whether the cell wall lipid profile was also affected as a result of the mutation. Total lipids were extracted from whole cells, separated into polar/apolar fractions and analyzed by two-dimensional T L C using 137 Figure 5.1 The disruption of mtrB affects colonial morphology and microscopic cording. (A) Colonies of M. tuberculosis (MTB) and the mutant were cultured on 7H10 agar for 4 weeks. B) The wild type and mutant strains were cultured in 7H9 broth in the absence of Tween-80 until late-logarithmic phase. Bacterial smears for each strain were heat-fixed to a glass slide, fixed in 10% formaldehyde in ethanol for 10 minutes and stained using Kinyoun's Carbol Fushcin (xlOOO magnification). 138 standard protocols. As shown in Figure 5.2A, the mtrB mutant synthesized an increased abundance of the cell wall mycocerosates as compared to the parental wild type. Further comparative analysis of the apolar fraction did not reveal any other differences in the levels of cord factor, sulfolipid, triacylglycerols or acylated trehaloses (data not shown). The polar lipid class of the M. tuberculosis cell envelope is comprised of a mixture of glycolipids that are based on phosphatidylinositol which has been further modified by a varying number of mannosyl residues and acyl chains. This lipid group is collectively referred to as the phosphatidylinositol mannosides (PIMs). When we compared the levels of the various PIM isoforms of the wild type and the mtrB mutant, we observed that certain forms of PIM were reduced within the cell envelope of the mutant strain. As shown in Figure 5.2B, the mtrB mutant produced significantly reduced amounts of hexamannoside lipids (PIM6) as compared to wild type while the levels of other PIM species, including the dimannoside acylated forms (PIM2) were unaffected. The observation that the disruption of mtrB coincides with differences in the distribution of cell wall lipids raises interesting questions about the role of the mtrA-mtrB two component system in the direct or indirect regulation of cell wall biosynthetic pathways. 5.6 Growth of the mtrB mutant in vivo To determine whether disease outcome would be affected by the disruption of the mtrB gene, mice were infected via aerosol with either the mutant or the parental wild type and bacterial growth was monitored over time in the lung and spleen. As shown in Figure 3, the mtrB displayed little to no replication within the murine lung and the infection was eventually cleared by day 80 post infection. Interestingly, there was no 139 Figure 5.2 Disruption of the mtrB gene affects the synthesis of cell wall lipids The apolar (A) and polar (B) lipid fractions were extracted from whole cells and separated using 2D-TLC as outlined in the Materials and Methods. A) The mtrB mutant synthesizes an increased level of phthiocerol dimycocerosates. Menaquinone (MK), phthiocerol dimycocerosates (PDIM) and Triacylglycerol (TAG). B) Disruption of mtrB results in a decreased production of the acylated forms of PIM6. Acylated forms of phosphatidylinositol dimannoside (Ac-PIM2), acylated forms of phosphatidylinositol hexamanoside (Ac-PIM6). The identity of the lipids was achieved by comparison with published standards for M. tuberculosis (36, 37). 140 Figure 5.3 The mtrB mutant fails to thrive within the mouse model of infection. Mice were infected with M. tuberculosis (A) and the mtrB mutant (•) via the aerosol route and the bacterial loads within the lung were monitored over time. Values shown are the means + S E M of cfu counts from four mice per strain for each time point. In some instances the error bars do not extend beyond the symbol. 141 evidence of dissemination to the spleen in mice infected with the mtrB mutant. This could be due to the lack of mutant replication within the murine lung. This is in contrast to the parental wild type, where the bacterial loads within spleen reached approximately 5 log C F U after 80 days post infection (data not shown). 5.7 Discussion In this study, we report on the initial characterization of an M. tuberculosis mutant that carries a disruption in the w^rS-encoded sensor kinase. The mtrA-mtrB system is found in several pathogenic species of mycobacteria (M. bovis, M. leprae, M. avium and M. tuberculosis) as well as in the saprophyte, M. smegmatis (17). The conservation of mtrA-mtrB across several mycobacterial species is intriguing, particularly in the case of M. leprae (48, 49) as this genome is largely regarded as the minimal gene set necessary for an intracellular lifestyle (50). One possibility is that the mtrA-mtrB locus is involved in sensing environmental cues as part of an essential or housekeeping process. This notion is supported by the recent studies of Sassetti et al. that have identified M. tuberculosis genes essential for growth under various environmental conditions using a ///mar-based mariner transposon library in combination with the Transposon Site Hybridization (TraSH) screening technique (51). When this mutant bank was cultured on 7H10 agar, there was a noted paucity of viable mutants with transposon insertions within the mtrB gene, leading the authors to conclude that under these defined growth conditions, the mtrB gene was essential for optimal growth (30). Interestingly, disruptions within the mtrA gene were not lethal under in vitro culture conditions but did impair mycobacterial replication within the spleens of mice (31). While we were able to 142 isolate and culture an mtrB mutant of M. tuberculosis, our results do not completely contradict these findings, as the mutant does exhibit excessive clumping in vitro and was slow to form colonies when cultured on 7H10 agar. Recently, a disruption of the mtrB gene was reported in the M. avium complex (MAC) (52). Characterization of the mutant indicates that the mtrA-mtrB system is involved in the morphotype switching that is associated with M A C . In addition, the mutant displayed an increased susceptibility to various antibiotics and was impaired for survival within macrophages (52). Similarly, deletion of the mtrA-mtrB genes in Corynebacterium glutamicum also affected the susceptibility of the bacteria to different antibiotics (53). Sensitivity to antibiotics that affected peptidoglycan synthesis was increased while there was a heightened resistance to ethambutol which acts on the synthesis of arabinogalactan. Furthermore, the mutation in C. glutamicum affected septum formation whereby the bacteria had several segmentations and an overall increase in length (53). Our studies did not explore the antibiotic susceptibility of the M. tuberculosis mtrB mutant primarily due to the excessive clumping of the mutant strain which precluded turbidity measurements and led to difficulties in obtaining accurate colony counts. However, we did note significant alterations to the colonial morphology and the surface characteristics of the M. tuberculosis mtrB mutant. The analysis of the cell-cell interactions revealed that the strong aggregative phenotype completely masked the cording ability of this strain. Collectively, the phenotypical similarities in our studies and those reported in M A C and C. glutamicum lend support to the view that the mtrA-mtrB system may exert some regulatory control over cell wall processes. 143 This is not the first example of an M. tuberculosis two-component system that has been shown to affect the surface properties of the bacillus. The initial characterization of a phoP disruption in M. tuberculosis reported that the mutant produced significantly smaller colonies, shorter bacillus rods during logarithmic phase and also exhibited cording differences as compared to wild type (18). Subsequent reports have noted differences in the lipid content of the phoP mutant affecting either the distribution of lipoarabinomannan isoforms (54) or the production of acylated forms of trehalose (di-and poly-acylated trehalose and sulfolipid) (55) depending on the parental strain of M. tuberculosis. The similarities between the phoP and mtrB mutants with regard to the regulation of lipid biosynthesis could indicate the existence of an overlap between the two signal transduction systems. Crosstalk between regulatory systems can provide the organism with a means to coordinate an even greater number of genes in response to stimuli (56). This effect has been extensively documented in the PhoP-PhoQ system of Salmonella which not only overlaps with other two-component systems with respect to target genes, but also directly regulates the expression of a second two-component system (57, 58). Future studies that examine the transcriptional differences of the mtrB mutant using microarrays should aid in deciphering the downstream gene targets and any overlap with other systems. The observation that the disruption of mtrB coincides with an increase in PDIM expression raises interesting questions about the regulated expression of this lipid throughout different growth phases and environmental conditions. The cell wall mycocerosates, particularly PDIM, have been the focus of intense study since PDIM has been confirmed as an important virulence determinant of M. tuberculosis (35, 59) with a 144 possible role in resistance against reactive nitric oxide intermediates within the phagosomal environment (60). To our knowledge, this is the first M. tuberculosis mutant describing an increased abundance of surface mycocerosate that is also compromised in vivo. When we conducted further analysis on the distribution of the various PIM isoforms, we noted that the mtrB mutant synthesized a reduced amount of PIM6 without affecting the levels of other PIM isoforms. A number of interesting biological effects have been attributed to the mycobacterial PIMs. Recently, the PIMs have been implicated in the aberrant trafficking of the mycobacterial phagosome within macrophages. Vergne et al. showed that PIM lipids stimulated phagosomal fusion with early endosomes (61) and others have suggested that PIMs bind galectin-3 on the phagosomal membrane (62) or can intercalate into host membranes (63). In addition, studies have also shown that PIMs can function as mycobacterial adhesins on the bacillus surface (64, 65). Whether the reduced production of PIM6 in the mtrB mutant affects macrophage infection at the level of surface interactions or phagosomal processing are both valid questions that will require further study. Unfortunately, these studies are greatly complicated by the tendency of the mutant to clump, even in the presence of Tween-80. This precludes the production of single cell suspensions that are essential for macrophage infection studies. Our results also demonstrate that the mtrB gene contributes to mycobacterial survival in vivo. Mice challenged with the mtrB mutant were able to successfully control and eliminate the infection within the confines of the lung, without any evidence of dissemination to the spleen. One possibility is that the cell wall alterations render the 145 mtrB mutant incapable of survival within the macrophage environment or alternatively, the differences in lipid composition may also affect the immune response to infection. The infectious cycle of M. tuberculosis is comprised of a number of obstacles; some are host-derived (macrophage defenses and adaptive immunity) while others are due to purely external environmental stresses (temperature, nutrient availability and oxygen tension). To date, studies in M. tuberculosis have found that two-component systems help regulate the onset of dormancy, maintain persistence within the mouse model of infection and contribute to intramacrophage survival. 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Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells. Infect Immun 65:3896. 153 Chapter 6 Discussion and Future Directions 6.1 Introduction When this project first began, genetic tools for use in M. tuberculosis were still relatively novel. Two groups had published separate protocols for random mutagenesis in M. tuberculosis (1, 2) using the Tn5367 transposon (3). Bardarov et al. developed a recombinant temperature-sensitive mycobacteriophage that could infect and deliver the transposon into the bacteria yet would fail to undergo a lytic lifecycle at the non-permissive temperature. Meanwhile, Pelicic et al. reported on a plasmid system that incorporated a counterselectable marker to prevent integration of the plasmid backbone into the genome. Initially, I began construction of the mutant bank for this project using phage delivery of Tn5367, which depends on kanamycin resistance as a selection marker. Unfortunately, these experiments were hampered by a high level of spontaneous resistance to the antibiotic that developed in M. tuberculosis Erdman strain. PCR analysis of kanamycin-resistant colonies from phage-treated cultures indicated that only 40-50% of the clones actually represented transposon mutants. Around this time, Cox et al. reported the use of signature-tagged mutagenesis in M. tuberculosis using the mini-Tn5370 where the kanamycin resistance marker was exchanged for a hygromycin resistance cassette (4). Using the mini-7«5370 and phage delivery, I successfully generated a transposon library of roughly 10000 mutants. As outlined in Chapter 2, the library was screened for mutants with a specific phenotype; an increased propensity to bind macrophages. Previous findings from our group (5) and others (6) suggested that the M. tuberculosis capsule could control the presentation of bacterial ligands. Our initial expectation was that these mutants would 154 enable the identification of M. tuberculosis genes that, in some way, contribute to capsule production. A total of five mutants were isolated from the library screening, all of which displayed increased binding to macrophage monolayers as compared to the parental wild type. However, B L A S T analysis of the transposon-disrupted genes or the corresponding predicted proteins did not signal any homology to genes or proteins involved in capsule synthesis from other organisms. In addition, a crude dry weight comparison of the capsular material that was released following sonication of the bacteria did not show any consistent differences between the wild type and the mutants. Therefore, it did not appear that the selected mutants were overtly deficient for capsule synthesis. Nevertheless, the increased propensity of the mutants to associate with macrophages suggested that some cell envelope alterations had incurred as a result of the transposon insertion. Four of the selected mutants were tested for virulence as assessed by their ability to induce signs of morbidity in infected mice. The pks6 and Rv3335c mutants exhibited moderate signs of attenuation in the mouse model, while the Rvl505c and fadD23 mutants displayed a more dramatic loss of virulence as none of the mice infected with these latter strains demonstrated any signs of disease. The focus of the thesis was then re-directed toward the characterization of the Rv1505c and fadD23 mutant strains during macrophage infection and to establish the replication kinetics of the mutants in vivo during the acute and chronic phases of infection. 6.2 Advances in the genetic tools for mycobacteria 155 Although we were able to isolate mutants from the library for further study, the design of the selection procedure did have some inherent limitations. The majority Of these disadvantages center on the use of large pools of mutants during screening which does not favour the selection of clones with reduced rates of in vitro replication. This is further compounded in our protocol since the selection relied on several rounds of macrophage infection that were interspersed with periods of broth culture in order to increase the bacterial population before the next passage. Therefore, a slow-growing mutant would likely be out-competed during the in vitro culture stage, even i f the mutant presented with the desired phenotype. Another disadvantage to the use of large mutant pools is the possibility that neighbouring bacteria within the pool will compensate for the deficiencies of the desired mutant. While these issues can be averted by assessing individual mutants in isolation (7-10), this typically reduces the total number of mutants that are assayed and can also increase the time required for screening. There are also noted disadvantages to the use of Tn5370 for mutagenesis. Shortly after the completion of the mutant library for this thesis, McAdam et al. provided a comprehensive analysis of the insertion pattern for Tn5370. The authors demonstrated that the transposon favoured regions of low G+C content for insertion, leading to hot-spots of insertion and reducing the overall complexity of Tn5370-based libraries (9). Sequence analysis of transposon junctions revealed that out of 1189 insertions within an open reading frame, disruption had occurred in only 351 separate genes (9). However, it is worth noting that we found a diverse insertion using Southern hybridization of randomly chosen library mutants. A n alternative mutagenesis construct is the Himar-based mariner transposon which inserts 156 within any TA dinucleotide, allowing for a more random pattern of insertion (11). The main drawback is that this necessitates the construction of much larger mutant banks to ensure complete genome coverage, which returns to the problematic issues with screening large mutant pools. Recently, alternative approaches to library screening have emerged that utilize D N A microarrays to highlight differences within a mutant population before and after selection (12). Typically these methods have been most useful when identifying mutants that are negatively selected due to a loss of function. The loss of a mutant clone in response to a selective pressure is readily apparent by a reduction in the corresponding fluorescent signal on a hybridized microarray. These high-throughput techniques have identified genes that are required for intramacrophage survival (13, 14) and for growth within the spleens of mice (15). A characteristic feature of macrophage infection with mycobacteria is the pathogen's ability to control the trafficking events of its phagosomal niche such that fusion event with late endosomes are limited. Stewart et al. recently reported a screening strategy that positively selected for M. bovis B C G mutants that has lost this capability and preferentially accumulated within the late endosomal and lysosomal compartments of the macrophages. Macrophages were infected with pools of mutants from a transposon library and flow cytometry was used to separate the early and late endosomes post infection. D N A microarrays were hybridized with differentially labeled genetic probes that were synthesized from the mutants in each vesicle population (14). Mutants that were unable to block maturation of the phagosome were represented by an increased intensity in the fluorescent signal from the genetic probes derived from the mutants in 157 late endosomes (14). Conceivably, a similar protocol could also be applied to address the goals of this thesis. After successive rounds of macrophage binding assays, a microarray-based comparison of the mutants associated with the cell monolayer as compared to the mutant population that remained in the overlying cell culture medium could provide a list of potential mutants for further study. This approach may have proceeded more quickly than the protocol that we employed; Southern blot hybridization to identify dominant siblings within the pools followed by macrophage binding assays to confirm the desired phenotype. In summary, the list of genetic tools available for mutagenesis in mycobacteria has rapidly expanded since the start of this project. Enormous advances have been made that reduce the time and difficulty associated with site-directed mutagenesis in M. tuberculosis and transposon mutagenesis. Furthermore, the availability of inexpensive D N A microarrays has increased our understanding of the transcriptional patterns of M. tuberculosis during environmental stress, macrophage infection and replication in vivo. Looking back, these advances certainly would have impacted the manner in which this project could have been approached today. However, at the time these studies were underway, these new techniques were only just coming to fruition. Thus, while we were able to isolate a number of mutants for further study, it is quite likely that additional mutants or perhaps, an entirely different set of mutants would have been identified using a microarray-based approach in conjunction with the mariner transposon mutagenesis. 6.3 Further Characterization of the fadD23 mutant 158 Since the early studies of Goren et al., it has been known that purified SL-1 exhibits strong immunomodulatory effects on macrophage function (16). The fact that SL-1 production is restricted to M. tuberculosis within the genus Mycobacteria but is lacking from H37Ra, the avirulent M. tuberculosis laboratory-adapted strain, also led to early speculation with respect to a role in virulence. However, the results obtained in the pks2 knockouts in M. tuberculosis suggest that the fully synthesized SL-1 is not a requirement for infection (17, 18). Our findings demonstrate that disruption of fadD23 impacts SL-1 production and impairs bacterial replication in both macrophages and mice. While studies with the mmpL8 mutants also showed losses of virulence in vivo (18, 19), a direct comparison of these studies with our findings in the fadD23 mutant are complicated by differences in the selected model of infection, the strains of bacteria and the lack of mutant complementation in these other studies (18, 19). For the future studies of the fadD23 mutant, the appearance of the unidentified lipid in the apolar lipid fraction of the mutant strain merits further study. The first step toward assigning a chemical structure would require the identification of the attached acyl chains using a gas-chromatography-mass spectrometry of the fatty acid methyl esters (20). Alternatively, the lipid analysis could be broadened to compare all sulfated metabolites that are produced by the fadD23 mutant and the wild type by metabolic labeling with separate sulfur isotopes and mass spectrometry (21). This technique was recently applied to isogenic mutants in mycobacteria to pinpoint differences within the distribution of sulfur-containing compounds (18, 21, 22). While FadD23 is predicted to work in concert with Pks2, the identity of the SL-1 precursors that are synthesized in the 159 absence of FadD23 would aid in defining the precise role of the enzyme during SL-1 production. Interestingly, a recent report suggests that certain SL-1 precursors may help to modulate the adaptive arm of the host immune response (23). Antigen presentation of a diacylated SL-1 precursor via CD- lb was sufficient to stimulate the bactericidal response of CD8+ CD lb-restricted T cells (23). It is important to note that the fully mature SL-1, which is tetra-acylated, could not be presented by CD- lb (23). Thus, one could surmise that the increased production of diacylated sulfatides, as is the case in mutants lacking mmpL8 (18, 19), may actually be disadvantageous to the bacteria. With regard to the fadD23 mutant, our findings showed reduced replication in vivo even during the very early stages of infection prior to the onset of adaptive immunity. Therefore, while an increase in antigen presentation as mediated by the CD1 system may contribute to clearance of mutant, there must also be other factors at work. SL-1 production as assessed by lipid analysis (24) and the transcription of SL-1 related genes (25) has been shown to vary across both laboratory and clinical isolates. Mature SL-1 is not an absolute requirement for virulence as there are clinical isolates of M. tuberculosis that do not synthesize the sulfatides however, it may be of interest to assess i f the disruption of SL-1 synthesis has a differential impact on virulence depending whether the parental strain was a low or abundant producer of sulfatides. 6.4 Further characterization of the Rvl505c mutant Our results show that disruption of the Rvl505c gene in M. tuberculosis results in a pronounced growth defect following macrophage infection. The intracellular doubling 160 time for the Rvl505c mutant was increased by 77% relative to the parental wild type. We conducted preliminary experiments to assess whether the mutant survival was affected within acidic environments or by the presence of acidified sodium nitrite. A hypersensitivity to toxic environments could be indicative of increased permeability in the cell envelope. Our results did not reveal any differences in survival following a 24h exposure to either condition; however it is possible that a different outcome could have been observed had we examined additional time points. While some studies report statistical differences in nitrite sensitivity at early time points (26), others have only noted differences after more prolonged exposure (up to 10 days) (27). Following internalization into the macrophage, pathogenic mycobacteria effectively halt the maturation of the phagosome which prevents acidification, restricts the accumulation of lysosomal hydrolases and yet, maintains access to other early endosomal compartments. The bacterial factors that can directly modulate these events include cell wall lipoglycans and proteins that interfere with endosomal traffic (28-30) and a secreted protein that affects host cell signaling through phosphatidylinositol 3-kinase (31). In addition, a number of other M. tuberculosis genes also contribute to intramacrophage survival, although further study will be needed to elucidate their mechanisms of action (13, 14, 32, 33). It is possible that the impaired replication of the Rvl505c mutant within the phagosome is not attributable to an increased sensitivity to harsh environments but rather reflects an increased exposure of the mutant to such conditions. A more in-depth analysis of the intracellular events that follow phagocytosis of the Rvl505c mutant would certainly complement our findings of the reduced replication within macrophages. For instance, an ongoing measurement of the pH levels 161 within the mutant phagosome and monitoring the phagosomal membrane for the acquisition of lysosomal markers would help to address this question. 6.5 Issues with Genetic Complementation Genetic complementation of a gene-disrupted mutant that is accompanied by a reversal of the mutant phenotype remains as the gold standard test for confirming gene function. Yet, mutant complementation in M. tuberculosis is frequently unreported, likely due to the difficulties with genetic manipulation. In this study, complementation of the fadD23 and Rvl505c mutants reversed the growth defects within the THP-1 infection model. There was also an indication of virulence restoration in the complemented fadD23 mutant during the acute phase of infection in mice, but no improvement was observed in the complemented Rvl505c mutant. Possible explanations for the discrepancy between the two models of infection could be dysregulated expression of the complementing gene, polar effects due to the transposon or a decrease in fitness due to the complementation vector. In the case of the Rvl505c mutant, a constitutive promoter was used to drive gene expression in the complemented mutant that could impart a disadvantage to the bacteria during in vivo infection. This notion is supported by the genome-wide expression analysis of M. tuberculosis during growth in vitro and in different in vivo models of infection (34). In this study, the expression of Rvl505c was only noted during M. tuberculosis infection of SCID mice and was not apparent during broth culture or during infection of immunocompetent mice (34). Thus there is the possibility that the constant expression of Rvl505c in the complemented mutant could have additional adverse effects. With 162 respect to polar effects, the Rvl505c gene is the second gene within an apparent operon that encompasses Rvl506c-Rvl503c. Thus, there is also the possibility that the transposon disruption in Rv1505c downregulates the transcriptional expression of downstream genes. The complementation construct for the fadD23 mutant did employ native sequence from upstream of the translational start site. However, there is the possibility that additional regulatory regions may have been omitted from the complementation construct. Given these potential issues, additional studies with the complemented strains should examine the transcriptional expression of the complementing genes using RT-PCR. In addition, RT-PCR analysis of the genes which lie downstream of Rvl505c would also provide assurance that phenotypical observations of the mutant are not due to polar effects from the mutation. It is evident that a complete restoration of the virulence is far more difficult to attain when the complementing strains are faced with the defense mechanisms of the host as presented in the in vivo setting compared to isolated cell types cultured in vitro. While at this time we cannot disregard that polar effects or suppressor mutations may have occurred within the complementing strains, there is also the possibility that survival within the mouse is hampered by the presence of an additional antibiotic resistance cassette from the complementation vector. 6.6 Further characterization of the mtrB mutant During the construction of the Tn5370 library, one mutant clone displayed remarkable changes in colonial morphology and was kept aside for further study. The site of transposon insertion was mapped to the mtrB gene that encodes the sensor kinase 163 of the mtrA-mtrB two component regulatory system. The recent findings of other groups (35, 36) support our contention that the mtrA-mtrB system is linked with cell wall-related processes. Future studies with the mtrB mutant will use D N A microarrays to probe the transcriptional differences between the wild type and mutant. Ideally, this will reveal a set of genes that are coordinately expressed (37, 38). However, as others have noted, it can be difficult to extract meaningful data from such microarray studies in absence of the activating signal that is recognized by the sensor kinase (39). An alternate approach is to define the recognition sequence for the response regulator and then search for promoters that harbour the motif (40, 41). Finally, the phenotype of the mtrB mutant would also need to be confirmed using a complemented mutant strain. As the mtrA-mtrB system is also linked with a gene that encodes a putative lipoprotein, IpqB, there is the possibility that some of the characteristics of the mtrB mutant may also be attributed to polar effects on the IpqB gene. This could be tested by comparing various complementation constructs that comprise one, or all three genes of the genes within the operon. 164 6.7 Literature cited 1. Bardarov, S., J. Kriakov, C. Carriere, S. Yu , C. Vaamonde, R. A . McAdam, B. R. Bloom, G. F. Hatfull, and W. R. Jacobs, Jr. 1997. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94:10961. 2. Pelicic, V . , M . Jackson, J. M . Reyrat, W. R. Jacobs, Jr., B. Gicquel, and C. Guilhot. 1997. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U SA 94:10955. 3. McAdam, R., T. Weisbrod, J. Martin, J. Scuderi, A . Brown, J. Cirillo, B . Bloom, and W. Jacobs, Jr. 1995. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis B C G generated by transposon mutagenesis. Infect. Immun. 63:1004. 4. Cox, J. S., B. Chen, M . McNeil, and W. R. Jacobs, Jr. 1999. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:79. 5. Stokes, R. W., R. Norris-Jones, D. E. Brooks, T. J. Beveridge, D. Doxsee, and L . M . Thorson. 2004. The Glycan-Rich Outer Layer of the Cell Wall of Mycobacterium tuberculosis Acts as an Antiphagocytic Capsule Limiting the Association of the Bacterium with Macrophages. Infect Immun 72:5676. 6. Cywes, C , H. C. Hoppe, M . Daffe, and M . R. Ehlers. 1997. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent. Infect Immun 65:4258. 7. Hsu, T., S. M . Hingley-Wilson, B . Chen, M . Chen, A . Z. Dai, P. M . Morin, C. B . Marks, J. Padiyar, C. Goulding, M . Gingery, D. 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Goren. 1988. Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis. J Immunol 140:634. 17. Rousseau, C , O. C. Turner, E. Rush, Y . Bordat, T. D. Sirakova, P. E. Kolattukudy, S. Ritter, I. M . Orme, B. Gicquel, and M . Jackson. 2003. Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs. Infect Immun 71:4684. 18. Converse, S. E., J. D. Mougous, M . D. Leavell, J. A . Leary, C. R. Bertozzi, and J. S. Cox. 2003. MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc Natl Acad Sci USA 100:6121. 19. Domenech, P., M . B . Reed, C. S. Dowd, C. Manca, G. Kaplan, and C. E. Barry, III. 2004. The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis. J Biol Chem 279:21257. 20. Slayden, R. A . a. B., C.E., III. 2001. Analysis of the Lipids of Mycobacterium tuberculosis. In Mycobacterium tuberculosis Protocols, Vol . 54. S. Parish T., N.G. , ed. Humana Press Inc., Totowa, NJ, p. 229. 21. Mougous, J. D., M . D. Leavell, R. H . Senaratne, C. D. Leigh, S. J. Williams, L . W. Riley, J. A . Leary, and C. R. Bertozzi. 2002. Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach. Proc Natl Acad Sci USA 99:17037. 166 22. Mougous, J. D., C. J. Petzold, R. H . Senaratne, D. H . Lee, D. L . Akey, F. L . Lin, S. E. Munchel, M . R. Pratt, L . W. Riley, J. A . Leary, J. M . Berger, and C. R. Bertozzi. 2004. Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis. Nat Struct Mol Biol 11:721. 23. Gilleron, M . , S. Stenger, Z. Mazorra, F. Wittke, S. Mariotti, G. Bohmer, J. Prandi, L . Mori, G. Puzo, and G. De Libera. 2004. Diacylated Sulfoglycolipids Are Novel Mycobacterial Antigens Stimulating CD 1-restricted T Cells during Infection with Mycobacterium tuberculosis. J Exp Med 199:649. 24. Goren, M . B. , O. Brokl, and W. B . 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Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 187:202. 

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