A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.     

Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> from THP1-derived macrophages

Background

Although mycobacterial glycolipids are among the first-line molecules involved in host–pathogen interactions, their contribution in virulence remains incomplete. Mycobacterium marinum is a waterborne pathogen of fish and other ectotherms, closely related to Mycobacterium tuberculosis. Since it causes tuberculosis-like systemic infection it is widely used as a model organism for studying the pathogenesis of tuberculosis. It is also an occasional opportunistic human pathogen. The M. marinum surface-exposed lipooligosaccharides (LOS) are immunogenic molecules that participate in the early interactions with macrophages and modulate the host immune system. Four major LOS species, designated LOS-I to LOS-IV, have been identified and characterized in M. marinum. Herein, we investigated the interactions between a panel of defined M. marinum LOS mutants that exhibited various degrees of truncation in the LOS structure, and human-derived THP-1 macrophages to address the potential of LOSs to act as pro- or avirulence factors.

Results

A moderately truncated LOS structure did not interfere with M. marinum invasion. However, a deeper shortening of the LOS structure was associated with increased entry of M. marinum into host cells and increased elimination of the bacilli by the macrophages. These effects were dependent on Toll-like receptor 2.

Conclusion

We provide the first evidence that LOSs inhibit the interaction between mycobacterial cell wall ligands and appropriate macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes.

     

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.KEY WORDS: Tuberculous meningitis, Tuberculosis, Zebrafish, Mycobacterium marinum, Blood-brain barrier, ESX-1 mutant     

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium‐containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid‐bound mycobactin (MBT) and the water‐soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe‐MBT or Fe‐cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe‐cMBT promoted the growth of wild‐type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild‐type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra‐ and intracellular growth of the pathogen.     

Mycobacterium marinum Lipooligosaccharides Are Unique Caryophyllose-containing Cell Wall Glycolipids That Inhibit Tumor Necrosis Factor-�� Secretion in Macrophages

Earlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I–IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-α in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response.A key feature of all members of the genus Mycobacterium is a cell wall of unique and complex structure, which plays an important role in antibiotic resistance and pathogenesis of mycobacteria by modulating the host immune system and phagocytic cell functions (1). The mycobacterial cell wall includes essentially two types of lipids, the mycolic acids, which are very long chain fatty acids covalently bound to the arabinogalactan polysaccharide attached to a peptidoglycan backbone (2), and a vast array of extractable lipids/glycolipids (3). The latter include the ubiquitous trehalose dimycolate (TDM)³ and phosphatidyl mannosides (PIM) (4) as well as a vast array of species-specific lipids such as phenol glycolipids (5), phthiocerol dimycocerosates (5), sulfolipids (4), glycopeptidolipids, and lipooligosaccharides (LOSs).LOSs were found and described in Mycobacterium kansasii (6–8), Mycobacterium gastri (8, 9), Mycobacterium szulgai (10), Mycobacterium malmoense (11), Mycobacterium gordonae (12), Mycobacterium butyricum (13), Mycobacterium mucogenicum (14), the Canetti variant of Mycobacterium tuberculosis (15) and, more recently in Mycobacterium marinum (Mma) (16). However, they remain among the less studied mycobacterial glycolipids at a biosynthetic, structural, and functional point of view. To date, only three genes have been experimentally demonstrated to be involved in the late steps of LOS biosynthesis in M. marinum (16, 17), and one gene encodes a polyketide synthase responsible for the synthesis of the polymethyl-branched fatty acid in the Mycobacterium smegmatis LOS (18).LOSs represent highly antigenic glycoconjugates exposed to the cell surface and useful target molecules for serotyping in a given mycobacterial species. Their precise role in mycobacteria virulence as well as in the colony morphology remains unclear (19, 20). Early studies demonstrated that rough variants of M. kansasii, devoid of all LOSs, induce chronic systemic infections in mice, whereas smooth variants containing LOSs are rapidly cleared from the organs of infected animals (19, 21). It was therefore proposed that LOSs may act as avirulence factors by masking other cell wall-associated virulence factors. Accordingly, LOSs are absent in most clinical isolates of M. tuberculosis as well as in the laboratory strain H37Rv. A recent genetically based comparison of the LOS biosynthetic cluster in M. marinum and M. tuberculosis revealed that only about one-third of the genes are conserved between the two species, with the genetic locus of M. tuberculosis H37Rv containing fewer genes (17). Although recent studies suggested a possible role of LOSs in sliding motility, biofilm formation, and infection of macrophages by M. marinum (17), the precise contribution of LOSs to M. marinum pathogenesis or virulence is seriously hampered by the restricted number of isogenic strains deficient in their production and the lack of precise structural data of LOS variants. LOSs from different mycobacterial species exhibit considerable variations in the glycan core. A previous work identified the presence of four major LOS variants in M. marinum, designated LOS-I to LOS-IV (16). Through partial characterization, the structure of LOS-I was previously established as 3-O-Me-Rhap-(1–3)-Glcp-(1–3)-Glcp-(1–4)-Glcp-(1–1)-Glcp. Although all LOSs were shown to contain this common oligosaccharidic core substituted by an additional Xylp unit, LOS-II, -III, and -IV are further substituted by other unidentified monosaccharides, designated X and YZ, which leave their exact sequence largely unknown (16).In this study, we report the identification of a natural mutant of M. marinum, devoid of LOS-IV production, which allowed the production of large amounts of LOS-III and the determination of the fine structure of all LOSs. In addition, the availability of all LOS variants with defined structures has opened the possibility to undertake structure-function relationship studies. These molecules were therefore used in in vitro assays to uncover their potent biological roles.     

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence of Mycobacterium marinum in multiple hosts

Despite intense research, PE_PGRS proteins still represent an intriguing aspect of mycobacterial pathogenesis. These cell surface proteins influence virulence in several pathogenic species, but their diverse and exact functions remain unclear. Herein, we focussed on a PE_PGRS member from Mycobacterium marinum, MMAR_0242, characterized by an extended and unique C‐terminal domain. We demonstrate that an M. marinum mutant carrying a transposon insertion in MMAR_0242 is highly impaired in its ability to replicate in macrophages and amoebae, because of its inability to inhibit lysosomal fusion. As a consequence, this mutant failed to survive intracellularly as evidenced by a reduced number of cytosolic actin tail‐forming bacteria and by quantitative electron microscopy, which mainly localized MMAR_0242::Tn within membrane‐defined vacuoles. Functional complementation studies indicated that the C‐terminus, but not the N‐terminal PE_PGRS domain, is required for intracellular growth/survival. In line with these findings, disruption of MMAR_0242 resulted in a highly attenuated virulence phenotype in zebrafish embryos, characterized by restricted bacterial loads and a failure to produce granulomas. Furthermore, expression of MMAR_0242 in Mycobacterium smegmatis, a non‐pathogenic species naturally deficient in PE_PGRS production, resulted in increased survival in amoebae with enhanced cytotoxic cell death and increased survival in infected mice with splenomegaly. Overall, these results indicate that MMAR_0242 is required for full virulence of M. marinum and sufficient to confer pathogenic properties to M. smegmatis.     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.     

Whole Genome Analyses of Marine Fish Pathogenic Isolate,Mycobacterium sp. 012931

Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.     

Role of the horizontal gene exchange in evolution of pathogenic Mycobacteria

Background

Mycobacterium tuberculosis is one of the most dangerous human pathogens, the causative agent of tuberculosis. While this pathogen is considered as extremely clonal and resistant to horizontal gene exchange, there are many facts supporting the hypothesis that on the early stages of evolution the development of pathogenicity of ancestral Mtb has started with a horizontal acquisition of virulence factors. Episodes of infections caused by non-tuberculosis Mycobacteria reported worldwide may suggest a potential for new pathogens to appear. If so, what is the role of horizontal gene transfer in this process?

Results

Availing of accessibility of complete genomes sequences of multiple pathogenic, conditionally pathogenic and saprophytic Mycobacteria, a genome comparative study was performed to investigate the distribution of genomic islands among bacteria and identify ontological links between these mobile elements. It was shown that the ancient genomic islands from M. tuberculosis still may be rooted to the pool of mobile genetic vectors distributed among Mycobacteria. A frequent exchange of genes was observed between M. marinum and several saprophytic and conditionally pathogenic species. Among them M. avium was the most promiscuous species acquiring genetic materials from diverse origins.

Conclusions

Recent activation of genetic vectors circulating among Mycobacteria potentially may lead to emergence of new pathogens from environmental and conditionally pathogenic Mycobacteria. The species which require monitoring are M. marinum and M. avium as they eagerly acquire genes from different sources and may become donors of virulence gene cassettes to other micro-organisms.

     

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense^1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores⁴. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.     

Phagocytosis of Cryptococcus neoformans by,and Nonlytic Exocytosis from,Acanthamoeba castellanii

Cryptococcus neoformans, an encapsulated, pathogenic yeast, is endowed with a variety of virulence factors, including a polysaccharide capsule. During mammalian infection, the outcome of the interaction between C. neoformans and macrophages is central to determining the fate of the host. Previous studies have shown similarities between the interaction of C. neoformans with macrophages and with amoebae, resulting in the proposal that fungal virulence for mammals originated from selection by amoeboid predators. In this study, we investigated the interaction of C. neoformans with the soil amoeba Acanthamoeba castellanii. Comparison of phagocytic efficiency of the wild type, nonencapsulated mutants, and complemented strains showed that the capsule was antiphagocytic for amoebae. Capsular enlargement was associated with a significant reduction in phagocytosis, suggesting that this phenomenon protects against ingestion by phagocytic predators. C. neoformans var. neoformans cells were observed to exit amoebae several hours after ingestion, in a process similar to the recently described nonlytic exocytosis from macrophages. Cryptococcal exocytosis from amoebae was dependent on the strain and on actin and required fungal viability. Additionally, the presence of a capsule was inversely correlated with the likelihood of extrusion in certain strains. In summary, nonlytic exocytosis from amoebae provide another parallel to observations in fungus-macrophage interactions. These results provide additional support for the notion that some mechanisms of virulence observed during mammalian infection originated, and were selected for, by environmental interactions.The encapsulated yeast Cryptococcus neoformans is an environmental organism that is capable of causing human disease. This fungus is a facultative intracellular pathogen with a unique pathogenic strategy, despite no obvious need for replication in an animal host as part of its life cycle (10). C. neoformans is known to interact with protozoa, some of which have been shown to be effective predators for this fungus (6, 26), and amoebae appear to be important for the control of C. neoformans in the environment (28). Previously, we reported that the interaction of C. neoformans with Acanthamoeba castellanii directly paralleled the interaction with human macrophages (33). Similarities between C. neoformans interactions with amoebae and macrophages included intracellular replication in a phagosome and the release of polysaccharide-containing vesicles into the cytoplasm (33). Furthermore, passage of avirulent C. neoformans and Histoplasma capsulatum through slime mold and amoebae was shown to increase virulence in mice (31, 32). On the basis of these observations, it was proposed that the capacity for mammalian virulence emerged from interactions with phagocytic predators, such as amoebae and slime mold, in the environment (7, 17, 30). Consequently, single-cell protists have emerged as important systems for the study of C. neoformans virulence, and subsequent studies have investigated the interaction of this fungus with slime mold and paramecia (9, 31). Additional evidence for this concept comes from studies of insect fungal pathogens, which suggest that the capacity for insect pathogenicity may follow preadaptation from interactions with amoebae in the environment (4). Understanding the mechanisms by which virulence emerges in environmental microbes is important considering that global warming has been hypothesized to bring about new fungal diseases in the coming century (13).Recent work in our laboratory and in that of Robin May simultaneously uncovered a novel strategy of avoiding macrophage killing whereby yeast cells were expulsed without lysis of the host cell (2, 19). The process is remarkable in that extrusion of the C. neoformans-filled phagosome is accompanied by the survival of both the host cells and the yeast cells. Phagosome extrusion or fungal exocytosis appears to be a C. neoformans-dictated event that is dependent on both the presence of the polysaccharide capsule and on the depolymerization of actin. A corollary of the hypothesis that C. neoformans virulence emerged from interactions with environmental predators is that phenomena observed with mammalian cells are likely to have a counterpart in free-living phagocytic cells. Consequently, the observation of an apparently unique event such as phagosomal extrusion from mammalian macrophages suggested a need to search for similar events in C. neoformans interactions with environmental phagocytic predators.In this study, we investigated parallels between the intracellular pathogenic strategy of C. neoformans in both macrophages and A. castellanii, focusing on characterizing the impact of the capsule on protozoan phagocytosis and on ascertaining whether fungal cells could also exit amoebae, including the role of the capsule in that possible mechanism. Using time-lapse microscopy, we observed the exocytosis of C. neoformans from A. castellanii. While there are significant differences in the nonlytic exocytosis process when comparing amoebae and macrophages, the observation of this phenomenon in amoebae provides additional support for the idea that the virulence of C. neoformans was selected for, and is maintained, by interactions in the environment with other soil organisms.(This research was conducted by Cara Chrisman in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY [awarded in 2010].)     

Oxidative Stress Response and Characterization of the oxyR-ahpC and furA-katG Loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H₂O₂, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.     

Contribution of GM-CSF on the enhancement of the T cell-stimulating activity of macrophages

Mycobacterium leprae is an intracellular parasitic organism that multiplies in macrophages (MØ). It inhibits the fusion of mycobacterial phagosome with lysosome and induces interleukin (IL)-10 production from macrophages. However, macrophages are heterogenous in various aspects. We examined macrophages that differentiated from monocytes using either recombinant (r) granulocyte-MØ colony-stimulating factor (GM-CSF) (these MØ are named as GM-MØ) or rMØ colony-stimulating factor (M-CSF) (cells named as M-MØ) in terms of the T cell-stimulating activity. Although both macrophages phagocytosed the mycobacteria equally, GM-MØ infected with M. leprae and subsequently treated with IFN-γ- and CD40 ligand (L) stimulated T cells to produce interferon-gamma (IFN-γ), but M-MØ lacked the ability to stimulate T cells. While M-MØ mounted a massive IL-10 production, GM-MØ did not produce the cytokine on infection with M. leprae. M. leprae-infected, IFN-γ- and CD40L-treated GM-MØ expressed a higher level of HLA-DR and CD86 Ags than those of M-MØ, and expressed one of the dominant antigenic molecules of M. leprae, Major Membrane Protein-II on their surface. These results indicate that GM-CSF, but not M-CSF, contributes to the up-regulation of the T cell-stimulating activity of M. leprae-infected macrophages.     

MKAN27435 Is Required for the Biosynthesis of Higher Subclasses of Lipooligosaccharides in Mycobacterium kansasii

Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.     

Oxadiazolone derivatives,new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.