Sudagar S. Gurcha

CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-β1-phosphodolichols. Responses to mannosyl-β1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

The evaluation of forty-three plant species for in vitro antimycobacterial activities ; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC=62.5 microg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC(50)=7.30 microg/ml [19.02 microM] and 29.0 microg/ml [75.56 microM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bovis BCG with compound (2) being the most active (M. bovis BCG, IC(50)=14.3 microg/ml [37.3 microM]). These results support the use of these plants in traditional medicine.

Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice

Mycobacterium tuberculosis, the causative agent of tuberculosis, has two distinguishing characteristics: its ability to stain acid-fast and its ability to cause long-term latent infections in humans. Although this distinctive staining characteristic has often been attributed to its lipid-rich cell wall, the specific dye-retaining components were not known. Here we report that targeted deletion of kasB, one of two M. tuberculosis genes encoding distinct β-ketoacyl- acyl carrier protein synthases involved in mycolic acid synthesis, results in loss of acid-fast staining. Biochemical and structural analyses revealed that the ΔkasB mutant strain synthesized mycolates with shorter chain lengths. An additional and unexpected outcome of kasB deletion was the loss of ketomycolic acid trans-cyclopropanation and a drastic reduction in methoxymycolic acid trans-cyclopropanation, activities usually associated with the trans-cyclopropane synthase CmaA2. Although deletion of kasB also markedly altered the colony morphology and abolished classic serpentine growth (cording), the most profound effect of kasB deletion was the ability of the mutant strain to persist in infected immunocompetent mice for up to 600 days without causing disease or mortality. This long-term persistence of ΔkasB represents a model for studying latent M. tuberculosis infections and suggests that this attenuated strain may represent a valuable vaccine candidate against tuberculosis.

A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis

The Mycobacterium tuberculosis TetR‐type regulator Rv3574 has been implicated in pathogenesis as it is induced in vivo, and genome‐wide essentiality studies show it is required for infection. As the gene is highly conserved in the mycobacteria, we deleted the Rv3574 orthologue in Mycobacterium smegmatis (MSMEG_6042) and used real‐time quantitative polymerase chain reaction and microarray analyses to show that it represses the transcription both of itself and of a large number of genes involved in lipid metabolism. We identified a conserved motif within its own promoter (TnnAACnnGTTnnA) and showed that it binds as a dimer to 29 bp probes containing the motif. We found 16 and 31 other instances of the motif in intergenic regions of M. tuberculosis and M. smegmatis respectively. Combining the results of the microarray studies with the motif analyses, we predict that Rv3574 directly controls the expression of 83 genes in M. smegmatis, and 74 in M. tuberculosis. Many of these genes are known to be induced by growth on cholesterol in rhodococci, and palmitate in M. tuberculosis. We conclude that this regulator, designated elsewhere as kstR, controls the expression of genes used for utilizing diverse lipids as energy sources, possibly imported through the mce4 system.

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes

The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14alpha-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0.2 and 0.3 micro M, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1.2 and 36.5 micro M, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K(d) values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor DeltaCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium–host interaction

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC‐SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose‐dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC‐SIGN‐transfected Raji cells, but no differences in IL‐10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose‐capped LAM does not dominate the Mycobacterium–host interaction.

Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis.

Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.

3‐Ketosteroid 9α‐hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3‐ketosteroid 9α‐hydroxylase (KSH). Consistent with their predicted roles, the ΔkshA and ΔkshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4‐androstene‐3,17‐dione as primary carbon and energy sources. Interestingly, ΔkshA and ΔkshB mutants were also unable to metabolize the steroid substrate 5α‐androstane‐3,17‐dione, whereas wild‐type M. tuberculosis H37Rv could. The deletion of either of these genes lead to rapid death of the microorganism in murine infection models and in macrophages, showing that kshA and kshB are essential factors for M. tuberculosis pathogenesis. Penta‐acylated trehalose (PAT) biosynthesis was altered in the ΔkshB mutant, but not the ΔkshA mutant. The ΔkshB mutant synthesizes all other types of lipids. The ΔkshB mutant had a thickened outer layer in its cell wall. KshB thus appears to be involved in multiple processes, probably as a reductase of different oxygenases. We conclude that an impaired 3‐ketosteroid 9α‐hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants.

Mycolic Acid Modification by the mmaA4 Gene of M. tuberculosis Modulates IL-12 Production

Mycobacterium tuberculosis has evolved many strategies to evade elimination by the host immune system, including the selective repression of macrophage IL-12p40 production. To identify the M. tuberculosis genes responsible for this aspect of immune evasion, we used a macrophage cell line expressing a reporter for IL-12p40 transcription to screen a transposon library of M. tuberculosis for mutants that lacked this function. This approach led to the identification of the mmaA4 gene, which encodes a methyl transferase required for introducing the distal oxygen-containing modifications of mycolic acids, as a key locus involved in the repression of IL-12p40. Mutants in which mmaA4 (hma) was inactivated stimulated macrophages to produce significantly more IL-12p40 and TNF-α than wild-type M. tuberculosis and were attenuated for virulence. This attenuation was not seen in IL-12p40-deficient mice, consistent with a direct linkage between enhanced stimulation of IL-12p40 by the mutant and its reduced virulence. Treatment of macrophages with trehalose dimycolate (TDM) purified from the ΔmmaA4 mutant stimulated increased IL-12p40, similar to the increase observed from ΔmmaA4 mutant-infected macrophages. In contrast, purified TDM isolated from wild-type M. tuberculosis inhibited production of IL-12p40 by macrophages. These findings strongly suggest that M. tuberculosis has evolved mmaA4-derived mycolic acids, including those incorporated into TDM to manipulate IL-12-mediated immunity and virulence.

Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component

SUMMARY The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobacterium marinum infection model to study initiation of granuloma formation and developed an in vivo screen to select for random M. marinum mutants that were unable to induce granuloma formation efficiently. Upon screening 200 mutants, three mutants repeatedly initiated reduced granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster. The ESX-1 cluster is disrupted in the Mycobacterium bovis BCG vaccine strain and encodes a specialized secretion system known to be important for granuloma formation and virulence. Although espL has not been implicated in protein secretion before, we observed a strong effect on the secretion of the ESX-1 substrates ESAT-6 and EspE. We conclude that our zebrafish embryo M. marinum screen is a useful tool to identify mycobacterial genes involved in the initial stages of granuloma formation and that we have identified a new component of the ESX-1 secretion system. We are confident that our approach will contribute to the knowledge of mycobacterial virulence and could be helpful for the development of new TB vaccines.