Sulfolipid accumulation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> disrupted in the <Emphasis Type="Italic">mce2</Emphasis> operon

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called nice (mce1–4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL₁₂₇₈ molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [³⁵S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [³⁵S] SL₁₂₇₈ in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL₁₂₇₈ at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL₁₂₇₈ contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.     

Characterization of Mce4A protein of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: role in invasion and survival

Background  

The mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.     

Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator

Mycobacterium tuberculosis causes a variety of clinical outcomes determined by host as well as bacterial factors. M. tuberculosis disrupted in the mce1 operon causes increased mortality in immunocompetent mice. This operon is negatively regulated by mce1R (Rv0165c). We studied the role of mce1R in infection outcome in mice. At 5 x 10(4) tail vein infectious dose, the median survival time (MST) of mice infected with the mce1R mutant M. tuberculosis H37Rv was 293 days, while mice infected with the wild-type H37Rv survived more than 350 days (P < 0.0001). At a higher dose (5 x 10(6)), the MST of mutant-infected mice was 32 days, compared with 127 days for wild type-infected mice (P < 0.0001). With either tail vein or aerosol infection, mutant-infected mice developed larger granulomatous lesions in their lungs than mice infected with the wild type. Mutant-infected mice were unable to control the bacterial burden in the first 4 weeks of infection, but even after achieving control later, these mice succumbed to granulomatous pneumonia. These observations suggest that the early deregulated expression of the mce1 operon products determines later granulomatous tissue response. mce1 operon may homeostatically regulate the cell wall architecture in vivo that elicits a steady-state granuloma tissue response permitting M. tuberculosis to establish a long-term infection.     

Impact of the deletion of the six mce operons in Mycobacterium smegmatis

The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4operon is involved in cholesterol transport in M. smegmatis.     

Molecular and functional analysis of the mce4 operon in Mycobacterium smegmatis

Mycobacterium smegmatis contains 6 homologous mce (mammalian cell entry) operons which have been proposed to encode ABC‐like import systems. The mce operons encode up to 10 different proteins of unknown function that are not present in conventional ABC transporters. We have analysed the consequences of individually deleting each of the genes of the mce4 operon of M. smegmatis, which mediates the transport of cholesterol. None of the mce4 mutants were able to grow in cholesterol suggesting that all these genes are required for its uptake and that none of them can be replaced by the homologous genes of the other mce operons. This result suggests that different mce operons do not provide redundant capabilities and that M. smegmatis, in contrast with Mycobacterium tuberculosis, is not able to use alternative systems to import cholesterol in the analysed culture conditions. Either deletion of the entire mce4 operon or single point mutations that eliminate the transport function cause a phenotype similar to the one observed in a mutant lacking all 6 mce operons suggesting a pleiotropic role for this system.     

Function of a mycobacterial major facilitator superfamily pump requires a membrane-associated lipoprotein

The lprG-Rv1410c operon is critical for the survival of Mycobacterium tuberculosis during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in Mycobacterium smegmatis is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from M. tuberculosis. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type M. smegmatis and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the lprG-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.     

Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species

Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.     

In silico comparisons of lipid-related genes between Mycobacterium tuberculosis and BCG vaccine strains

Despite highly variable efficacy, BCG (Bacillus Calmette-Guérin) is the only vaccine available to prevent the tuberculosis (TB). Genomic heterogeneity between attenuated BCG strains and virulent Mycobacterium tuberculosis might help to explain this vaccine’s impaired capacity to induce long-term protection. Here, we investigate the lipid-related genes absent in attenuated BCG strains in order to correlate changes in both lipid metabolism and cell-wall lipid content to vaccine impairment. Whole genome sequences of M. tuberculosis H37Rv and the six most used BCG strains worldwide were aligned and the absent regions functionally categorized. Genomes of the BCG strains showed a total of 14 non-homologous lipid-related genes, including those belonging to mce3 operon, as well as the gene echaA1, which encodes an enoyl-CoA hydratase, and the genes encoding phospholipases PlcA, PlcB and PlcC. Taken together, the depletion of these M. tuberculosis H37Rv genomic regions were associated with marked alterations in lipid-related genes of BCG strains. Such alterations may indicate a dormant-like state and can be determining factors to the vaccine’s inability to induce long-term protection. These lipids can be further evaluated as an adjuvant to boost the current BCG-based vaccine.     

Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope

The antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non-covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall-linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.     

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications.     

Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG

Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Deltanat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.     

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Background

Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/Principle Findings

We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/Significance

This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.     

Functional analysis of an intergenic non-coding sequence within <Emphasis Type="Italic">mce1</Emphasis> operon of <Emphasis Type="Italic">M.tuberculosis</Emphasis>

Background  

The mce operons play an important role in the entry of M. tuberculosis into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of mce mutants. Recently, mce1 operon was found to extend over 13 genes, fadD5 (Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to mce1 among the four mce operons of M.tuberculosis. We have examined the function of this region.     

Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant

Mycobacterium tuberculosis causes a variety of host clinical outcomes. We previously showed that M. tuberculosis disrupted in an operon called mce1 proliferates unchecked in BALB/c mouse lungs. The observed outcome could be attributed either to the mutant bacterial burden or to the host immunopathologic response. To differentiate these possibilities, we studied the outcomes of infection in a mouse strain (C57BL/6) less susceptible to M. tuberculosis than BALB/c. We found that the mutant infection reached a plateau in the lungs at a rate similar to that of the wild type. All mice infected with the mutant, but only half of the groups of mice infected with the wild type or complemented strain, died by 40 weeks (p<0.05). At 12-21 weeks of infection, histological examination of the lungs of mice infected with the mutant showed a diffuse pattern of lymphocyte infiltration, while that of mice infected with the other strains exhibited a nodular cellular infiltration pattern. Surprisingly, the number of bacilli recovered from the lungs was similar in all three groups. These observations suggest that rather than the bacterial burden, products of the mce1 operon may directly or indirectly modulate the host immune response that is protective to both the tubercle bacilli and the host.     

Benzothiazinones Mediate Killing of Corynebacterineae by Blocking Decaprenyl Phosphate Recycling Involved in Cell Wall Biosynthesis

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.     

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.