Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E_MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E_MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E_MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.     

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741‐1743 (highly homologous to a Pseudomonas aeruginosa acyl‐CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886‐MSMEG_1741‐1743 strictly required oleic acid for growth. The ΔMSMEG_1886‐ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742‐43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.     

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.     

How do antibiotic‐producing bacteria ensure their self‐resistance before antibiotic biosynthesis incapacitates them?

Acquired antibiotic resistance among dangerous bacterial pathogens is an increasing medical problem. While in Mycobacterium tuberculosis this occurs by mutation in the genes encoding the targets for antibiotic action, other pathogens have generally gained their resistance genes by horizontal gene transfer from non‐pathogenic bacteria. The ultimate source of many of these genes is almost certainly the actinomycetes that make the antibiotics and therefore need self‐protective mechanisms to avoid suicide. How do they ensure that they are resistant at the time when intracellular antibiotic concentrations reach potentially lethal levels? In this issue of Molecular Microbiology, Tahlan et al. describe a solution to this problem in which an antibiotically inactive precursor of a Streptomyces coelicolor antibiotic induces resistance – in this example by means of a trans‐membrane export pump – so that the organism is already primed for resistance at the time when it is needed. The authors generalize their interpretation to other cases where antibiotic resistance depends on export, but it will be interesting to find out whether it could in fact apply more widely, to include the other major mechanisms of resistance: target modification and the synthesis of antibiotics via a series of chemically modified intermediates, with removal of the protective group at the time of secretion into the outside medium.     

Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis

The C‐19 steroids 4‐androstene‐3,17‐dione (AD), 1,4‐androstadiene‐3,17‐dione (ADD) or 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD), which have been postulated as intermediates of the cholesterol catabolic pathway in Mycobacterium smegmatis, cannot be used as sole carbon and energy sources by this bacterium. Only the ΔkstR mutant which constitutively expresses the genes repressed by the KstR regulator can metabolize AD and ADD with severe difficulties but still cannot metabolize 9OH‐AD, suggesting that these compounds are not true intermediates but side products of the cholesterol pathway. However, we have found that some M. smegmatis spontaneous mutants mapped in the PadR‐like regulator (MSMEG_2868) can efficiently metabolize all C‐19 steroids. We have demonstrated that the PadR mutants allow the expression of a gene cluster named C‐19+ (MSMEG_2851 to MSMEG_2901) encoding steroid degrading enzymes, that are not expressed under standard culture conditions. The C‐19+ cluster has apparently evolved independently from the upper cholesterol kstR‐regulon, but both clusters converge on the lower cholesterol kstR2‐regulon responsible for the metabolism of C and D steroid rings. Homologous C‐19+ clusters have been found only in other actinobacteria that metabolize steroids, but remarkably it is absent in Mycobacterium tuberculosis.     

Unravelling a new catabolic pathway of C‐19 steroids in Mycobacterium smegmatis

In this work, we have characterized the C‐19+ gene cluster (MSMEG_2851 to MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis, we have identified the genes encoding enzymes involved in the modification of the A/B steroid rings during the catabolism of C‐19 steroids in certain M. smegmatis mutants mapped in the PadR‐like regulator (MSMEG_2868), that constitutively express the C‐19+ gene cluster. By using gene complementation assays, resting‐cell biotransformations and deletion mutants, we have characterized the most critical genes of the cluster, that is, kstD2, kstD3, kshA2, kshB2, hsaA2, hsaC2 and hsaD2. These results have allowed us to propose a new catabolic route named C‐19+ pathway for the mineralization of C‐19 steroids in M. smegmatis. Our data suggest that the deletion of the C‐19+ gene cluster may be useful to engineer more robust and efficient M. smegmatis strains to produce C‐19 steroids from sterols. Moreover, the new KshA2, KshB2, KstD2 and KstD3 isoenzymes may be useful to design new microbial cell factories for the 9α‐hydroxylation and/or Δ1‐dehydrogenation of 3‐ketosteroids.     

Identification of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> genes associated with antibiotic susceptibility

Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.