Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence

The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a Delta pstA1 Delta pstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, Delta pstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a Delta pstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions

Unique Roles of DosT and DosS in DosR Regulon Induction and Mycobacterium tuberculosis Dormancy ▿ †

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state

Mycobacterium tuberculosis Requires Phosphate-Responsive Gene Regulation To Resist Host Immunity

Mycobacterium tuberculosis persists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-gamma). We identified the M. tuberculosis phosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-gamma-dependent immunity. A Delta pstA1 mutant was fully virulent in IFN-gamma(-/-) mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-gamma-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that Delta pstA1 bacteria are sensitized to an IFN-gamma-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. In M. tuberculosis, we found that Delta pstA1 bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of the regX3 gene suppressed the replication and virulence defects of Delta pstA1 bacteria in NOS2(-/-) mice, suggesting that M. tuberculosis requires the Pst system to negatively regulate activity of RegX3 in response to available phosphate in vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controlling M. tuberculosis gene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response

The Mycobacterium tuberculosis DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy ▿ †

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability

Virulence of <i>M</i>. <i>tuberculosis</i> Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> mutants in immune-deficient mice.

<p>Groups of immune-deficient mice (n = 8), all on the C57BL/6 background, were aerosol-infected with ~100 CFU of wild-type <i>M</i>. <i>tuberculosis</i> (WT, squares), Δ<i>pstA2</i> (circles), or Δ<i>pstA1</i>Δ<i>pstA2</i> (triangles) and monitored for signs of illness. Moribund mice were euthanized and scored as dead. <b>(A</b>) IFN-γ^-/-, (<b>B</b>) Irgm1^-/-, (<b>C</b>) NOS2^-/-.</p

Growth kinetics of <i>M</i>. <i>tuberculosis</i> Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> mutants in aerosol-infected mice.

<p>Mice were aerosol-infected with ~100 CFU of <i>M</i>. <i>tuberculosis</i> WT (squares), Δ<i>pstA2</i> (circles), or Δ<i>pstA1</i>Δ<i>pstA2</i> (triangles). Groups of mice (n = 4) were sacrificed at the indicated time points. Bacterial CFU were enumerated by plating serially diluted lung homogenates on 7H10 agar and incubating 3–4 weeks at 37°C. All mouse strains were on the C57BL/6 background. Symbols represent means; error bars indicate standard error of the mean. Asterisks indicate statistically significant differences between WT and Δ<i>pstA1</i>Δ<i>pstA2</i>: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. No significant differences in pulmonary CFU were observed between the WT and Δ<i>pstA2</i> strains. (<b>A</b>) IFN-γ^-/-, (<b>B</b>) Irgm1^-/-, (<b>C</b>) NOS2^-/-, (<b>D</b>) C57BL/6.</p

Deletion of <i>pstA2</i> causes increased resistance to acidified medium.

<p><i>M</i>. <i>tuberculosis</i> WT, Δ<i>pstA1</i>, Δ<i>pstA2</i>, Δ<i>pstA1</i>Δ<i>pstA2</i>, Δ<i>pstA2</i> pMV<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> pMV<i>pstA2</i> strains were grown to mid-exponential phase in P_i-rich 7H9 medium and then subjected to stress conditions, as indicated. Bacterial CFU were enumerated by plating serially diluted cultures on 7H10 agar and incubating for 3 to 4 weeks at 37°C. Percent survival was calculated as (CFU poststress)/(CFU pre-stress)x100. Data shown are means ± standard deviations of three independent cultures from one experiment, and are representative of at least three independent experiments. Asterisks indicate statistically significant differences between the deletion mutant and WT control for panels A-C or between the deletion mutant and both the WT and complemented strains for panels D and E: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. <b>(A)</b> Cell wall stress (sodium dodecyl sulfate, SDS) and reactive oxygen stress (hydrogen peroxide, H₂O₂). Either 0.5% SDS or 5 mM H₂O₂ was added to each culture. CFU were enumerated at 0 and 24 hours and percent survival was calculated. <b>(B)</b> P_i starvation. Bacteria were shifted to P_i-free medium and CFU were enumerated at 0, 3, 5, 7, and 10 days. <b>(C-E)</b> Acidic pH stress. Bacteria were shifted to acidified 7H9 medium pH 4.5 containing 0.1% Tween-80 and CFU were enumerated at 0, 3, 7, and 10 days.</p

Deletion of <i>pstS1</i> causes increased resistance to acidified medium.

<p>(<b>A</b>) <i>M</i>. <i>tuberculosis</i> WT, Δ<i>pstS1</i>, and Δ<i>pstS1</i> pMV<i>pstS1</i> were grown to mid-exponential phase in P_i-rich 7H9 medium and then shifted to acidified 7H9 medium pH 4.5 containing 0.1% Tween-80. Bacterial CFU were enumerated on days 0, 3, 7, and 10 by plating serially diluted cultures on 7H10 agar and incubating for 3 to 4 weeks at 37°C. Data shown are means ± standard deviations of three independent cultures from one experiment and are representative of at least three independent experiments. Asterisks indicate statistically significant differences from the WT control: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. (<b>B</b>) PstS1 protein production. Whole cell lysate proteins (4 μg) of WT, Δ<i>pstS1</i>, Δ<i>pstS1</i> pMV<i>pstS1</i> were subjected to SDS-PAGE and Western blot analysis. Mouse monoclonal anti-PstS1 and anti-GroEL2 antibodies were used to detect PstS1 and GroEL2, a cell-associated protein that serves as a loading control. Results shown are representative of two independent experiments.</p

Confirmation of the Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> deletions by Southern blotting.

<p><b>(A</b>) Genetic organization of the <i>M</i>. <i>tuberculosis pst</i> locus. Genes are indicated by arrows; probes used for Southern blotting are indicated by the gray rectangles. Locations of relevant restriction enzyme sites are shown. (<b>B</b>) Southern blot of wild-type Erdman (WT) and Δ<i>pstA2</i> genomic DNA digested with either SacI or XhoI, as indicated, and probed with probe2. Molecular weight standards are indicated at the right. The Δ<i>pstA2</i> deletion removes 0.9 kb. (<b>C</b>) Southern blot of WT and Δ<i>pstA1</i>Δ<i>pstA2</i> genomic DNA digested with PstI and probed with probe1 to detect the Δ<i>pstA1</i> deletion. Molecular weight standards are indicated at the right. The Δ<i>pstA1</i> deletion removes 0.89 kb. (<b>D, E</b>) Phthiocerol dimycocerosate (PDIM) production by the Δ<i>pstA2</i> (<b>D</b>) and Δ<i>pstA1</i>Δ<i>pstA2</i> (<b>E</b>) mutants. ¹⁴C propionate-labeled apolar lipid fractions were extracted and analyzed by thin-layer chromatography. Two spots that correspond to the phthiocerol (methoxy) and phthiodiolone (keto) forms of PDIM are indicated.</p