PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence

Abstract

Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The M. tuberculosis PhoP regulon.

The PhoP regulon was identified by comparing transcriptional profiles of the M. tuberculosis wild type and the phoP mutant using DNA microarrays. Some of the more relevant genes to virulence and intracellular survival are listed and grouped by function. Green and red arrows indicate genes whose expression is positively or negatively regulated by PhoP, respectively.

Figure 2. Protein expression patterns of M. tuberculosis and the phoP mutant.

Areas of 2D-polyacrylamide gels show differences in the protein expression patterns between the wild type strain and the phoP mutant. Spots that showed at least three-fold differential expression across triplicate gels were selected for identification by mass spectrometry. EspB and Hsp65 are more expressed in the wild type strain while ICL shows a higher expression in the phoP mutant. The vertical arrows indicate decreased (↓) or increased (↑) expression in the M. tuberculosis phoP mutant relative to the parent strain.

Figure 3. Quantification of gene expression by qRT-PCR.

Relative expression levels of the dosR, nuoB, lipF, pks3 and icl genes. The relative quantity (RQ) for each gene in the phoP mutant and the complemented strain were calculated with respect to the gene expression levels in the wild type strain. The expression levels of each gene in each strain were normalized to the levels of sigA mRNA. Primers and probe sequences for the aforementioned genes as well as for the endogenous control sigA are listed in Table S3.

Figure 4. Schematic representation of the PhoP-regulated genes within the extRD1 region.

The extRD1 region includes genes essential for ESAT-6/CFP10 secretion (blue), genes essential for ESAT-6/CFP10 expression (green) and genes implicated in RD1-mediated virulence (red). Genes identified as positively regulated by PhoP are indicated (+).

Figure 5. Determination of sensitivity to the ICL inhibitor 3-NP.

The wild type, phoP mutant and complemented strains were tested for their ability to grow in 7H9 medium supplemented with glucose or propionate as sole carbon sources in the presence of 3-NP. A change from blue to pink coloration is indicative of resazurin reduction and consequently it correlates with bacterial viability. No differences in 3-NP sensitivity were observed when the wild type, the phoP mutant or the complemented strains were grown in glucose as unique carbon source. The phoP mutant is less sensitive to 3-NP than the parental strain when grown in propionate as sole carbon supplier, indicating higher levels of ICL expression in the mutant. Complementation of the mutant with the phoPR operon renders bacteria as susceptible to 3-NP as the parental strain when propionate is the unique carbon source.

Figure 6. Hsp65- and Ag85A-specific responses exhibited by mice immunised with M. tuberculosis phoP mutant and BCG.

Cells from spleen, lungs and lymph nodes from mice immunised with either BCG or the phoP mutant were stimulated with Hsp65 or Ag85A (p 85) and IFNγ production was measured by ELISA. Bars represent mean and SD from two separate experiments. Asterisks indicate significant differences in IFN-γ production. A higher percentage of Hsp65-specific cells is found in spleen and lymph nodes from mice immunized with SO2 when compared with BCG-immunised mice. Lymph nodes from SO2-immunised mice contained a higher fraction of cell responding to Ag85 in comparison with BCG-immunised mice.

Figure 7. Persistence of BCG and the M. tuberculosis phoP mutant in BALB/c mice.

Animals were intravenously infected with either BCG or the SO2 phoP mutant. Bars represent mean and SD of log10 CFUs recovered from spleen and lungs of inoculated animal at 1 and 3 months after the initial infection. Asterisks indicate significant differences in CFU counts. We were unable to compare persistence of the SO2 phoP mutant with the wild type since the latter strain kills animals by day 60 post infection (data not shown).