Mycobacterium tuberculosis Requires Regulation of ESX-5 Secretion for Virulence in Irgm1-Deficient Mice

ABSTRACT The Mycobacterium tuberculosis type VII secretion system ESX-5, which has been implicated in virulence, is activated at the transcriptional level by the phosphate starvation-responsive Pst/SenX3-RegX3 signal transduction system. Deletion of pstA1 , which encodes a Pst phosphate transporter component, causes constitutive activation of the response regulator RegX3, hypersecretion of ESX-5 substrates and attenuation in the mouse infection model. We hypothesized that constitutive activation of ESX-5 secretion causes attenuation of the Δ pstA1 mutant. To test this, we uncoupled ESX-5 from regulation by RegX3. Using electrophoretic mobility shift assays, we defined a RegX3 binding site in the esx-5 locus. Deletion or mutation of the RegX3 binding site reversed hypersecretion of the ESX-5 substrate EsxN by the Δ pstA1 mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-type M. tuberculosis. The esx-5 RegX3 binding site deletion (Δ BS ) also suppressed attenuation of the Δ pstA1 mutant in Irgm1 −/− mice. These data suggest that constitutive ESX-5 secretion sensitizes M. tuberculosis to an immune response that still occurs in Irgm1 −/− mice. However, the Δ pstA1 Δ BS mutant remained attenuated in both NOS2 −/− and C57BL/6 mice, suggesting that factors other than ESX-5 secretion also contribute to attenuation of the Δ pstA1 mutant. In addition, a Δ pstA1 Δ esxN mutant lacking the hypersecreted ESX-5 substrate EsxN remained attenuated in Irgm1 −/− mice, suggesting that ESX-5 substrates other than EsxN cause increased susceptibility to host immunity. Our data indicate that while M. tuberculosis requires ESX-5 for virulence, it tightly controls secretion of ESX-5 substrates to avoid elimination by host immune responses.