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 ΔpstA1 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 ΔpstA1 mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-type M. tuberculosis. The esx-5 RegX3 binding site deletion (ΔBS) also suppressed attenuation of the ΔpstA1 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 ΔpstA1 ΔBS mutant remained attenuated in both NOS2-/- and C57BL/6 mice, suggesting that factors other than ESX-5 secretion also contribute to attenuation of the ΔpstA1 mutant. In addition, a ΔpstA1 ΔesxN 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.
Bibliographical noteFunding Information:
We thank Alyssa Brokaw and Leanne Zhang for expert technical assistance with animal experiments and the staff of the University of Minnesota BSL-3/ABSL-3 core facility. Antisera against GroEL2 (monoclonal clone IT-70; catalog no. NR-13657) and ModD (polyclonal anti-Mpt32; catalog no. NR-13807) were obtained from BEI Resources, NIAID, NIH. This work was supported by NIH Director's New Innovator Award DP2AI112245 (A.D.T.), start-up funding from the University of Minnesota (A.D.T.), and the Dennis W. Watson Fellowship (D.W.W.).
This work was supported by NIH Director’s New Innovator Award DP2AI112245 (A.D.T.), start-up funding from the University of Minnesota (A.D.T.), and the Dennis W. Watson Fellowship (D.W.W.).
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- ESX secretion
- Gene regulation
- Mycobacterium tuberculosis
- Pst system
- Two-component regulatory systems
- Type VII secretion