15The Mycobacterium tuberculosis Type VII secretion system ESX-5, which has been implicated 16 in virulence, is activated at the transcriptional level by the phosphate starvation responsive 17 Pst/SenX3-RegX3 signal transduction system. Deletion of pstA1, which encodes a Pst 18 phosphate transporter component, causes constitutive activation of the response regulator 19 RegX3, hyper-secretion of ESX-5 substrates and attenuation in the mouse infection model. We 20 hypothesized that constitutive activation of ESX-5 secretion causes attenuation of the ∆pstA1 21 mutant. To test this, we uncoupled ESX-5 from regulation by RegX3. Using electrophoretic 22 mobility shift assays, we defined a RegX3 binding site in the esx-5 locus. Deletion or mutation of 23 the RegX3 binding site reversed hyper-secretion of the ESX-5 substrate EsxN by the ∆pstA1 24 mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-25 type M. tuberculosis. Deletion of the esx-5 RegX3 binding site (∆BS) suppressed attenuation of 26 the ∆pstA1 mutant in Irgm1 -/mice, suggesting that constitutive ESX-5 secretion limits M.27 tuberculosis evasion of host immune responses that are independent of Irgm1. However, the 28 ∆pstA1∆BS mutant remained attenuated in both NOS2 -/and C57BL/6 mice, suggesting that 29 factors other than ESX-5 secretion also contribute to attenuation of the ∆pstA1 mutant. In 30 addition, a ∆pstA1∆esxN mutant lacking the hyper-secreted ESX-5 substrate EsxN remained 31 attenuated in Irgm1 -/mice, suggesting that ESX-5 substrates other than EsxN cause increased 32 susceptibility to host immunity. Our data indicate that while M. tuberculosis requires ESX-5 for 33 virulence, it tightly controls secretion of ESX-5 substrates to avoid elimination by host immune 34 responses. 35 INTRODUCTION 36Pathogenic bacteria often regulate the activity of specialized protein secretion systems 37 that are required for virulence to ensure release of secreted effectors only at the appropriate 38 stage of infection. Tight control of secretion system activity may limit recognition by the host 39 immune system or prevent expression of complex secretion machines that restrict growth (1, 2). 3Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes five Type VII or ESX 41 specialized protein secretion systems, of which ESX-1, ESX-3 and ESX-5 have been shown to 42 promote pathogenesis (3). M. tuberculosis regulates activity of each of these secretion systems 43 in response to signals encountered in the host. Iron limitation activates ESX-3 (4), which plays a 44 role in both iron scavenging and inhibiting phagosome maturation (5, 6). ESX-1 permeabilizes 45 the phagosomal membrane to allow bacterial access to the host cell cytoplasm (7-9). ESX-1 46 secretion is regulated by two signal transduction systems, PhoPR and MprAB, that respond to 47 acidic pH and cell wall stress, respectively, signals that M. tuberculosis encounters in the 48 phagosome (10-13). We recently demonstrated that M. tuberculosis activates ESX-5 ...