In vivo Reporters of Mycobacterium tuberculosis ESX-5 Secretion

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PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) requires the specialized Type VII protein secretion system ESX-5 for growth in vitro, but little is known about the expression or activity of ESX-5 during infection. A recent report suggested ESX-5 genes are transcriptionally induced at the acute stage of infection in mice. However, the regulatory networks and host signals that stimulate ESX-5 expression as well as the stage(s) of infection at which ESX-5 is induced remain to be comprehensively identified. The overall objective of this proposal is to characterize how Mtb regulates ESX-5 secretion during infection. The proposed research will test the central hypothesis that ESX-5 is activated in response to specific nutritional cues or environmental stressors to counteract growth restriction in host interstitial macrophages. Prior studies implicated phosphate limitation as one signal that induces ESX-5 activity in vitro, but disrupting phosphate-dependent ESX-5 regulation did not impact Mtb growth or persistence in mice, suggesting that other uncharacterized regulators control ESX-5 in the host. Preliminary data show that over-expression of two different transcriptional regulators can induce ESX-5 secretion in vitro. Specific Aim 1 will test the hypothesis that specific transcription factors activate ESX-5 in response to nutritional cues and stress conditions that Mtb encounters in host macrophages. In Specific Aim 2, fluorescent ESX-5 transcriptional reporters will be developed and tested in macrophage and mouse infection models using flow cytometry to determine when and in what cell types ESX-5 expression is induced during infection. Fluorescent reporters of ESX-5 substrate export will also be developed for analysis of ESX-5 activity during macrophage infection. The innovative fluorescent reporters developed will enable future screens for small molecules that disrupt ESX-5 expression and/or activity. The proposed research is significant because it will fill important gaps in knowledge of ESX-5 function by identifying the host cues and regulatory factors that control ESX-5 expression and establishing the stage(s) of infection when Mtb will be most vulnerable to drugs targeting ESX-5 or its regulation.