Carbon dioxide regulates <i>Mycobacterium tuberculosis</i> PhoPR signaling and virulence

The <i>Mycobacterium tuberculosis</i> (Mtb) two-component regulatory system PhoPR is implicated in pH sensing within the macrophage because it is strongly induced by acidic pH both <i>in vitro</i> and the macrophage phagosome. The carbonic anhydrase (CA) inhibitor ethoxzolamide inhibits PhoPR signaling supporting the hypothesis that CO₂ may also play a role in regulating PhoPR. Here, we show that increasing CO₂ concentration induces PhoPR signaling, at both pH 7.0 and pH 5.7. At acidic pH 5.7, a normally strong inducer of PhoPR signaling, increasing CO₂ from 0.5% to 5% further induces the pathway, showing CO₂ acts synergistically with acidic pH to induce the PhoPR regulon. Based on these findings, we propose that PhoPR functions as a CO₂ sensor. Mtb has three CA (CanA, CanB, and CanC), and using CRISPR interference knockdowns and gene deletion mutants, we assessed which CAs regulate PhoPR signaling and macrophage survival. We first examined if CA played a role in Mtb pathogenesis and observed that CanB was required for survival in macrophages, where the knockdown strain had ~1-log reduction in survival. To further define the interplay of CO₂ and Mtb signaling, we conducted transcriptional profiling experiments at varying pH and CO₂ concentrations. As hypothesized, we observed that the induction of PhoPR at acidic pH is dependent on CO₂ concentration, with a subset of core PhoPR regulon genes dependent on both 5% CO₂ and acidic pH for their induction, including expression of the ESX-1 secretion system. Transcriptional profiling also revealed core CO₂-responsive genes that were differentially expressed independently of the PhoPR regulon or the acidic pH-inducible regulon. Notably, genes regulated by a second two-component regulatory system, TrcRS, are associated with adaptation to changes in CO₂.