Carbon dioxide regulates Mycobacterium tuberculosis PhoPR signaling and virulence

The Mycobacterium tuberculosis (Mtb) two-component regulatory system, PhoPR, is implicated in pH-sensing within the macrophage because it is strongly induced by acidic pH both in vitro and the macrophage phagosome. The carbonic anhydrase (CA) inhibitor ethoxzolamide (ETZ) 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, and its induction is independent of medium pH. We also show that at acidic pH 5.7, a normally strong inducer of PhoPR signaling, that increasing CO2 from 0.5% to 5% further induces the pathway. 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 virulence. We first examined if CA played a role in Mtb pathogenesis and observed that CanB was required for virulence in macrophages, where the knockdown strain had ~1 log reduction in virulence. 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 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. ### Competing Interest Statement RBA is the founder of Tarn Biosciences, Inc. a company developing new antimycobacterial drugs. RBA and BKJ are inventors on a patent (US10,653,679B2) describing the use of ETZ to treat mycobacterial infections.