Biology subjects

Dechow, S. J.

Publications and source records attributed to Dechow, S. J..

3 recordsLinked to original sources

bioRxiv2022

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.

microbiology↗

bioRxiv2021

ppe51 variants promote non-replicating Mycobacterium tuberculosis to grow at acidic pH by selectively promoting glycerol uptake

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acidic growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we conducted forward genetic screens that identified several Mtb mutants that could grow under these restrictive conditions. All of the mutants were mapped to the ppe51 gene and resulted in three amino acid substitution - S211R, E215K, and A228D. Expression of the PPE51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, enhanced acid growth (eag) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting ppe51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced in virulence in activated macrophages as compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the WT or S211R variants in the presence of glycerol, supporting the eag substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis.

microbiology↗

bioRxiv2021

AC2P20 selectively kills M. tuberculosis at acidic pH by depleting free thiols

Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a [~]220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.

microbiology↗