Biology subjects

Dechow, S. J.

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

5 recordsLinked to original sources

bioRxiv2026

Combined lactate- and phosphate-dependent cytoplasmic acidification drives Mycobacterium tuberculosis growth arrest at acidic pH

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

microbiology↗

bioRxiv2025

Mycobacterium tuberculosis growth arrest on propionate at acidic pH is suppressed by mutations in phoPR and pyrazinamide treatment

Mycobacterium tuberculosis (Mtb) arrests its growth at acidic pH, when grown on specific single carbon sources, including propionate. However, Mtb grows well on propionate at pH 7.0, supporting that propionate can support growth as a sole carbon source. To understand the basis of the propionate-driven growth arrest at acidic pH, we performed a forward genetic selection for mutants that enable growth on propionate at pH 5.7. All the selected mutants had insertions in the two-component regulatory genes phoR or phoP. We hypothesized that growth arrest at acidic pH is caused by PhoPR diverting carbon from central carbon metabolism towards lipid anabolism and that when PhoPR is inactivated, growth is promoted through metabolizing propionate by the methyl citrate cycle (MCC) into pyruvate, a permissive carbon source for growth at acidic pH. Using chemical inhibition and mutants of the MCC pathway, we demonstrate that the enhanced growth is dependent on the MCC. Furthermore, stimulating lipid synthesis via the methylmalonyl-CoA pathway by adding vitamin B12 restricts growth in the{Delta} phoPR mutant and, conversely, restricting lipid anabolism by inhibiting the triacylglycerol (TAG) synthase tgs1 enhances growth of the{Delta} phoPR mutant. Notably, CoA pools increased in the{Delta} phoPR mutant grown on propionate, directly supporting our model. Given the role of CoA metabolism in pyrazinamide sensitivity, we examined Mtb sensitivity to pyrazinamide on propionate at acidic pH and, surprisingly, observed that pyrazinamide treatment of WT Mtb suppresses growth arrest on propionate at acidic pH. In contrast, the phoPR mutant has enhanced sensitivity to pyrazinamide. Together, these findings support that propionate-driven growth arrest at acidic pH is caused by metabolic remodeling that is regulated by PhoPR and is associated with pyrazinamide sensitivity. ImportanceWhen grown on certain single carbon sources, such as propionate, Mtb arrests its growth at acidic pH and establishes a state of non-replicating persistence (NRP). To understand the genetic basis of this growth restriction, a genetic selection was performed to identify mutants unable to arrest growth at acidic pH with propionate as a sole carbon source. The selection exclusively identified mutants in the PhoPR two-component regulatory system, which functions to modulate cell envelope lipids and redox homeostasis through the upregulation of lipid synthesis at acidic pH. Using genetic and chemical inhibition studies, we demonstrate that PhoPR arrests growth at acidic pH by diverting carbon away from the methyl citrate cycle towards lipid anabolism. Surprisingly, treatment of Mtb with pyrazinamide at acidic pH on propionate, also enabled growth. Therefore, this study defines new mechanisms by which Mtb integrates environmental signaling to regulate growth, metabolism, and drug susceptibility. These findings are relevant to pathogenesis, as PhoPR is essential for growth in macrophages and animals, environments with varying pH and carbon source availability, depending on immune pressures. These data suggest that drug susceptibility may be impacted by enhanced growth and metabolic capacity of Mtb in acidic and propionate-rich environments, such as the within the macrophage or the granuloma.

microbiology↗

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↗