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Sensing and response to environmental cues, such as pH and chloride (Cl−), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl− levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure–activity relationship studies on the hit compound “C6,” or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl− response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl− response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl−-responsive genes in the simultaneous presence of cholesterol and high external Cl− concentration, versus transcript levels observed during exposure to high external Cl− concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery., Responding to environmental cues such as pH and chloride is critical in enabling Mycobacterium tuberculosis to colonize its host. A chemical screen using an M. tuberculosis strain bearing a fluorescent reporter identifies a compound that perturbs the bacterial response to chloride and inhibits its growth in a murine infection model.

Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl-) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K+) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K+ levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K+, Cl-, and pH. We demonstrate that intraphagosomal K+ levels increase during macrophage phagosome maturation, and find using a novel fluorescent K+-responsive reporter Mtb strain that K+ is not limiting during macrophage infection. Disruption of Mtb K+ homeostasis by deletion of the Trk K+ uptake system results in dampening of the bacterial response to pH and Cl-, and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb., Author summary Tuberculosis is the leading cause of death from infectious diseases globally, and knowledge of the fundamental biology underlying successful host colonization by Mycobacterium tuberculosis (Mtb) is critical for understanding its pathogenicity. Our study focuses on one such important facet—the ability of Mtb to sense and respond to changing environmental ionic signals, and its relation to maintenance of bacterial ionic homeostasis. Potassium (K+) is the most abundant ion in both mammalian and bacterial cells, but much remains unknown about how it may affect host-pathogen interactions. We show here that Mtb has distinct gene expression changes in response to variation in environmental K+ levels. Importantly, disruption of a Mtb K+ uptake system reduces the ability of Mtb to respond to other key ionic cues, and results in diminished host colonization. Our study reveals novel roles of K+ during Mtb-host interactions, and suggests the broader importance of abundant ions in bacterial pathogenicity.

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor., Author summary The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE -based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δ erm . Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δ erm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δ erm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA. IMPORTANCE Clostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Clostridium difficile is a spore-forming obligate anaerobe that is a leading cause of healthcare-associated infections. C. difficile infections begin when its metabolically dormant spores germinate in the gut of susceptible individuals. Binding of bile salt germinants to the Csp family pseudoprotease CspC triggers a proteolytic signaling cascade consisting of the Csp family protease CspB and the cortex hydrolase SleC. Conserved across many of the Clostridia, Csp proteases are subtilisin-like serine proteases that activate pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the protective cortex layer, allowing spores to resume metabolism and growth. This signaling pathway, however, is differentially regulated in C. difficile, since CspC functions both as a germinant receptor and regulator of CspB activity. CspB is also produced as a fusion to a catalytically inactive CspA domain that subsequently undergoes interdomain processing during spore formation. In this study, we investigated the role of the CspA pseudoprotease domain in regulating C. difficile spore germination. Mutational analyses revealed that the CspA domain controls CspC germinant receptor levels in mature spores and is required for optimal spore germination, particularly when CspA is fused to the CspB protease. During spore formation, the YabG protease separates these domains, although YabG itself is dispensable for germination. Bioinformatic analyses of Csp family members suggest that the CspC-regulated signaling pathway characterized in C. difficile is conserved in related Peptostreptococcaceae family members but not in the Clostridiaceae or Lachnospiraceae. Our results indicate that pseudoproteases play critical roles in regulating C. difficile spore germination and highlight that diverse mechanisms control spore germination in the Clostridia.