Rita Tamayo

Clostridioides difficile is a major cause of antibiotic-associated diarrhea. This pathogen produces toxins essential for disease and flagella that promote intestinal colonization. Flagella and toxins are coregulated in an ON/OFF manner leading to a heterogeneous population of toxigenic, flagellated cells ( flg -ON) and aflagellate cells with attenuated toxin production ( flg -OFF). How selective pressures affect ON/OFF switching dynamics and overall population composition is unknown. Here, we use a mouse model of disease to evaluate the ON/OFF makeup of the C. difficile population in vivo. Two key results emerged: (i) Directional inversion of the flg switch to the “ON” orientation occurs during spore germination, and (ii) toxin-induced inflammation enriches for a flg -OFF population during infection. These findings reveal a cycle in which predominantly flg -OFF spores are disseminated into the environment, and spore germination in the next host resets C. difficile to a virulent population of motile, toxigenic cells at infection onset.

Many Clostridioides difficile strains can form two colony morphotypes: rough and smooth. The rough and smooth morphotypes differ in multiple phenotypes, including cell length and chaining, motility, biofilm production, and virulence in the hamster model of C. difficile infection (CDI). Colony morphology undergoes phase variation and is determined by the ON/OFF expression of cmrRST , which encodes a signal transduction system. Here, we test the hypothesis that differences in colony morphology and the associated phenotypes influence pathogenesis and the host response to infection. We first compared the rough and smooth colony variants of wild-type C. difficile in a mouse model of CDI. However, CmrRST phase varied during infection such that the C. difficile populations became indistinguishable in feces and tissues, and no differences in disease were observed in mice inoculated with these variants. We next circumvented phase variation using mutants that form only rough or only smooth colonies. Co-infection of mice with these phenotypically locked strains revealed that the smooth colony mutant has greater fitness than the rough mutant, which is outcompeted during late infection. In addition, NanoString analyses showed a higher number of differentially expressed pro-inflammatory genes and overall higher expression levels in mice infected with the rough colony mutant, independent of bacterial burden and toxin levels. Our results indicate that in a mouse model of CDI, cells from rough colonies are more immunostimulatory during early murine infection, potentially leading to reduced relative fitness compared to cells from smooth colonies.

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile , employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis , an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase-variable CmrRST signal transduction system in C. difficile . The CmrRST system controls multiple phenotypes, including colony morphology, cell elongation, and cell chaining in C. difficile . Here, we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr- ON state, leading to chaining and a rough colony morphology. This phenomenon is most pronounced with E. faecalis , as other enterococcal species and select Gram-negative enteric bacteria do not show a similar effect. These results suggest that the composition of the polymicrobial environment in the gut is critical to influencing C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. IMPORTANCE: Clostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile ; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis , an opportunistic pathogen known to co-colonize the gut with C. difficile , influences phase variation and downstream growth phenotypes in C. difficile . This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

Many Clostridioides difficile strains can form two colony morphotypes: rough and smooth. The rough and smooth morphotypes differ in multiple phenotypes, including cell length and chaining, motility, biofilm production, and virulence in the hamster model of C. difficile infection (CDI). Colony morphology undergoes phase variation and is determined by the ON/OFF expression of cmrRST , which encodes a signal transduction system. Here, we test the hypothesis that differences in colony morphology and the associated phenotypes influence pathogenesis and the host response to infection. We first compared the rough and smooth colony variants of wild-type C. difficile in a mouse model of CDI. However, CmrRST phase varied during infection such that the C. difficile populations became indistinguishable in feces and tissues, and no differences in disease were observed in mice inoculated with these variants. We next circumvented phase variation using mutants that form only rough or only smooth colonies. Co-infection of mice with these phenotypically locked strains revealed that the smooth colony mutant has greater fitness than the rough mutant, which is outcompeted during late infection. In addition, NanoString analyses showed a higher number of differentially expressed pro-inflammatory genes and overall higher expression levels in mice infected with the rough colony mutant, independent of bacterial burden and toxin levels. Our results indicate that in a mouse model of CDI, cells from rough colonies are more immunostimulatory during early murine infection, potentially leading to reduced relative fitness compared to cells from smooth colonies. IMPORTANCE: Clostridioides difficile is a major cause of antibiotic-associated diarrhea and one of the most common hospital-acquired infections. These infections are often recurrent and recalcitrant to antibiotic treatment. Bacteria use diverse mechanisms to adapt to stressful host environments, including the development of different subpopulations to help ensure survival of the whole population. C. difficile produces two different colony types, rough and smooth, that have multiple distinguishing traits. We interrogated the effect of these different forms of C. difficile on pathogenesis and the host immune response. We show that the rough colony phenotype results in a more robust immune response and has reduced fitness during infection compared to the smooth colony phenotype. This work sheds light on the impact of these phenotypic subpopulations on disease, leading to a deeper understanding of C. difficile -host interactions.

Phase variation of C. difficile colony morphology occurs via modulation of transcription of cmrRST , which encodes a three-protein signal transduction system. Response regulators CmrR and CmrT promote rough colony development, cell elongation and chaining, surface motility, and disease in the hamster model of infection, while impairing swimming motility and biofilm formation. Using RNA-Seq, we identified the CmrR and CmrT-dependent transcriptional differences in rough and smooth colonies. Further analysis showed that CmrT, but not CmrR, is required for differential expression of most of the genes. Two CmrT-regulated genes, herein named mrpA and mrpB , were together sufficient for restoring all CmrT-dependent in vitro phenotypes in a cmrT mutant and alleviating selection of cmr phase ON cells during growth on an agar surface. MrpA and MrpB are uncharacterized proteins with no known function but are highly conserved in C. difficile . Using immunoprecipitation and mass spectrometry to identify interacting partners, we found that MrpA interacts with the septum site-determining protein MinD and several other proteins involved in cell division and cell shape determination. Ectopic expression of mrpAB resulted in atypical cell division, consistent with MrpAB interference with MinD function. Our findings reveal a potential mechanism by which phase variation of CmrRST modulates colony morphology and motility: in cmr phase ON cells, CmrT-mediated expression of mrpAB interferes with normal cell division resulting elongated cells that enable expansion of the population across a surface while limiting swimming motility.

Two-component regulatory systems typically consist of a sensor kinase and a response regulator. Phosphorylation of the receiver domain controls response regulator activity. Pseudo-receivers (PsRs) are identified computationally as receivers but lack key residues to catalyze phosphotransfer reactions. Although PsRs are common, molecular mechanisms that activate and inactivate bacterial PsRs remain a mystery. We untangled four potentially related but distinct concepts: bacterial PsRs, receivers with regulatory mechanisms in addition to phosphorylation, receivers that are active without phosphorylation, and orphan receivers without an obvious partner sensor kinase. We also analyzed bacterial PsR sequences and structures to identify regions of likely functional significance.

As an obligate anaerobe, Clostridioides difficile grows exclusively in the host intestinal tract, necessitating mechanisms to sense and respond to the gut environment. The atypical signal transduction system encoded by cmrRST includes two OmpR-family response regulators, CmrR and CmrT. These regulators control multiple important phenotypes in C. difficile , including cell chaining, colony morphology, swimming motility, and biofilm formation. CmrR has a typical receiver domain with key conserved residues for phosphotransfer and conformational change, while CmrT is missing conserved residues and is likely a pseudoreceiver. Here, we used multiple methods to investigate dimer formation, including bacterial two-hybrid systems in both Escherichia coli and C. difficile and pull-down assays in E. coli . We detected CmrR homodimers and found that CmrR recognizes a specific DNA sequence found in multiple places in the genome, including upstream of cmrRST . CmrT formed homodimers in multiple assays, and mutation analysis of residues in its unusual active site suggests multiple mutations are needed to maximally reduce CmrT activity. Our data also suggest that CmrR and CmrT can form heterodimers, though the biological relevance of CmrR-CmrT heterodimers remains unclear. Our results suggest that CmrR and CmrT work together in transcriptional responses to stimuli. IMPORTANCE: Two-component systems are nearly ubiquitous among bacteria and are one of the primary ways that bacteria respond to their environment. Atypical two-component proteins and systems are being identified in diverse bacteria, and studying these proteins helps us to understand the underlying mechanisms of these systems. CmrRST is an unusual two-component system that regulates many important phenotypes in Clostridioides difficile . This work begins to untangle the complex regulatory mechanisms by which CmrRST controls gene expression and furthers our understanding of the fundamental biology of C. difficile .

Phase variation of C. difficile colony morphology occurs via modulation of transcription cmrRST , which encodes a three-protein signal transduction system. Response regulators CmrR and CmrT promote rough colony development, cell elongation and chaining, surface motility, and disease in the hamster model of infection, while impairing swimming motility and biofilm formation. Using RNA-Seq, we identified the CmrR and CmrT-dependent transcriptional differences in rough and smooth colonies. Further analysis showed that CmrT, but not CmrR, is required for differential expression of most of the genes. Two CmrT-regulated genes, herein named mrpA and mrpB , were together sufficient for restoring all CmrT-dependent in vitro phenotypes in a cmrT mutant and alleviating selection of cmr phase ON cells during growth on an agar surface. MrpA and MrpB are uncharacterized proteins with no known function but are highly conserved in C. difficile . Using immunoprecipitation and mass spectrometry to identify interacting partners, we found that MrpA interacts with the septum-site determining protein MinD and several other proteins involved in cell division and cell shape determination. Ectopic expression of mrpAB resulted in atypical cell division, consistent with MrpAB interference with MinD function. Our findings reveal a potential mechanism by which phase variation of CmrRST modulates colony morphology and motility: in cmr phase ON cells, CmrT-mediated expression of mrpAB interferes with normal cell division resulting elongated cells that enable expansion of the population across a surface while limiting swimming motility. AUTHOR SUMMARY: C. difficile can reversibly switch between two colony morphology variants that differ in multiple additional phenotypes. Previous work determined that the phenotypic switch occurs through phase variation of the CmrRST signal transduction system, however the CmrRST-regulated genes that mediate these phenotypes were unknown. Here, we identified the CmrR- and CmrT-regulated genes that are differentially expressed between rough and smooth colonies and identified two genes, mrpA and mrpB , that together are sufficient to confer phenotypes associated with cmrRST expression. Protein interaction studies revealed that MrpA interacts with MinD, a protein that helps ensure symmetric cell division in bacteria. Based on these findings, we propose that MrpA disrupts MinD function leading to aberrant cell division, resulting in elongated cells that form rough colonies. This study reveals previously uncharacterized proteins that affect C. difficile cell division and broadly impact the physiology of this pathogen.

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile, employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that Enterococcus faecalis , an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase variable CmrRST signal transduction system in C. difficile . The CmrRST system controls multiple phenotypes including colony morphology, cell elongation, and cell chaining in C. difficile . Here we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr- ON state leading to chaining and a rough colony morphology. This phenomenon preferentially occurs with E. faecalis among the enterococci, as other enterococcal species do not show a similar effect, suggesting that the composition of the polymicrobial environment in the gut is likely critical to shaping C. difficile population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens. IMPORTANCE STATEMENT: Clostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile ; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis , an opportunistic pathogen known to co-colonize the gut with C. difficile , influences phase variation and downstream growth phenotypes in C. difficile . This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

Two-component regulatory systems typically consist of a sensor kinase and a response regulator. All response regulators contain a receiver domain; most also contain an output domain. Response regulator activity is controlled by the phosphorylation state of the receiver. Receivers contain five conserved active site residues that catalyze phosphorylation and dephosphorylation reactions. Some protein domains identified computationally as receivers (PF00072) lack one or more of the five key conserved residues and are termed pseudoreceivers (PsRs). Because receivers are among the most abundant protein domains in nature, PsRs are also common. PsRs are especially common in plants, where they control circadian rhythms. Little is known about PsRs in fungi and archaea. Here, we focused on bacterial PsRs. We created representative datasets of 9,153 PsR and 143,116 true receiver domain sequences from bacteria. Comparison of amino acid composition of PsR and true receiver domains at each position showed (i) many differences at positions known to be important for phosphorylation-mediated signaling in true receiver domains, consistent with diminished importance in PsRs; and (ii) greatest differences between PsR and true receiver domains in the β3α3 and β4α4 loops, potentially highlighting functionally important regions of PsRs. We also performed covariation analyses of PsR and true receiver domains, which suggested six networks of linked residues that may be important for PsR function. Our analyses lay the foundation for rational experimental approaches to investigate molecular mechanisms of signaling by bacterial PsRs.