Biology subjects

Tamayo, R.

Publications and source records attributed to Tamayo, R..

14 recordsLinked to original sources

bioRxiv2026

Phase variable colony morphotypes of Clostridioides difficile elicit distinct host responses during acute infection

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. IMPORTANCEClostridioides 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.

microbiology↗

bioRxiv2025

Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division

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 SUMMARYC. 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.

microbiology↗

bioRxiv2025

Enterococcus faecalis modulates phase variation in Clostridioides difficile

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 STATEMENTClostridioides 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.

microbiology↗

bioRxiv2025

A Clarifying Perspective on Bacterial Pseudo-Receiver Domains

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 {beta}33 and {beta}44 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.

bioinformatics↗

bioRxiv2024

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches. Author SummaryThe Clade 5 strains of the Clostridioides difficile species are so phylogenetically divergent that they almost meet the threshold of being a distinct species. Although these strains are ubiquitously isolated from agricultural and environmental settings and an important source of zoonotic and community-acquired infections, it is unclear whether they have distinct phenotypic properties that allow them to colonize diverse hosts or persist in the environment. By combining a novel anaerobic time-lapse microscopy method with automated image analysis, we discovered that Clade 5 strains grow faster than strains from other C. difficile clades and that they frequently form long chains. These chaining properties are driven by the environmentally responsive expression of a non-canonical signal transduction system, which our analyses suggest is selected against during murine infection. Collectively, our analyses reveal that Clade 5 strains have distinct growth properties that may promote their persistence in the environment.

microbiology↗

bioRxiv2024

Clostridioides difficile-mucus interactions encompass shifts in gene expression, metabolism, and biofilm formation

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides diffiicile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight a flexibility in metabolism that may influence pathogenesis. IMPORTANCEClostridioides difficile results in upwards of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood, but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand dynamics of C. difficile-mucus interactions in the context of the human gut.

microbiology↗

bioRxiv2021

Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or pdcB switch, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes. These results indicate that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

microbiology↗

bioRxiv2021

Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the cmr switch, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCEClostridioides difficile is a leading cause of hospital-acquired intestinal infections in the U.S. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here we define the complex, multi-level regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

microbiology↗

bioRxiv2021

Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection

Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

microbiology↗

bioRxiv2021

c-di-GMP inhibits early sporulation in Clostridioides difficile

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside of the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One bacterial second messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. Overexpression of a gene encoding a diguanylate cyclase, dccA, decreased sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630{Delta}erm strains. Expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630{Delta}erm, suggesting that DccA may contribute to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. C-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCEMany bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. C-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogens life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic R20291 and the historical 630{Delta}erm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a potential mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.

microbiology↗

bioRxiv2020

Genome-scale metabolic reconstruction analysis of Clostridioides difficile identifies conserved patterns of virulence-related metabolic reprogramming

The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the US. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hyper-virulent isolate (str. R20291) and a historic strain (str. 630), validating both with in vitro and in vivo datasets. Growth simulations revealed significant correlations with measured carbon source usage (PPV [≥] 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the Pentose Phosphate Pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in bacterial pathogenesis. ImportanceClostridioides difficile is a Gram-positive, sporulating anaerobe that has become the leading cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curated C. difficile GENREs for both a well-studied laboratory strain (str. 630) as well as a more recently characterized hyper-virulent isolate (str. R20291). In silico validation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization datasets. Subsequent exploration of context-specific metabolism during both in vitro growth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differential C. difficile virulence is associated with distinct metabolic programs related use of carbon sources and provides a platform for identification of novel therapeutic targets.

bioengineering↗

bioRxiv2019

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies, and that bacteria derived from the isolates display opposing surface and swimming motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein CmrRST, which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of Type IV pili, suggesting a novel mechanism of surface expansion in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration. In a hamster model of acute C. difficile disease, colony morphology correlates with virulence, and the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.\n\nSignificance StatementPhenotypic heterogeneity within a genetically clonal population allows many mucosal pathogens to survive within their hosts, balancing the need to produce factors that promote colonization and persistence with the need to avoid the recognition of those factors by the host immune system. Recent work suggests that the human intestinal pathogen Clostridium difficile employs phase variation during infection to generate a heterogeneous population differing in swimming motility, toxin production, and more. This study identifies a signal transduction system that broadly impacts C. difficile physiology and behavior in vitro and in an animal model. Phase variation of this system is therefore poised to modulate the coordinated expression of multiple mechanisms influencing C. difficile disease development.

microbiology↗

bioRxiv2018

Epigenomic landscape of the human pathogen Clostridium difficile

Clostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed the first comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ~300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.

genomics↗

bioRxiv2018

Characterization of flagellar and toxin phase variation in Clostridium difficile ribotype 012 isolates

Clostridium difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a \"flagellar switch\" upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting the strain is phase-locked. To determine whether a \"phase locked\" flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase OFF, non-motile, and attenuated for toxin production. We isolated low frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production, and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase ON derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains, and perhaps other ribotypes, which could impact disease progression and diagnosis.\n\nImportanceClostridium difficile causes diarrheal disease resulting in significant morbidity and mortality in many countries. C. difficile produces flagella that enhance bacterial motility, and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins are co-regulated via a flippable DNA element termed the \"flagellar switch\", which mediates the phase variable production of these factors. C. difficile can exist as flagellated, motile, toxigenic bacteria (\"flg ON\") or aflagellate, non-motile, nontoxigenic bacteria (\"flg OFF\") due in part to the flagellar switch orientation. Here we evaluate multiple isolates of C. difficile ribotype 012 strains and find them to be primarily flg OFF. Some, but not all, of these isolates showed the ability to switch between flg ON and OFF states. These findings suggest heterogeneity in the ability of C. difficile ribotype 012 strains to phase vary flagellum and toxin production, which may broadly apply to pathogenic C. difficile.

microbiology↗