C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

C. difficile exploits a host metabolite produced during toxin-mediated disease.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.

A conserved switch controls virulence, sporulation, and motility in C. difficile.

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.

Dietary trehalose enhances virulence of epidemic Clostridium difficile.

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Clostridioides difficile infection promotes gastrointestinal dysfunction in human and mice post-acute phase of the disease.

OBJECTIVES: In the US, Clostridioides difficile (C. difficile) infection (CDI) is the 8th leading cause of hospital readmission and 7th for mortality among all gastrointestinal (GI) disorders. Here, we investigated GI dysfunction post-CDI in humans and mice post-acute infection. MATERIALS AND METHODS: From March 2020 to July 2021, we reviewed the clinical records of 67 patients referred to the UVA Complicated C. difficile clinic for fecal microbiota transplantation (FMT) eligibility. C57BL/6 mice were infected with C. difficile and clinical scores were determined daily. Stool samples from mice were collected to measure the shedding of C. difficile and myeloperoxidase (MPO) levels. On day 21 post-infection, Evans's blue and FITC-70kDa methods were performed to evaluate GI motility in mice. RESULTS: Of the 67 patients evaluated at the C. difficile clinic, 40 patients (59.7%) were confirmed to have CDI, and 22 patients (32.8%) with post-CDI IBS (diarrhea-type, constipation-type, and mixed-type). In infected mice, levels of MPO in stools and clinical score were higher on day 3. On day 21, mice recovered from body weight loss induced by CDI, and fecal MPO was undetectable. The total GI transit time (TGITT) and FITC-70kDa levels on the proximal colon were increased in infected mice (p = 0.002), suggesting a constipation phenotype post-acute phase of CDI. A positive correlation intestinal inflammation on day 3 and TGITT on day 21 was observed. CONCLUSION: In conclusion, post-infection intestinal dysfunction occurs in humans and mice post-CDI. Importantly, we have validated in the mouse model that CDI causes abnormal GI transit in the recovery phase of the disease, indicating the potential utility of the model in exploring the underlying mechanisms of post-infectious IBS in humans.

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence.

Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease.

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

Fusobacteriumnucleatum Adheres to Clostridioides difficile via the RadD Adhesin to Enhance Biofilm Formation in Intestinal Mucus.

BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

Fecal Microbiota Transplantation Influences Procarcinogenic Escherichia coli in Recipient Recurrent Clostridioides difficile Patients.

BACKGROUND & AIMS: Patients with multiple recurrent Clostridioides difficile infection (rCDI) have a disturbed gut microbiota that can be restored by fecal microbiota transplantation (FMT). Despite extensive screening, healthy feces donors may carry bacteria in their intestinal tract that could have long-term health effects, such as potentially procarcinogenic polyketide synthase-positive (pks+) Escherichia coli. Here, we aim to determine whether the pks abundance and persistence of pks+E coli is influenced by pks status of the donor feces. METHODS: In a cohort of 49 patients with rCDI treated with FMT and matching donor samples-the largest cohort of its kind, to our knowledge-we retrospectively screened fecal metagenomes for pks+E coli and compared the presence of pks in patients before and after treatment and to their respective donors. RESULTS: The pks island was more prevalent (P = .026) and abundant (P < .001) in patients with rCDI (pre-FMT, 27 of 49 [55%]; median, 0.46 reads per kilobase per million [RPKM] pks) than in healthy donors (3 of 8 donors [37.5%], 11 of 38 samples [29%]; median, 0.01 RPKM pks). The pks status of patients post-FMT depended on the pks status of the donor suspension with which the patient was treated (P = .046). Particularly, persistence (8 of 9 cases) or clearance (13 of 18) of pks+E coli in pks+ patients was correlated to pks in the donor (P = .004). CONCLUSIONS: We conclude that FMT contributes to pks+E coli persistence or eradication in patients with rCDI but that donor-to-patient transmission of pks+E coli is unlikely.

Gastrointestinal microbiota-mediated control of enteric pathogens.

The gastrointestinal (GI) microbiota is a complex community of microorganisms residing within the mammalian gastrointestinal tract. The GI microbiota is vital to the development of the host immune system and plays a crucial role in human health and disease. The composition of the GI microbiota differs immensely among individuals yet specific shifts in composition and diversity have been linked to inflammatory bowel disease, obesity, atopy, and susceptibility to infection. In this review, we describe the GI microbiota and its role in enteric diseases caused by pathogenic Escherichia coli, Salmonella enterica, and Clostridium difficile. We discuss the central role of the GI microbiota in protective immunity, resistance to enteric pathogens, and resolution of enteric colitis.

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B.

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women's Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

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 discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration 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 on surfaces. In a hamster model of acute C. difficile disease, 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.

Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.

Frizzled proteins are colonic epithelial receptors for C. difficile toxin B.

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

Signal Versus Noise: How to Analyze the Microbiome and Make Progress on Antimicrobial Resistance.

Antimicrobial resistance has become a worldwide medical challenge [1], so impactful that vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have entered the common vernacular. We have attempted to reduce the selective pressure through antimicrobial stewardship, curtail the spread by identifying and isolating carriers and individuals with symptomatic infection, and treat antibiotic-resistant organisms (AROs) by developing novel antimicrobials. Despite these extraordinary measures, the challenge of AROs continues to grow. The gut microbiome, the ecosystem of microbes (ie, the microbiota) and metabolites present upon and within all humans, is an emerging target for both the risk for colonization and defense against infection with AROs. Here, informed from experiences and successes with understanding the role of the microbiome in mediating risk of Clostridioides difficile infection (CDI), we (1) review our understanding of the risk from ARO acquisition; (2) review our current understanding of the gut microbiome's ability to resist colonization with AROs; (3) describe how experimental model systems can test these initial, global insights to arrive at more granular, mechanistic ones; and (4) suggest a path forward to make further progress in the field.

Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15-25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host-pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

Murine Intrarectal Instillation of Purified Recombinant Clostridioides difficile Toxins Enables Mechanistic Studies of Pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474-4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.