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Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR', coding for SinR (112 aa) and SinR' (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus's role in C. difficile physiology. Transcriptome analysis of the sinRR' mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR' acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system.

Background Clostridium difficile infection (CDI) is a leading cause of diarrhoea in health care settings with symptoms ranging from mild and self-limiting to life threatening. SMT19969 is a novel, non-absorbable antibiotic currently under development for the treatment of CDI. Here we report the results from a Phase I study. Methods A double-blind, randomized, placebo-controlled study assessing safety and tolerability of single and multiple oral doses of SMT19969 in healthy volunteers. Pharmacokinetic assessments included blood and faecal sampling. The effect of food on systemic exposure and analysis of the gut microbiota were also included. Results Fifty-six healthy male subjects were enrolled. Following single oral doses of up to 2,000 mg in the fasted state, plasma concentrations of SMT19969 were generally below the lower limit of quantification. In the fed state levels ranged from 0.102 to 0.296 ng/mL after single dosing and after repeat dosing at Day 10 from 0.105 to 0.305 ng/mL. Following single and multiple oral doses of SMT19969, mean daily faecal concentrations increased with increasing dose level and were significantly above the typical MIC range for C. difficile (0.06-0.5 μg/mL). At 200 mg BID, mean (± SD) faecal concentrations of 1,466 (±547) μg/g and 1,364 (±446) μg/g were determined on days 5 and 10 of dosing respectively. No notable metabolites were detected in faeces. Overall, all doses of SMT19969 were well tolerated both as single oral doses or BID oral doses for 10 days. The majority (88%) of adverse events (AEs) were classified as gastrointestinal disorders and were mild in severity, resolving without treatment. The gut microbiota was analysed in the multiple dose groups with minimal changes observed in the bacterial groups analysed except for total clostridia which were reduced to below the limit of detection by day 4 of dosing. Conclusions Oral administration of SMT19969 was considered safe and well tolerated and was associated with negligible plasma concentrations after single and multiple doses. In addition, minimal disruption of normal gut microbiota was noted, confirming the highly selective spectrum of the compound. These results support the further clinical development of SMT19969 as an oral therapy for CDI. Trial registration Current Controlled Trials. ISRCTN10858225 .

Antibiotics can have significant and long-lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile . Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including the primary bile acid taurocholate for germination, and carbon sources such as mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favours C. difficile germination and growth. Antibiotics alter the intestinal microbiota and facilitate colonization of pathogens such as Clostridium difficile . Here, the authors show that antibiotic-induced shifts in the mouse gut microbiome are correlated with changes in levels of certain metabolites that C. difficile can use for germination and growth.

Dietary zinc supplements are in common use, but their effect on infection is unclear. New findings now show that excess dietary zinc reduces the diversity of the gut microbiota and increases the susceptibility of antibiotic-treated mice to Clostridium difficile infection. Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and is an urgent public health concern worldwide 1 . Over the past decade, incidence, severity and costs associated with C. difficile infection (CDI) have increased dramatically 2 . CDI is most commonly initiated by antibiotic-mediated disruption of the gut microbiota; however, non-antibiotic-associated CDI cases are well documented and on the rise 3 , 4 . This suggests that unexplored environmental, nutrient and host factors probably influence CDI. Here we show that excess dietary zinc (Zn) substantially alters the gut microbiota and, in turn, reduces the minimum amount of antibiotics needed to confer susceptibility to CDI. In mice colonized with C. difficile , excess dietary Zn severely exacerbated C. difficile– associated disease by increasing toxin activity and altering the host immune response. In addition, we show that the Zn-binding S100 protein calprotectin has antimicrobial effects against C. difficile and is an essential component of the innate immune response to CDI. Taken together, these data suggest that nutrient Zn levels have a key role in determining susceptibility to CDI and severity of disease, and that calprotectin-mediated metal limitation is an important factor in the host immune response to C. difficile .

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 year 1 ; 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. RNA-sequencing experiments determine that sorbitol, a metabolite produced by the host enzyme aldose reductase, is exploited by Clostridium difficile in its adaptation to inflammatory conditions in the gut.

Iron is indispensable for almost all forms of life but toxic at elevated levels 1 – 4 . To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis 1 , 5 , 6 . Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules 7 , 8 . However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA 9 . Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P 1B6 -ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection. Ferrosome organelles produced by Clostridioides difficile are required to support colonization of the inflamed gut, highlighting the potential of targeting ferrosome formation as an antimicrobial strategy against this important pathogen.

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C . difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C . difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C . difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C . difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C . difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1 -/- (cholic acid-deficient) mutant mice and infected them with C . difficile spores. We show that 7α-dehydroxylation ( i . e ., secondary bile acid generation) is dispensable for protection against C . difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C . difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C . difficile -resistant environment but the microbiome protects against C . difficile infection in bile acid-independent mechanisms.