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is a Gram-positive, spore-forming, toxin-producing anaerobe, and an important nosocomial pathogen. Due to the strictly anaerobic nature of the vegetative form, spores are the main morphotype of infection and transmission of the disease. Spore formation and their subsequent germination play critical roles in infection (CDI) progress. Under suitable conditions, spores will germinate and outgrow to produce the pathogenic vegetative form. During CDI, produces toxins (TcdA and TcdB) that are required to initiate the disease. Meanwhile, it also produces spores that are responsible for the persistence and recurrence of in patients. Recent studies have shed light on the regulatory mechanisms of sporulation and germination. This review is to summarize recent advances on the regulation of sporulation/germination in and the corresponding therapeutic strategies that are aimed at these important processes.

Clostridioides difficile infection (CDI) presents a major public health threat by causing frequently recurrent, life-threatening cases of diarrhea and intestinal inflammation. The ability of C. difficile to express antibiotic resistance and to form long-lasting spores makes the pathogen particularly challenging to eradicate from healthcare settings, raising the need for preventative measures to curb the spread of CDI. Since C. difficile utilizes the fecal–oral route of transmission, a mucosal vaccine could be a particularly promising strategy by generating strong IgA and IgG responses that prevent colonization and disease. This mini-review summarizes the progress toward mucosal vaccines against C. difficile toxins, cell–surface components, and spore proteins. By assessing the strengths and weaknesses of particular antigens, as well as methods for delivering these antigens to mucosal sites, we hope to guide future research toward an effective mucosal vaccine against CDI.

Clostridioides difficile is a leading cause of nosocomial antibiotic-associated diarrhea in developed countries with many known virulence factors. In several pathogens, motility and virulence are intimately linked by regulatory networks that allow coordination of these processes in pathogenesis and physiology. Regulation of C. difficile toxin production by FliC has been demonstrated in vitro and in vivo and has been proposed to link motility and virulence. Here, we show that clinically important, non-motile C. difficile strains have conserved FliC and regulatory partners FliW and CsrA, despite lacking the rest of the machinery to produce functional flagella. Our work highlights a novel role for flagellin outside of its role in motility and FliW in the pathogenesis and physiology of C. difficile .

Treatment options for infection are limited, with very high rates of recurrence. Active vaccination provides an attractive opportunity to prevent infection (CDI) and recurrence. In a search for potential surface-exposed antigens involved in colonization, two putative lipoproteins, designated LP1 and LP2, were identified from R20291. Lipoprotein sequences were aligned, analyzed, and evaluated for their immune properties. The antigenic characteristics of both LP1 and LP2 were assessed in silico and in a mouse model of immunization and CDI. Multiple sequence alignments showed that the lipoprotein sequences were highly conserved among various ribotypes. analysis predicted that LP1 and LP2 possess cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes with antigenic and immunogenic properties. Immune simulation provided insights into the ability of LP1 and LP2 to stimulate humoral and cellular immune responses. These properties were further examined in a mouse model of immunization and CDI. After three immunizations at 12-day intervals, significant amounts of IgG and IgA antibodies were detected in sera and feces. LP1 and LP2 immunizations provided mice with intermediate and higher levels of protection, respectively, against R20291 infection, and significantly reduced spore and toxin levels in feces. Furthermore, anti-LP1 and anti-LP2 sera significantly inhibited adhesion of R20291 vegetative cells to HCT-8 gut epithelial cells. These results indicate that both lipoproteins play a significant role in adhesion and that LP1 and LP2 are promising immunogens for preventing colonization.

Background. Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years. Methods. Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI. Results. We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI. Conclusion. Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.

Treatment with β-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to β-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for β-lactams in C. difficile . PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with β-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn 2+ -binding motif that influences β-lactam binding and protein stability. The Zn 2+ -binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn 2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments. Antibiotics of the β-lactam class inhibit bacterial cell wall synthesis by targeting penicillin-binding proteins (PBPs). Here, Sacco et al. study the four PBPs present in the pathogen C. difficile , revealing unique structural features and shedding light on the mechanisms underlying β-lactam resistance in this organism.

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.

Background/Objectives: Clostridioides difficile is a Gram-positive, spore-forming enteric pathogen that causes intestinal disorders, including inflammation and diarrhea, primarily through toxin production. Standard treatment options for C. difficile infection (CDI) involve a limited selection of antibiotics that are not fully effective, leading to high recurrence rates. Vaccination presents a promising strategy for preventing both CDI and its recurrence. Cell wall protein 2 (Cwp2), a highly immunogenic and abundant surface-exposed C. difficile cell wall protein, plays an important role in the bacterium’s adherence in vitro. In this study, we aimed to analyze the homology and immunogenicity of Cwp2 and its protection efficacy as a vaccine candidate against CDI in mice. Methods: we conducted in silico analyses to assess the homology and immunogenicity of Cwp2, and we evaluated its potential as a vaccine candidate against CDI using a mouse model of immunization and infection. Results: Our in silico analyses predicted the immunogenic region (functional domain) of Cwp2 and revealed its high homology among various toxinotypes and ribotypes (R.T.s) or sequence types (S.T.s). Immunizations of mice with the Cwp2 functional domain (Cwp2_A) induced potent IgG/A antibody responses against Cwp2_A, protected mice from CDI, and reduced C. difficile spore and toxin levels in feces post-infection. Additionally, anti-Cwp2_A sera inhibited the binding of C. difficile vegetative cells to HCT8 cells. Conclusions: Our report demonstrates for the first time the potential of Cwp2_A as an effective vaccine candidate against CDI in mice.

Chemical investigation of an Antarctic deep-water octocoral has led to the isolation of four new compounds, including three illudalane sesquiterpenoids (1–3) related to the alcyopterosins, a highly oxidized steroid, alcyosterone (5), and five known alcyopterosins (4, 6–9). The structures were established by extensive 1D and 2D NMR analyses, while 9 was verified by XRD. Alcyopterosins are unusual for their nitrate ester functionalization and have been characterized with cytotoxicity related to their DNA binding properties. Alcyopterosins V (3) and E (4) demonstrated single-digit micromolar activity against Clostridium difficile, an intestinal bacterium capable of causing severe diarrhea that is increasingly associated with drug resistance. Alcyosterone (5) and several alcyopterosins were similarly potent against the protist Leishmania donovani, the causative agent of leishmaniasis, a disfiguring disease that can be fatal if not treated. While the alcyopterosin family of sesquiterpenes is known for mild cytotoxicity, the observed activity against C. difficile and L. donovani is selective for the infectious agents.

infection (CDI) is the leading cause of world-wide nosocomial acquired diarrhea in adults. Active vaccination is generally accepted as a logical and cost-effective approach to prevent CDI. In this paper, we have generated two novel chimeric proteins; one designated Tcd169, comprised of the glucosyltransferase domain (GT), the cysteine proteinase domain (CPD), and receptor binding domain (RBD) of TcdB, and the RBD of TcdA; the other designated Tcd169FI, which contains flagellin (sFliC) and Tcd169. Both proteins were expressed in and purified from . Point mutations were made in the GT (W102A, D288N) and CPD (C698) of TcdB to ensure that Tcd169 and Tcd169FI were atoxic. Immunization with Tcd169 or Tcd169Fl induced protective immunity against TcdA/TcdB challenge through intraperitoneal injection, also provided mice full protection against infection with a hyper-virulent strain (BI/NAP1/027). In addition, inclusion of sFlic in the fusion protein (Tcd169Fl) enhanced its protective immunity against toxin challenge, reduced numbers in feces from Tcd169Fl-immunized mice infected . Our data show that Tcd169 and Tcd169FI fusion proteins may represent alternative vaccine candidates against CDI.