Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

Resistance of Clostridioides difficile spores from the NAP CR1 strain (Clostridiales: Peptostreptococcaceae) to sodium dichloroisocyanurate / Resistencia de esporas de Clostridioides difficile de la cepa NAPCR1 (Clostridiales: Peptostreptococcaceae) al dicloroisocianurato de sodio

Abstract Introduction: Clostridioides difficile is a significant cause of diarrhea in hospitals and the community. This bacterial pathogen is transmitted through the ingestion of endospores, which are challenging to eliminate due to intrinsic resistance to a variety of chemical disinfection agents. The well-characterized laboratory strain CD630 displays low virulence, has not caused outbreaks, and is highly susceptible to disinfectants. Nonetheless, a closely related strain termed NAPCR1 caused outbreaks in Costa Rica and later became endemic in many hospitals from this country. This strain causes disease through unusual mechanisms and is genotypically distinct from CD630. Consequently, its epidemic potential could be influenced by as yet unknown spore phenotypes, such as increased resistance to disinfectants. Objective: To determine whether the NAPCR1 strain is more resistant to a conventional and highly effective C. difficile sporicidal agent than strain CD630 and to identify potential explanatory mechanisms at the genomic level. Methods: We used an in vitro dilution-neutralization method to calculate the sporicidal activity of sodium dichloroisocyanurate (DCC) against purified spores from three subtypes of NAPCR1 isolates (LIBA-2945, LIBA-5761, and LIBA-6276), CD630, and a representative of the highly virulent and epidemic NAP1 strain (LIBA-5758). This phenotypic characterization was complemented with a genomics-steered search of polymorphisms in 15 spore- or sporulation-related genes. Results: Whereas DCC at a final concentration of 0.1 % (w/v) eradicated CD630 endospores with high efficacy (log10 reduction factor (LFR) ≥ 5), it only partially inactivated NAPCR1 (average LFR range: = 1.77-3.37) and NAP1 endospores (average LRF = 3.58). As hypothesized, the three NAPCR1 subtypes tested were more resistant to DCC than strain CD630 (ANOVA, P < 0.05), with LIBA-5761 showing the highest level of DCC resistance overall (ANOVA, P < 0.05). All three NAPCR1 isolates showed large deletions in bclA1. Besides, isolates LIBA-5761 and LIBA-6276 had deletions in bclA2. Conclusions: Our in vitro tests revealed a differential resistance of spores from the C. difficile NAPCR1 strain to DCC. They highlight the importance of continuously evaluating the efficacy of deployed disinfection agents against circulating strains and hint to a potential role of structural proteins from the exosporium in resistance to disinfectants in C. difficile.

Resumen Introducción: Clostridioides difficile es una causa importante de diarrea a nivel hospitalario y comunitario. Esta bacteria se transmite por medio de la ingestión de endosporas, las cuales son difíciles de erradicar por su resistencia intrínseca a diferentes agentes químicos de desinfección. La cepa de referencia CD630 está bien caracterizada, es poco virulenta, no ha causado brotes, y es altamente susceptible a los desinfectantes. Además, pertenece al mismo clado MLST y es filogenéticamente muy cercana a la cepa NAPCR1. Sin embargo, solo la última ha causado brotes en Costa Rica y se ha convertido en una cepa endémica en varios hospitales locales. La cepa NAPCR1 causa enfermedad por mecanismos poco usuales y es genotípicamente diferente a la cepa CD630. Por lo tanto, su potencial epidémico podría estar influenciado por cambios fenotípicos en sus esporas, como una resistencia incrementada a los desinfectantes. Objetivo: Determinar si la cepa NAPCR1 presenta mayor resistencia que CD630 a un desinfectante de alta eficacia utilizado a nivel hospitalario y dilucidar posibles mecanismos a nivel genómico. Métodos: Se utilizó el método de dilución-neutralización para evaluar la actividad esporicida in vitro del dicloroisocianurato de sodio (DCC) contra esporas de 3 subtipos de la cepa NAPCR1 (LIBA-2945, LIBA-5761, y LIBA-6276), CD630 y un aislamiento representativo de la cepa epidémica e hipervirulenta NAP1 (LIBA-5758). Esta caracterización fenotípica fue complementada con una búsqueda genómica de polimorfismos en 15 genes relacionados con la estructura de la endospora o el proceso de esporulación. Resultados: El DCC a una concentración final de 0.1 % (p/v) erradicó las endosporas de la cepa CD630 con gran eficacia (factor de reducción logarítmica; FRL ≥ 5) y eliminó parcialmente las de las cepas NAPCR1 (FRL promedio = 1.77-3.64) y NAP1 (FRL promedio = 3.58). El perfil de susceptibilidad del aislamiento NAPCR1 LIBA-5761 fue único, ya que mostró un mayor nivel de resistencia hacia el DCC que los otros aislamientos NAPCR1 y la cepa NAP1 examinada (ANOVA, P < 0.05). Los tres aislamientos NAPCR1 mostraron deleciones en bclA1 y los aislamientos LIBA-5761 y LIBA-6276 tenían deleciones adicionales en bclA2. Conclusiones: Nuestros experimentos in vitro confirman la resistencia incrementada a los desinfectantes de la cepa NAPCR1 y una susceptibilidad diferencial en sus tres subtipos. Adicionalmente, señalan la importancia de evaluar continuamente la eficacia de los desinfectantes contra cepas circulantes y asignan un posible papel en la resistencia a los desinfectantes gracias a las proteínas del exosporio de C. difficile.

The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli.

Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.

Characterization of Exosporium Layer Variability of Clostridioides difficile Spores in the Epidemically Relevant Strain R20291.

Clostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea. C. difficile spore is a dormant state which acts as a vehicle of transmission and infection. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that spore-coat laminations are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC, and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

Identification of Escherichia coli strains for the heterologous overexpression of soluble Clostridium difficile exosporium proteins.

Clostridium difficile infections are one of the leading causes of hospital-acquired infections. C. difficile spores are considered the morphotype of transmission and recurrent infection due to its natural spore resistance properties. The outermost spore layer, the exosporium, provides the first contact with the environment and the host. However, molecular biology studies on exosporium proteins are lacking primarily due to difficulties in over-expressing these proteins under soluble conditions. In this work, we have developed a protocol to express soluble exosporium proteins of C. difficile spores in the heterologous Escherichia coli host. We found that the optimum soluble expression conditions may vary between 21, 30 and 37 °C, depending on the protein, and at least CdeC, BclA1 and BclA3, required E. coli strains that provided an oxidative environment such as Shuffle T7. These results will allow further studies with recombinant proteins of the exosporium of C. difficile spores.

Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease.

Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro. In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro. Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.

Updates on Clostridium difficile spore biology.

Clostridium difficile is a Gram-positive, anaerobic spore former, and an important nosocomial pathogenic bacterium. C. difficile spores are the morphotype of transmission and recurrence of the disease. The formation of C. difficile spores and their subsequent germination are essential processes during the infection. Recent in vitro and in vivo work has shed light on how spores are formed and the timing of in vivo sporulation in a mouse model. Advances have also been made in our understanding of the machineries involved in spore germination, and how antibiotic-induced dysbiosis affects the metabolism of bile salts and thus impacts C. difficile germination in vivo. Studies have also attempted to identify how C. difficile spores interact with the host's intestinal mucosa. Spore resistance has also been revisited by several groups highlighting the extreme resistance of this morphotype to traditional food processing regimes and disinfectants used in clinical settings. Therefore, the aim of this review is to summarize recent advances on spore formation/germination in vitro and in vivo, spore-host interactions, and spore resistance that contribute to our knowledge of the role of C. difficile spores in the infectious process.

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa.

Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.

Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. There is a lack of information on the composition of the outermost exosporium layer of C. difficile spores. Using recently developed exosporium removal methods combined with MS/MS, we have established a gel-free approach to analyze the proteome of the exosporium of C. difficile spores of strain 630. A total of 184 proteins were found in the exosporium layer of C. difficile spores. We identified 7 characterized spore coat and/or exosporium proteins; 6 proteins likely to be involved in spore resistance; 6 proteins possibly involved in pathogenicity; 13 uncharacterized proteins; and 146 cytosolic proteins that might have been encased into the exosporium during assembly, similarly as reported for Bacillus anthracis and Bacillus cereus spores. We demonstrate through Flag-fusions that CotA and CotB are mainly located in the spore coat, while the exosporium collagen-like glycoproteins (i.e. BclA1, BclA2 and BclA3), the exosporium morphogenetic proteins CdeC and CdeM, and the uncharacterized exosporium proteins CdeA and CdeB are mainly located in the exosporium layer of C. difficile 630 spores. This study offers novel candidates of C. difficile exosporium proteins as suitable targets for detection, removal and spore-based therapies. BIOLOGICAL SIGNIFICANCE: This study offers a novel strategy to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores and spore coat since it defines the proteome of the outermost layer of C. difficile spores, the exosporium. This study suggests that C. difficile spores have several proteins involved in protection against environmental stress as well as putative virulence factors that might play a role during infection. Spore exosporium structural proteins were also identified providing the ground basis for further functional studies of these proteins. Overall this work provides new protein target for the diagnosis and/or therapeutics that may contribute to combat C. difficile infections.

Characterization of the collagen-like exosporium protein, BclA1, of Clostridium difficile spores.

Spores of Clostridium difficile are essential for infection, persistence and transmission of C. difficile infections (CDI). Proteins of the surface of C. difficile spores are thought to be essential for initiation and persistence of CDI. In this work, we demonstrate that three C. difficile collagen-like exosporium proteins (BclA) encoded in the C. difficile 630 genome are expressed during sporulation and localize to the spore via their N-terminal domains. Using polyclonal antibodies against the N- and C-terminal domains and full length BclA1 we demonstrate that BclA1 is likely to be localized to the exosporium layer, presumably undergoes post-translational cleavages and might be cross-linked with other exosporium proteins. The collagen-like region of recombinant BclA1 and BclA2 was susceptible to collagenase degradation. Collagenase digestion assay of C. difficile spores suggests that, similarly as in Bacillus anthracis BclA, the N-terminal domain and the C-terminal domain of BclA1 might be buried in the basal layer and oriented to the exosporium surface, respectively. We also demonstrate that the collagen-like BclAs proteins do not contribute to the spore hydrophobicity and its absence slightly increased the adherence of spores to Caco-2 cells. BclA1 was also shown to have poor immunogenic properties. These results provide the first study on the BclA1 collagen-like proteins of C. difficile spores.