RESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Clostridioides difficile/metabolismo , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Esporos Bacterianos/química , Proteínas de Bactérias/genética , Parede Celular/química , Parede Celular/genética , Clostridioides difficile/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Esporos Bacterianos/genética , TemperaturaRESUMO
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.
Assuntos
Aderência Bacteriana/fisiologia , Clostridioides difficile/fisiologia , Enterocolite Pseudomembranosa/microbiologia , Esporos Bacterianos/fisiologia , Animais , Proteínas de Bactérias/genética , Células CACO-2/microbiologia , Parede Celular , Clostridioides difficile/patogenicidade , Modelos Animais de Doenças , Fibronectinas/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Camundongos , Microscopia Eletrônica de Transmissão , Microvilosidades/microbiologia , Mucinas , Vitronectina/metabolismoRESUMO
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.