Publicação
Role of cysteine-rich proteins in the assembly of the spore surface layers in the intestinal pathogen Chlostridium difficile
| Resumo: | Clostridium difficile is currently the main cause of intestinal nosocomial disease associated with antibiotic therapy in adults, and a growing concern in the community. C. difficile is a gram-positive, obligate anaerobe and endospore-forming bacterium. The spores are the main vehicle for transmission and persistence of the organism, and have been associated with disease recurrence. Ingested spores germinate in the anaerobic colon to establish a population that will produce more spores and the two main C. difficile virulence factors, the TcdA and TcdB cytotoxins. The surface layers contribute to the overall resistance of spores, and in addition are thought to play an important role during in the adherence of spores to the colonic mucosa during the initial stages of infection. However, the functional architecture and composition of the spore surface layers remains poorly characterized. The most abundant protein of C. difficile spores is Sp17. Sp17 is a 17 kDa cysteine-rich protein essential for the morphogenesis of the spore surface layers and of a spore polar appendage. sp17 is expressed at very high levels in vivo during infection of axenic mouse and is required for colonization. The spore polar appendage was proposed to mediate an interaction with a putative receptor located on the colonic mucosa. Interestingly, this appendage is found only in 24% of spores formed by a wild type strain. The bifurcation of the population in two spore morphotypes suggest diferent roles in the host, possibly in persistence and transmission of the organism. Here, we aimed at dissecting the role of Sp17 in spore assembly. First, we used a fluorescent reporter to show that sp17 is expressed later than other σK -target genes, such as the cotE gene. We then replaced the sp17 promoter by the stronger and earlier-activated PcotE and found that the level and timing of sp17 expression influences both the fraction of spores with appendage as well as the lenght of this structure. Hence, Sp17 is directly involved in assembly of the spore polar appendage. We demonstrated that spores with an appendage contain more Sp17 and in a multimeric form. Immunofluorescence revealed that Sp17 accumulates in arcs or patches along spores that lack a visible appendage, and at the spore pole on the edge of the appendage, when this struture is formed. Sp17 appears to have a dynamic behavior at the spore surface, nucleating assembly of the appendage when it accumulates at the spore poles. We also examined the role of another cysteine-rich protein, CdeC, in the assembly of the spore polar appendage. While CdeC seems to be required for assembly of Sp17, expression of sp17 is not a requirement for assembly of CdeC. Moreover, overexpression of cdeC alone has no effect on the frequence of appendage formation, and coexpression with sp17 does not enhances the effect of Sp17. Our data suggests that CdeC has a more internal localization than Sp17 in spores, lending support to a model in which CdeC forms a basal layer upon which Sp17 is assembled. |
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| Autores principais: | Feliciano, Carolina Alves |
| Assunto: | Clostridium difficile Infecção nosocomial Teses de mestrado - 2015 |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Clostridium difficile is currently the main cause of intestinal nosocomial disease associated with antibiotic therapy in adults, and a growing concern in the community. C. difficile is a gram-positive, obligate anaerobe and endospore-forming bacterium. The spores are the main vehicle for transmission and persistence of the organism, and have been associated with disease recurrence. Ingested spores germinate in the anaerobic colon to establish a population that will produce more spores and the two main C. difficile virulence factors, the TcdA and TcdB cytotoxins. The surface layers contribute to the overall resistance of spores, and in addition are thought to play an important role during in the adherence of spores to the colonic mucosa during the initial stages of infection. However, the functional architecture and composition of the spore surface layers remains poorly characterized. The most abundant protein of C. difficile spores is Sp17. Sp17 is a 17 kDa cysteine-rich protein essential for the morphogenesis of the spore surface layers and of a spore polar appendage. sp17 is expressed at very high levels in vivo during infection of axenic mouse and is required for colonization. The spore polar appendage was proposed to mediate an interaction with a putative receptor located on the colonic mucosa. Interestingly, this appendage is found only in 24% of spores formed by a wild type strain. The bifurcation of the population in two spore morphotypes suggest diferent roles in the host, possibly in persistence and transmission of the organism. Here, we aimed at dissecting the role of Sp17 in spore assembly. First, we used a fluorescent reporter to show that sp17 is expressed later than other σK -target genes, such as the cotE gene. We then replaced the sp17 promoter by the stronger and earlier-activated PcotE and found that the level and timing of sp17 expression influences both the fraction of spores with appendage as well as the lenght of this structure. Hence, Sp17 is directly involved in assembly of the spore polar appendage. We demonstrated that spores with an appendage contain more Sp17 and in a multimeric form. Immunofluorescence revealed that Sp17 accumulates in arcs or patches along spores that lack a visible appendage, and at the spore pole on the edge of the appendage, when this struture is formed. Sp17 appears to have a dynamic behavior at the spore surface, nucleating assembly of the appendage when it accumulates at the spore poles. We also examined the role of another cysteine-rich protein, CdeC, in the assembly of the spore polar appendage. While CdeC seems to be required for assembly of Sp17, expression of sp17 is not a requirement for assembly of CdeC. Moreover, overexpression of cdeC alone has no effect on the frequence of appendage formation, and coexpression with sp17 does not enhances the effect of Sp17. Our data suggests that CdeC has a more internal localization than Sp17 in spores, lending support to a model in which CdeC forms a basal layer upon which Sp17 is assembled. |
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