RESUMEN
Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Salmonella typhimurium/genética , Fenómenos Fisiológicos Bacterianos , Escherichia coli/genética , Salmonella typhimurium/citología , Regulación hacia ArribaRESUMEN
Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogen's virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors.
Asunto(s)
Proteínas de Escherichia coli/genética , Expresión Génica , Chaperonas Moleculares/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Factores de Virulencia/genética , Animales , Línea Celular , Proteínas de Escherichia coli/inmunología , Femenino , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Chaperonas Moleculares/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/fisiología , Virulencia , Factores de Virulencia/inmunologíaRESUMEN
Most bacteria, planktonic and sessile, are encapsulated inside loosely bound extracellular polymeric substance (EPS) in their physiological environment. Imaging a bacterium with its capsule requires lengthy sample preparation to enhance the capsular contrast. In this study, Salmonella typhimurium was investigated using atomic force microscopy for a practical means of imaging an encapsulated bacterium in air. The investigation further aimed to determine the relation between the buffers used for preparing the bacterium and the preservation of the capsular material surrounding it. It was observed that rinsing bacteria with HEPES buffer could stabilize and promote capsule formation, while rinsing with PBS, Tris, or glycine removes most of the capsular EPS. For bacteria rinsed with HEPES and air-dried, the height images showed only the contour of the capsular material, while the phase and amplitude images presented the detailed structures of the bacterial surface, including the flagella encapsulated inside the capsular EPS. The encapsulation was attributed to the cross-linking of the acidic exopolysaccharides mediated by the piperazine moiety of HEPES through electrostatic attraction. This explanation is supported by encapsulated bacteria observed for samples rinsed with N,N'-bis(2-hydroxyethyl)-piperazine solution and by the presence of entrapped HEPES within the dry capsular EPS suggested by micro-Raman spectroscopy.