Proteomic identification of novel adhesins in enterohaemorrhagic E. coli O157 : H7 in a strategy for preventing intestinal colonisation

Abstract

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC colonizes the large intestine and causes diarrhea, hemorrhagic colitis and in some cases, life-threatening hemolytic-uremic syndrome (HUS). The lack of an effective clinical treatment, sequelae after infection and mortality rate in humans, allied with high prevalence of antibiotic resistance make this pathogen as priority for the world health agencies. Initial adherence to host cells is the crucial and first step of the infection of enterohaemorrhagic Escherichia coli (EHEC). The attaching/effacing lesions are considered to be important in EHEC. However, although EHEC produce this lesion on animal mondels and cultured human cells, this phenotype has not been demonstrated on human intestinal mucosal surfaces. The aim of the present research work was to identify some novel adhesins in EHEC O157:H7 in a strategy for preventing intestinal colonisation. Rather than classical reverse vaccinology approach solely based on genomic analysis aiming at identifying potential antigen candidates based on the prediction of their subcellular location at the bacterial cell surface, the identification of candidates was here primarily based on subproteomic analysis. To investigate the cell surface proteome, an original approach based on the use of biotinylation reagents of different sizes was first developed. This method highlighted that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. Following the secrectome concept, three subproteomes were considered and characterised by relative quantification analysis, namely, the proteosurfaceome (proteome at the cell surface), exoproteome (proteome in the extracellular milieu) and proteovesiculome (proteome of the membrane vesicles). This secretome analysis revealed unique proteins expression depending on growth conditions and related to different secretion pathways from EHEC O157:H7, particularly Type III secretion system. Proteomics analysis was combined with functional protein characterisation to identify a group of proteins potentially involved in EHEC O157:H7 adhesion to extracellular matrix and to intestinal cell line. A group of outer membrane proteins revealed important role in the adherent phenotype of EHEC O157:H7 as well promoting bacterial aggregation. Moreover antibodies raised against selected recombinant proteins showed to effectively reduce the adhesion of EHEC to an intestinal cell model.