Functional characterization of ssle, a novel protective antigen involved in escherichia coli translocation of mucosal surfaces

Abstract

Pathogenic Escherichia coli are responsible for a heterogeneous group of disorders, including diarrhoea, urinary tract infections, sepsis and neonatal meningitis, that collectively cause significant morbidity, lost productivity and high healthcare costs. Considering the incidence and also the increasing antibiotic resistance of E. coli strains, the prevention of infections is of pressing concern from both the public health and economic perspectives. Since conventional attempts to develop a highly immunogenic, safe and polyvalent vaccine had failed, we decided to apply the reverse vaccinology approach for the identification of protective and broadly conserved vaccine antigens. Although some of the protective candidates have been previously described, most of them have just putative or hypothetical functions assigned and, therefore, their characterization could contribute to the understanding of E. coli pathogenesis. In this study, vaccine antigen EKO_K1 3385, formally known as SslE (Secreted and surface-associated lipoprotein from Escherichia coli) has been characterized. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. SslE can be divided into two main variants, we proved that antibodies raised against SslE variant I are able to inhibit translocation of E. coli strains expressing a different variant through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well established animal models and demonstrated that immunization with SslE variant I significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Furthermore by exploiting a human in vitro model of mucus-secreting cells, we demonstrated that bacteria expressing SslE have a metabolic benefit which results in an increased growth rate postulating the importance of this antigen in enhancing E. coli fitness. The results presented in this work conclusively designate SslE as an important colonization factor favouring E. coli access to both metabolic substrates and target cells and reinforce the use of this antigen as a component of a protective vaccine against pathogenic E. coli species.