Date Published: May 8, 2014
Publisher: Public Library of Science
Author(s): Barbara Nesta, Maria Valeri, Angela Spagnuolo, Roberto Rosini, Marirosa Mora, Paolo Donato, Christopher J. Alteri, Mariangela Del Vecchio, Scilla Buccato, Alfredo Pezzicoli, Isabella Bertoldi, Lapo Buzzigoli, Giovanna Tuscano, Maria Falduto, Valentina Rippa, Yaqoub Ashhab, Giuliano Bensi, Maria Rita Fontana, Kate L. Seib, Harry L. T. Mobley, Mariagrazia Pizza, Marco Soriani, Laura Serino, Vanessa Sperandio.
SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now 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. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants 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 SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
Pathogenic E. coli can be broadly classified as either extraintestinal pathogenic E. coli (ExPEC), the main cause of urinary tract infection (UTI), newborn meningitis and sepsis, or as intestinal pathogenic E. coli (InPEC) causing diarrhoeagenic infections. Among the intestinal pathogens there are at least six well-described groups: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC) . The plasticity of the E. coli genomes, due to the ability to gain or lose virulence attributes by horizontal gene transfer, allows these organisms to colonize different sites. Indeed, E. coli possesses an array of virulence factors which include various adhesins, capsule, iron-transporters, toxins and proteases (reviewed in ). However, recent studies have suggested that the pathogenesis of E. coli is considerably more complex than previously appreciated involving additional virulence factors , . The absence of a broadly protective vaccine against pathogenic E. coli is a major problem for modern society since diseases caused by these bacteria are associated with significant human suffering and high healthcare costs. The overall problem is exacerbated by the rising rates of multi-drug resistant strains and by the emergence of new sequence types and hypervirulent strains –. We have recently proposed ECOK1_3385 as a promising vaccine candidate able to confer protection in a murine model of sepsis , . This protein, described as SslE (for secreted and surface-associated lipoprotein from E. coli) and formerly known as YghJ , , appears to be required for biofilm formation and for virulence of EPEC strains , although more recent evidence indicates that SslE has no effect on adherence and biofilm formation in atypical EPEC strains . Thus, the function of SslE remains to be fully elucidated. However, it is known that SslE is secreted through a type II secretion system (T2SS), an exporting apparatus typically used by Gram-negative bacteria to secrete virulence determinants . Two T2SSs exist in E. coli, designated as alpha (T2SSα) and beta (T2SSβ) . The T2SSβ operon is composed of three genes (yghJ, pppA, and yghG) upstream of gspCβ. The first gene, yghJ, encodes for the SslE protein. A functional T2SSβ secreting a cognate SslE protein was recently studied in the non-pathogenic E. coli W strain . Recently, it was reported that SslE belongs to a new sub-family of extracellular zinc-metallopeptidases, characterized by a M60-like zinc-metalloprotease domain HEXXHX(8,24)E , that is distantly related to known viral enhancin zinc-metallopeptidases. The baculovirus enhancin protein Vef is able to digest intestinal mucins, facilitating the attachment and entry of the virus into epithelial cells .
E. coli is a well-adapted human pathogen which uses the gut as a preferential niche and, as for other intestinal microorganisms, it persists in this region due to its ability to exploit a number of metabolic substrates and to stay in the outer mucus layer where commensal bacteria normally reside. Recent studies –, including those reported by our group , , have postulated that this microorganism has adapted to the human body by developing a sophisticated network of virulence and colonization factors. Among these adhesins, iron-uptake systems and IgA binding proteins may allow E. coli to out-compete the many species occupying an overcrowded environment such as the intestine. In this scenario, our finding that SslE contributes to E. coli mucinase activity suggests the involvement of this antigen in landscaping the E. coli territory allowing the establishment of a long lasting colonization. Indeed, shaping of the intestinal microbial community by the mucosa does not only depend on goblet cells secreting antimicrobial proteins, but also on a number of metabolic substrates vital to mucus-degrading bacteria –. In our study, the diminished capacity of the sslE mutant strain to translocate through a mucin-rich matrix in vitro suggests that SslE activity may facilitate bacterial penetration of the mucosal surface, including the inner mucus layer, to reach the underlying host epithelium. Although these data do not exclude that the catabolism of such glycoproteins may also contribute to an increased fitness of E. coli in the outer mucus layer, the pathogenic strains that are armed with immune evasion virulence factors may use SslE as a spearhead to penetrate the sterile inner mucus layer so as to intimately adhere to the epithelial cells of the host.