Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae.

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low-molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a α/ß, ATP-binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae.

Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low-molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a alpha/beta, ATP-binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae

Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay.

The main and common virulence factor expressed by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is intimin, a 94-kDa outer membrane protein, which is a product of the eae gene, and, thus, an excellent target for the detection of these pathogens. Among the methods for detection of virulence factor expression, immunoassays can be considered the first alternative to either animal use or in vitro culture cells assays, for which polyclonal and/or monoclonal antibodies are raised. In the present work, we evaluated the sensitivity and specificity of an intimin recombinant antibody (scFv-intimin) using immunofluorescence assay. The scFv-intimin detected typical EPEC, atypical EPEC, and EHEC isolates (100% sensitivity) with no detection of eae- isolates (100% specificity). Thus, immunofluorescence is an effective and rapid method, and scFv-intimin, an excellent tool for the diagnosis of diarrhea caused by EPEC and EHEC and also can be employed in case-control epidemiological surveys.

Interaction between Shiga toxin and monoclonal antibodies: binding characteristics and in vitro neutralizing abilities.

Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.