ABSTRACT
BACKGROUND AND OBJECTIVES: Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTIs are caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new targets to develop an ideal vaccine against UTI. In this study, we constructed fusion fimH/fliC of UPEC as a novel vaccine candidate against UTI. MATERIAL AND METHODS: PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot. RESULTS: The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed correctly. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition. CONCLUSION: Urinary tract infection is a huge burden on healthcare system in many countries. UPEC is isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement for a vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolates. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.
ABSTRACT
BACKGROUND AND OBJECTIVES: Enteropathogenic Escherichia coli (EPEC) strains can be detected by serogrouping and the presence of enterocyte attaching- effacing (eae) gene. Most EPEC strains belong to a certain O antigenic group. Locus of enterocyte effacement (LEE) Pathogenicity Island contains the eae gene and secretory proteins (ESPs) that introduce the attaching-effacing lesion. LEE inserted in tRNA genes include the SelC, PheU and PheV sites. The aim of the present study was to genetically characterize EPEC strains isolated from children with diarrhea. MATERIALS AND METHODS: Serogrouping was performed by EPEC antisera in 321 E. coli isolates. The presence of eae, stx, espB, and eaf genes and detection of insertion sites of LEE was done by PCR using specific primers. RESULTS: Seventeen (5.3%) isolates belonging to 7 EPEC serogroups were identified among the whole material and all carried the eae gene. None of the 321 isolates showed presence of stx gene indicating that all 17 isolates classified as EPEC by O serogrouping did not belong to the enterohaemorrhagic E. coli (EHEC) group. Of these, 8 (53%) isolates carried the eaf and 16 (94.1%) carried the espB gene. The insertion sites of LEE in serogrouped isolates were selC (in 6 isolates), pheU (in 7 isolates) and pheV (in 2 isolates). The insertion site in 2 isolates was not determined by PCR. CONCLUSION: Serogrouping and detection of the eae gene showed to be reliable for detection of EPEC strains. No Shigatoxin- producing E. coli (STEC) strain was found among the isolates. Detection of the insertion site of LEE showed that selC, pheU or PheV are insertion sites of LEE in the EPEC strains.
