Molecular characterization of Shigella species isolated from diarrheal patients in Tehran, Iran: phylogenetic typing and its association with virulence gene profiles and a novel description of Shigella invasion associated locus.

The present study aims to employ a multiplex PCR-based method for phylogenetic typing of Shigella and determine the frequency of several virulence genes among Shigella phylogenetic clades and species. Species identification, phylogenetic typing of 44 previously diagnosed Shigella isolates, and frequency of virulence genes and loci, virA, virB, virF, ipaBCD, ial, sen, and set1A were investigated through performing several PCR assays. Distribution of virulence genes among Shigella phylogenetic clades and species was determined by the statistical analysis. The identities of 40 isolates out of 44 were confirmed as Shigella, and these isolates were classified in four phylogenetic clades, S1 (7.5%), S2 (52.5%), S3 (20%), and S5 (20%) and 4 species, S. sonnei (52.5%), S. flexneri (22.5%), S. dysenteriae (20%), and S. boydii (5%). The prevalence of virA, virB, virF, ipaBCD, ial, sen, and set1A was determined as 67.5%, 72.5%, 72.5%, 65%, 75%, 40%, and 5%, respectively. The presence of sen, uidA, or set1A was found to be statistically correlated with either of Shigella phylogenetic clades or species. A significant statistically association was also determined between set1A and Shigella phylogenetic clades. Furthermore, the nucleotide sequence of invasion-associated locus (ial) was determined and mapped on Shigella genome through in silico analysis. The current study shows the distribution of Shigella isolates and its key virulence genes within the five recently described phylogenetic clades for the first time in the Asia. This is also the first description of ial nucleotide sequence and its exact location on Shigella genome after its initial identification.

In-silico design and production of a novel antigenic chimeric Shigella IpaB fused to C-terminal of Clostridium perfringens enterotoxin.

The emergence of antibiotic-resistant phenotypes in Shigella serotypes and the high mortality rate, approximately one million dead annually, in affected patients announce a global demand for an effective serotype-independent vaccine against Shigella. This study aims to design, express, and purify a novel chimeric protein, as a serotype-independent vaccine candidate against Shigella containing full-length Shigella invasion plasmid antigen B (IpaB) and a C-terminal fragment (residues 194-319) of Clostridium perfringens enterotoxin (C-CPE) as a mucosal adjuvant. Several online databases and bioinformatics software were utilized to design the chimeric protein and the relative recombinant gene. The recombinant gene encoding IpaB-CPE194-319 was synthesized, cloned into pACYCDuet-1 expression vector, and transferred to E. coli Bl21 (DE3) cells. IpaB-CPE194-319 was then expressed in auto-induction medium, purified and characterized using MALDI-TOF-TOF mass spectrometry. Followed by subcutaneous injection of the purified IpaB-CPE194-319 to BALB/c mice, antigenicity of this chimeric protein was determined through performing dot-blot immunoassay on nitrocellulose membrane using mice sera. The outcomes of this study show the successful design, efficient expression, and purification of IpaB-CPE194-319 divalent chimeric protein under mentioned conditions. The obtained results also demonstrate the intrinsic antigenic property of IpaB-CPE194-319.

In silico design of a novel chimeric shigella IpaB fused to C terminal of clostridium perfringens enterotoxin as a vaccine candidate.

This study aimed to design a novel chimeric protein in silico to serve as a serotype-independent vaccine candidate against Shigella. The chimera contains amino acid residues 240-460 of Shigella invasion plasmid antigen B (IpaB) and the C-terminus of Clostridium perfringens enterotoxin (C-CPE). Amino acid sequences of 537 peptide linkers were obtained from two protein linker databases. 3D structures of IpaB-CPE290-319, IpaB-CPE184-319, IpaB-CPE194-319 and 537 newly designed IpaB-linker-CPE290-319 constructs with varying linker regions were predicted. These predicted 3D structures were merged with the 3D structures of native IpaB240-460, CPE194-319, CPE184-319 and CPE290-319 to select the structure most similar to native IpaB and C-CPE. Several in silico tools were used to determine the suitability of the selected IpaB-C-CPE structure as a vaccine candidate. None of the 537 linkers was capable of preserving the native structure of CPE290-319 within the IpaB-linker-CPE290-319 structure. In silico analysis determined that the IpaB-CPE194-319 3D structure was the most similar to the 3D structure of the respective native CPE domain and that it was a stable chimeric protein exposing multiple B-cell epitopes. IpaB-CPE194-319 was designed for its capability to bind to human intestinal epithelial and M cells and to accumulate on these cells. The predicted B-cell epitopes are likely to be capable of inducing a mucosal antibody response in the human intestine against Shigella IpaB. This study also showed that the higher binding affinities of CPE184-319 and CPE194-319 to claudin molecules than those of CPE290-319 is the result of preserving the 3D structures of CPE184-319 and CPE194-319 when they are linked to the C-termini of other proteins.

In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-ß-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.

Virulence Characteristics and Antibiotic Resistance Patterns among Various Phylogenetic Groups of Uropathogenic Escherichia coli Isolates.

The aim of this study was to determine the resistance patterns of uropathogenic Escherichia coli (UPEC) isolates and to investigate the frequency of several virulence genes, including fimH, papA, hlyD, cnf-1, sitA, and tsh, among various phylogenetic groups of UPEC isolates. A total of 85 E. coli isolates were recovered from urine samples from outpatients with a clinical diagnosis of uncomplicated urinary tract infections. A molecular approach to examine the antimicrobial resistance patterns was employed using PCR and the disc diffusion method. The detected frequencies of the virulence factor genes determined using PCR were: fimH (34.1%), papA (9.4%), hlyD (21.2%), cnf-1 (3.5%), sitA (15.3%), and tsh (27.1%). These results revealed that the isolates were resistant to trimethoprim-sulfamethoxazole (SXT) (74.1%), cefotaxime (CTX) (68.2%), and amoxicillin-clavulanic acid (AMC) (94.1%), and they were relatively less resistant to N (56.5%). According to these results, further investigation is needed to determine exactly whether or not SXT, CTX, and AMC are appropriate antibiotics for the treatment of UPEC infections in southern Iran. Although these results demonstrate that fimH is the most frequent virulence gene among UPEC isolates, the high prevalence of isolates that do not encode fimH (75.9%) and the relatively low frequency of isolates that carry other virulence genes require further investigation to clarify the role of the other potential virulence factors in the pathogenesis of these isolates.