Horizontal Gene Transfer and the Diversity of <i>Escherichia coli</i>

Escherichia coli (E. coli) strains are normal flora of human gastrointestinal tract. The evolution encoded by horizontally-transferred genetic (HGT) elements has been perceived in several species. E. coli strains have acquired virulence potential factors by attainment of particular loci through HGT, transposons or phages. The heterogeneous nature of these strains is because of HGT through mobile genetic elements. These genetic exchanges that occur in bacteria provide the genetic diversity

In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

Purpose At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. Materials and Methods In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. Results The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 µg/mL of the culture. Conclusion Our results indicate that Omp2b protein has a potential to induce both B-cell– and T-cell–mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis. Keywords: Brucella; Omp2b; In silico approach; Epitope prediction; Protein expressio

Antibiotic resistance, virulence and genetic diversity of Klebsiella pneumoniae in community- and hospital-acquired urinary tract infections in Iran

Klebsiella pneumoniae is among the most important causes of urinary tract infection (UTI). The aim of this study was to investigate the prevalence and correlation of antibiotic resistance with virulence characteristics and genetic diversity in K. pneumoniae isolated from UTIs in Iran. Phenotypic tests and antibiotic susceptibility were carried out on the isolates. Detection of the virulence and extended-spectrum β-lactamase (ESBL) genes was performed by polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) was used for exploring the genomic relatedness. Hemolysin, biofilm, and hypermucoviscosity formation were observed in 87.1%, 86.4%, and 12.1% of isolates, respectively. The antibiotic resistance rate of K. pneumoniae isolates ranged from 12.1% for meropenem to 100% for amoxicillin. The prevalence of virulence genes ranged from 1.4% for cnf-1 to 100% for mrkD, fimH, kpn, and entB genes. In this study, 91.7%, 33.3%, and 4.2% of phenotypically ESBL-producers were positive for blaCTX-M, blaTEM, and blaSHV genes, respectively. An association was observed between the presence of traT, fyuA, or cnf-1 genes with antibiotic resistance. Two clone types were obtained by PFGE that indicate different K. pneumoniae clones in community- and hospital-acquired UTIs. The findings of this study are valuable in development of treatment strategies against UTIs in Iran

A-NGR fusion protein induces apoptosis in human cancer cells

The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 μg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells

In silico study of ligand binding site of toll-like receptor 5

Background: Toll-like receptor-5 (TLR-5) is a member of TLRs family and responsible for bacterial flagellin recognition. The activation of TLR-5 with flagellin leads to initiation of signaling cascades, which in turn results in transcription of pro-inflammatory cytokines. Regarding the critical role of TLR-5 agonists and antagonists in activation of innate immune responses, an increasing number of studies have focused on their therapeutic applications in drug and vaccine design. In this study, to identify the most critical region and residues of TLR-5 for interaction with flagellin, different truncated forms of TLR-5 were designed and subjected to protein-protein interaction studies. Materials and Methods: The interactions of the full native TLR-5 and its truncated forms with bacterial flagellin (FliC) were evaluated using Hex docking server and molecular interaction analysis was performed using Dimplot analysis. Results: According to our in silico results, truncated form C (an amino acid sequence containing residues 174-401 of TLR-5) has the most suitable interaction with FliC and seven amino acids within this region were found to be crucial for the interaction with flagellin. Conclusions: These results provide new insights in to potential drug target sites of TLR-5, which may guide future TLR-5 targeting studies

Escherichia coli: a brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran.

International audienceDiarrheagenic Escherichia coli have developed different strategies for establishment of infection in their host. Understanding these pathogenic mechanisms has led to the development of specific diagnostic tools for identification and categorization of E. coli strains into different pathotypes. This review aims to provide an overview of the various categories of diarrheagenic Escherichia coli and the data obtained in Iran pertaining to these pathotypes

Modulation of Molecular Biomarker Expression in Response to Chemotherapy in Invasive Ductal Carcinoma

Breast cancer (BC) has varied morphological and biological features and is classified based on molecular and morphological examinations. Molecular classification of BC is based on biological gene-expression profiling. In this study, biomarker modulation was assessed during BC treatment in 30 previously untreated patients. Heterogeneity among patients was pathologically diagnosed and classified into luminal and basal-like immunohistochemical profiles based on estrogen, progesterone, and human epidermal growth factor receptor (ER/PR/HER2) status. Marker heterogeneity was compared with mRNA biomarker expression in patients with BC before and after therapy. Reverse transcription-polymerase chain reaction was performed for molecular characterization. Expression and modulation of biological markers, CK19, hMAM, CEA, MUC, Myc, Ki-67, HER2/neu, ErbB2, and ER, were assessed after treatment, where the expression of the biomarkers CK19, Ki-67, Myc, and CEA was noted to be significantly decreased. Marker expression modulation was determined according to different stages and pathological characteristics of patients; coexpression of three markers (CK19, Ki-67, and Myc) was specifically modulated after therapy. In the histopathologically classified basal-like group, two markers (CK19 and Ki-67) were downregulated and could be considered as diagnostic biomarkers. In conclusion, pathological characteristics and marker variation levels can be evaluated to decide a personalized treatment for patients

Signature Prediction Modeling in Cytolethal Distending Toxin-Producing Strains

In this study, cytolethal distending toxin (CDT) producer isolates genome were compared with genome of pathogenic and commensal Escherichia coli strains. Conserved genomic signatures among different types of CDT producer E. coli strains were assessed. It was shown that they could be used as biomarkers for research purposes and clinical diagnosis by polymerase chain reaction, or in vaccine development. cdt genes and several other genetic biomarkers were identified as signature sequences in CDT producer strains. The identified signatures include several individual phage proteins (holins, nucleases, and terminases, and transferases) and multiple members of different protein families (the lambda family, phage-integrase family, phage-tail tape protein family, putative membrane proteins, regulatory proteins, restriction-modification system proteins, tail fiber-assembly proteins, base plate-assembly proteins, and other prophage tail-related proteins). In this study, a sporadic phylogenic pattern was demonstrated in the CDT-producing strains. In conclusion, conserved signature proteins in a wide range of pathogenic bacterial strains can potentially be used in modern vaccine-design strategies

Genotype Cluster Analysis in Pathogenic Escherichia coli Isolates Producing Different CDT Types

Diarrheagenic and uropathogenic E. coli types are mainly characterized by the expression of distinctive bacterial virulent factors. stx1, stx2 (Shiga toxins), and cdt (cytolethal distending toxin) genes have been acquired by horizontal gene transfer. Some virulent genes such as espP (serine protease), etpD (part of secretion pathway), and katP (catalase-peroxidase), or sfpA gene (Sfp fimbriae), are on plasmids and the others like fliC (flagellin) and the fimH gene (fimbriae type-I) are located on chromosome. Genomic pathogenicity islands (PAIs) carry some virulent genes such as hly gene. To determine the existence of virulence genes in cdt clinical isolates, genes including stx1, stx2, cdt, hly, espP, katP, sfpA, etpD, fliC, and fimH were assessed by Polymerase Chain Reaction (PCR). The most prevalent isolates for etpD and katP genes were 85.7% in cdtII. katP gene was also observed 83.3% in cdtI. However, in 42.85% of cdtIII isolates, espP gene was the most detected. Moreover, hly gene was also the most prominent gene in cdtIII (71.42%). sfpA gene was observed in 66.6% of cdtV. stx1 gene was detected in 100% of cdtII, cdtIV, and cdtV types. Presence and pattern of virulence genes were considered among cdt positive isotypes and used for their clustering and profiling