ABSTRACT
Resumen Introducción. Los factores de virulencia de las cepas de Vibrio cholerae no-O1, no-O139 no son claramente conocidos. La cepa de origen septicémico NN1 Vibrio cholerae no-O1, no-O139 fue secuenciada previamente mediante la plataforma Illumina, detectándose en su genoma un fragmento de la isla de patogenicidad VPaI-7 de V. parahaemolyticus. Objetivo: detectar los genes de virulencia vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF en cepas chilenas clínicas de V. cholerae no-O1, no-O139. Material y Métodos: Un total de 9 cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 aisladas entre 2006-2012 fueron analizadas mediante ensayos de reacción de polimerasa en cadena (RPC, en inglés PCR) convencional para los genes de secreción tipo III codificados en dicha isla: vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF. Adicionalmente se determinó la presencia de los genes de virulencia hylA y rtxA. Además, se realizaron ensayos de repetitive element palindromic PCR (REP-PCR) y Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Resultados: la mayoría (6/9) de las cepas chilenas de V. cholerae no-O1, no-O139 contiene todos los genes de secreción tipo III vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF, codificados en una isla de patogenicidad. Además, el total de las cepas (9/9) contiene los genes de virulencia hylA y rtxA. Conclusión: Estos resultados sugieren fuertemente la posibilidad que dichas cepas posean un potencial de virulencia importante en seres humanos.
Backgound: The virulence factors of the Vibrio cholerae non-O1, non-O139 strains are not clearly known. The strain of septicemic origin NN1 Vibrio cholerae non-O1, non-O139 was sequenced previously by the Illumina platform. A fragment of the pathogenicity island VPaI-7 of V. parahaemolyticus was detected in its genome. Aim: To detect the virulence genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF in Chilean strains of V. cholerae non-O1, non-O139. Methods: A total of 9 Chilean strains of clinical origin of Vibrio cholerae non-O1, non-O139 isolated between 2006-2012 were analyzed by conventional PCR assays for type III secretion genes encoded on that island: vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF. Additionally, the presence of the virulence genes hylA and rtxA was determined. In addition, REP-PCR and ERIC-PCR assays were performed. Results: most (6/9) Chilean V. cholerae non-O1, non-O139 strains contain the type III secretion genes vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF, encoded in an island of pathogenicity. In addition, all (9/9) the strains contain the virulence genes hylA and rtxA. Conclusion: These results strongly suggest the possibility that those strains possess an important virulence potential in humans.
Subject(s)
Humans , Bacterial Proteins/genetics , Transcription Factors/genetics , Vibrio cholerae/genetics , Virulence Factors/genetics , Vibrio cholerae non-O1/genetics , Genomic Islands/genetics , DNA-Binding Proteins/genetics , Type III Secretion Systems/genetics , Bacterial Toxins/genetics , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicity , Chile , Polymerase Chain Reaction , Sequence Analysis, DNA , Vibrio cholerae non-O1/isolation & purification , Vibrio cholerae non-O1/pathogenicity , Hemolysin Proteins/geneticsABSTRACT
Objective@#To investigate the distribution and expression of T3SS virulence genes in clinically isolated Pseudomonas aeruginosa (P. aeruginosa) strains and its correlation with drug resistance. @*Methods@#A total of 68 P. aeruginosa isolates were collected from the First Affiliated Hospital of Wenzhou Medical University in 2015. The antimicrobial susceptibility was detected by the agar dilution method. The distribution of virulence genes such as exoU, exoS, exoT and exoY from different isolates was detected by PCR. The expression levels of transcriptional regulator genes (ptrA and exsA) and effector-related genes (exoT and exoS) in some isolates were determined by real-time fluorescence quantitative PCR, and the Pearson correlation analysis was used to analyze the results. @*Results@#The detection rates of exoT and exoY in 68 P. aeruginosa isolates were higher, accounting for 79.4% and 75.0%, respectively. The detection rate of exoT in wound-sourced isolates was significantly higher than that in sputum (97.0% vs 61.8%, P＜0.01). In addition, the genotype of exoU - /exoS + was the most common, accounting for 51.5% (35/68). The resistance rates of sputum-sourced isolates to imipenem and meropenem were significantly higher than that of wound-sourced isolates (47.1% vs 8.8%, 47.1% vs 14.7%, P＜0.01). The resistance rates of isolates carrying exoU gene to carbapenems, aminoglycosides and fluoroquinolones were higher than those of isolates carrying exoS, exoT or exoY genes. Pearson correlation analysis showed that the expression level of ptrA gene was negatively correlated with those of exoT, exoS and exsA genes (P＜0.05). @*Conclusion@#The P. aeruginosa isolates from our hospital carrying T3SS virulence genes exoT and exoY are common, and the virulence genes are related to the drug resistance of P. aeruginosa. In addition, ptrA may be a potential negative regulatory gene for the expression of T3SS virulence genes.
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Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .
ABSTRACT
Salmonella is a pathogenic bacteria to human and animals ,which can cause seriously complication and death . Salmonella pathogenicity is from the reactions of SP‐1 and SP‐2 T3SS effector proteins .In this paper ,the functions of different T3ss effector proteins in different infection periods is reviewed to provide a reference for further understanding the pathogenesis mechanisms of Salmonella .
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Objective To construct the typeⅢ secretion system (T3SS) deficient mutant of O157∶H7 EDL933, and to detect its ability of attachment after infection with HeLa cells.Methods The recombinant DNA fragments obtained kana-mycin resistant gene were constructed by overlap extension PCR and transferred into EDL933/pKD46 to replace escR gene by homologous recombination.Results The T3SS-deficient mutant (ΔescR) was successfully construted.Compared with the wild-type EDL933, the growth rate and attachment on HeLa cells of ΔescR were significantly decreased.Conclusion The deletion of escR gene, which encodes T3SS structural protein EscR, affects the attachment of EDL933, suggesting a better basis for future studies of T3SS.
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Objective To investigate the effects of shiga toxin ( Stx ) phage on the expression of type Ⅲsecretion system (T3SS) in E.coli O157 ∶H7.Methods A standard E.coli O157 ∶H7 strain, EDL933 and a natural Stx phage defective mutant of EDL 933 strain, TUV93-0 were used for this study .The expression of T3SS proteins was compared between EDL 933 and TUV93-0 strains.The expression of five operons ( LEE1-LEE5 ) was evaluated by measuring the green fluorescent protein ( GFP ) in five different plasmids with LEE1-LEE5 promoters, respectively.Results The expression of T3SS proteins in TUV93-0 mutant were significantly increased than those in EDL 933 strain.Moreover, the expression of LEE1, LEE2 and LEE5 were also increased in TUV93-0 mutant.Conclusion The deletion of Stx phage might enhance T3SS expression through the regulation of LEE 1.
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Many gram-negative bacteria use the type Ⅲ secretion system (T3SS) to inject virulence proteins (effectors) directly into host cells.These effectors play a central role in pathogen interactions,and their molecular structures are highly variable.Previous researchers have used particular motifs common to all effectors in order to identify a full suite of candidate effectors of T3SS in the genome of Pseudomonas syringae.Here,we present a program called EFFECTORSEARCH that synthesizes and extends previous work by allowing users to identify candidate effectors based on any combination of the following five criteria:protein length,proximity to the hrp promoter,n-terminal region,similarity to known effectors,and dissimilarity to housekeeping genes.We demonstrate the utility of the program in allowing users either to search broadly for candidates or to restrict attention to the candidates with the highest confidence in their assignment.
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Objective To investigate the effect of FNR on type Ⅲ secretion system (T3SS) in E.coli O157 ∶ H7.Methods fnr mutant was constructed by λRed recombineering technology promoted by Bet and Exo proteins using PCR products.Results Through bacterial infection assays and immunofluorescence microscopy,it was found that the adhesion ability was decreased insignificantly infnr mutant compared to the wild type ZAP198.However,the secreted proteins were reduced significantly in the mutant from the secretion profile.Conclusion The reason might be that high ClpXP protein caused by the deletion of fnr degraded GrlA resulting in the inhibition of LEE(locus of enterocyte effacement) and T3SS.