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Objective:To identify new host substrate of SseK3 and study its biological function.Methods:A yeast two hybrid system (Y2H) was used to identify the potential binding proteins of SseK3 from the Hela cDNA library; the arginine N-acetylglucosamine (Arg-GlcNAc) modification of the substrate protein by SseK3 was detected by co-expression in 293T cells and in vitro activity test; the modification sites of the substrate protein by SseK3 were detected by point mutation; the effect of Arg-GlcNAc modification of the substrate protein on its interaction protein binding ability was detected by immunoprecipitation test. Results:Results of Y2H and gene sequencing showed that Snapin was a new substrate of SseK3. Snapin could be Arg-GlcNAc-modified by SseK3 in vivo and in vitro; the modification sites of Snapin were arginine 119 and arginine 120; Arg-GlcNAc-modified Snapin inhibited its binding with SNAP25. Conclusions:Snapin, a new host substrate protein of SseK3, was successfully screened in this study. The Arg-GlcNAc modification of Snapin by SseK3 was studied, and the effect of this modification on Snapin function was preliminarily studied, which provided theoretical basis for further understanding the function of Arg-GlcNAc modification of bacteria and the mechanism of action in the process of pathogen infection.
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Objective To analyze the virulence genes expression and drug resistance situation of type three secretion system (TTSS) of pseudomonas aeruginosa in Zhongshan area to provide a basis for clinical anti-infection treatment .Methods Seventy-six clinically isolated strains of Pseudomonas aeruginosa were collected from the Zhongshan Municipal People′s Hospital from July to September 2016 .Four virulence genes exoU ,exoS ,exoT and exoY were isolated from the strains by PCR .The Vitek2 Compact au-tomatic microbiological identification instrument was used to detect the sensitivity of antibacterial drugs .The enumeration data were processed with chi-square test .Results The detection rates of gene exoS and exoY were highest ,which were 52 .6% (40/76) and 63 .2% (48/76) respectively ;genotype exoU(-)exoS(+ )exoT (-)exoY(+ ) were predominant and accounted for 59 .26% .The positive rates of 4 virulence genes had no statistical difference between the multiple resistant and non-multiple resistant strains of pseudomonas aeruginosa .The resistance rates in the TTSS positive group to 13 kinds of drugs were commonly lower than those in the TTSS negative group ,the difference was statistically significant .Conclusion The geographic difference exists in virulence genes carrier of pseudomonas aeruginosa .The virulence genes carrying situation in both the multiple resistant and non-multiple resistant strains of pseudomonas aeruginosa is similar .
ABSTRACT
Las nuevas tecnologías para la edición de genomas, como los TALEN y el sistema CRISPR/Cas9, representan una gran oportunidad para mejorar características deseables en diferentes organismos. Los TALEN son el resultado del acoplamiento de nucleasas a los TALE (Transcription Activator-Like Effectors), los cuales son efectores naturales de gran importancia en la patogénesis de las especies de Xanthomonas. Xanthomonas axonopodis pv. manihotis (Xam) es el agente causal del añublo bacteriano de la yuca, quien durante el proceso patogénico es capaz de translocar sus efectores a la célula vegetal mediante el sistema de secreción tipo tres (SSTT). Actualmente no hay protocolos estándar para la edición de genomas en yuca. En este estudio se exploró la posibilidad de translocar efectores sobre callo embriogénico friable (CEF) a través de la inoculación con Xam, con el fin de determinar el potencial de este patógeno como sistema de entrega de TALEN. El CEF de dos variedades de yuca susceptibles (COL2215 y cv. 60444) se cocultivaron con la cepa Xam668 a diferentes tiempos. Posteriormente, se evaluó la expresión de marcadores correspondientes a los genes blanco conocidos para los TALE presentes en esta cepa bacteriana. Aunque no se logró demostrar la translocación de los mismos en el tejido embriogénico, sí se lograron establecer condiciones adecuadas de cocultivo con Xam y el efecto que la infección bacteriana tiene sobre la regeneración de embriones a partir de este tejido.
New technologies for genome edition, such as TALENs and CRISPR/Cas9 system, are a great opportunity to improve desirable features in different organisms. TALENs are the result from coupling nucleases and TALEs (Transcription Activator-Like Effectors), which are natural effectors with an important role in pathogenicity for the Xanthomonas species. Xanthomonas axonopodis pv. manihotis (Xam) is the cassava bacterial blight causal agent, and this pathogen is able to translocate its effectors to the plant cell during pathogenesis by using the type-three secretion system (TTSS). Currently, there are no standard protocols for genome edition in cassava. In this study, we explored the possibility to translocate effectors to friable embryogenic calli (FEC) through Xam inoculation, in order to establish the potential of this pathogen as a TALEN delivery system. Friable embryogenic calli derived from two different susceptible varieties (COL2215 and cv. 60444) were co-cultured with the strain Xam668 using different culture times. Subsequently, we evaluated the expression of makers corresponding to reported target genes for TALEs present in this bacterial strain. Although we were not able to demonstrate effector translocation, we established the conditions for co-culturing cassava calli and Xam and determined the effects derived from bacterial infection on embryo regeneration from FECs.
ABSTRACT
P. aeruginosa é um importante agente de infecções relacionadas à assistência em saúde. Habitualmente, o estabelecimento de infecções agudas é precedido pela colonização das mucosas dos pacientes. Não se sabe, porém, se os processos infecciosos são causados pelas próprias cepas bacterianas colonizadoras ou por outras com que os pacientes entrem em contato, dotadas ou não de maior potencial de virulência ou de resistência a antimicrobianos que as tornem mais eficientes como agentes infecciosos. Assim, este estudo teve como objetivos i) investigar a existência de potenciais diferenças entre amostras de P. aeruginosa que causaram apenas colonização e aquelas responsáveis por infecção, isoladas de um mesmo paciente, quanto a seus fenótipos de virulência e de não susceptibilidade a antimicrobiamos; ii) pesquisar a existência de associação entre características dos paciente, incluindo o tipo de evolução clínica, com as demais variáveis estudadas. No estudo foram incluídos 21 pacientes que desenvolveram infecção por P. aeruginosa durante sua internação no Centro de Terapia Intensiva do Hospital Universitário Clementino Fraga Filho, entre abril de 2007 e abril de 2008. De cada paciente foram selecionadas duas amostras bacterianas: a primeira isolada durante o episódio de infecção e a amostra colonizadora obtida imediatamente antes da ocorrência da infecção. As amostras selecionadas foram estudadas quanto a i) expressão de três mecanismos de virulência (citotoxicidade, aderência a células epiteliais respiratórias humanas e capacidade de formação de biofilme); ii) presença de genes codificadores das proteínas efetoras do sistema de secreção do tipo 3 (SST3 - exoS, exoT, exoU e exoY); iii) perfil de susceptibilidade a antimicrobianos, iv) perfil de fragmentação do DNA cromossômico por eletroforese em gel de campo pulsado (PFGE). As amostras bacterianas obtidas de infecções agudas foram significativamente mais citotóxicas que aquelas obtidas de colonização...
P. aeruginosa is an important agent of healthcare-associated infections. The establishment of acute infectious episodes is usually preceded by colonization of patient mucosa. However, it remains unknown whether the infectious processes are caused by bacterial strains previously colonizing the patient or by additional strains the patient may come into contact. These new isolates may carry greater virulence potential or antibiotic resistance that makes them more efficient as an infecting agent. Thus, the objetives of the present study were i) to investigate the existence of potential differences between P. aeruginosa isolates obtained from a colonized mucosa and isolates accounting for infectious processes, recovered from the same patient, with respect to virulence phenotypes and non-susceptibility to antimicrobial agents; ii) to investigate the existence of association between patient features, including the type of clinical outcome, with bacterial characteristics. The study included 21 patients who developed P. aeruginosa infection during their stay in the Intensive Care Unit of the University Hospital Clementino Fraga Filho, from April 2007 to April 2008. Two P. aeruginosa isolates were selected from each patient: the first isolate recovered from the infectious episode and the colonizing isolate obtained immediately before the onset of the infection. Features from the isolates investigated included: i) expression of three virulence mechanisms (cytotoxicity, adherence to human respiratory epithelial cells and biofilm formation); ii) presence of the genes encoding type III secretion system effector proteins (TTSS, exoS , exoT , exoU and exoY); iii) antimicrobial susceptibility profile; iv) profile of the bacterial chromossomic DNA fragmentation following analysis by pulsed-field gel electrophoresis (PFGE). The bacterial isolates obtained from acute infections were significantly more cytotoxic than colonizing strains...