Research progress on the structure and function of the type IX secretion system of Porphyromonas gingivalis / 口腔疾病防治

@#The Porphyromonas gingivalis type IX secretion system (T9SS) is a recently discovered protein secretion system that is widely distributed in Bacillus cereus. The T9SS is structurally complex and powerful. More than 20 T9SS components have been verified, and more than 30 virulence factors can be secreted by Porphyromonas gingivalis alone, which contributes significant to the pathogenicity of Porphyromonas gingivalis. T9SS is a large protein complex spanning the inner cell membrane, periplasm, and outer cell membrane. Through the structural and functional connections among its components, it forms a sophisticated functional complex that includes power provision, energy transduction, inner and outer membrane translocation, outer membrane modification, and regulatory systems to recognize, translocate, shear, and modify cargo proteins and translocate bacterial intracellular cargo proteins to the cell surface. In recent years, with advancements in X-ray diffraction and in situ cryoelectron microscopy, the exploration of T9SS has evolved from the functional study of single components to the in situ structural study of multiprotein complexes. Still, the structural resolution of the protein still has shortcomings such as low resolution and an inability to capture dynamic functional structures. Future research directions should focus more on exploring how T9SS interacts and functions with cargo proteins. In this paper, we review the research progress on Porphyromonas gingivalis T9SS on X-ray diffraction and cryoelectron microscopy structure resolution in order to gain a deeper understanding of the transport mechanism of T9SS.

Mycolicibacterium fortuitum genomic epidemiology, resistome and virulome

BACKGROUND Mycolicibacterium fortuitum is an opportunistic pathogen associated with human and animal infection worldwide. Studies concerning this species are mainly represented by case reports, some of them addressing drug susceptibility with a focus on a specific geographic region, so there is a gap in relation to the global epidemiological scenario. OBJECTIVES We aimed determine the global epidemiological scenario of M. fortuitum and analyse its traits associated with pathogenicity. METHODS Based on publicly available genomes of M. fortuitum and a genome from Brazil (this study), we performed a genomic epidemiology analysis and in silico and in vitro characterisation of the resistome and virulome of this species. FINDINGS Three main clusters were defined, one including isolates from the environment, human and animal infections recovered over nearly a century. An apparent intrinsic resistome comprises mechanisms associated with macrolides, beta-lactams, aminoglycosides and antitubercular drugs such as rifampin. Besides, the virulome presented Type VII secretion systems (T7SS), including ESX-1, ESX-3, ESX-4 and ESX-4-bis, some of which play a role on the virulence of Mycobacteriaceae species. MAIN CONCLUSIONS Here, M. fortuitum was revealed as a reservoir of an expressive intrinsic resistome, as well as a virulome that may contribute to its success as a global opportunist pathogen.

Identification of the host substrate of a Salmonella typhimurium type three secretion system effector SseK3 and preliminary investigation on its biological function / 中华微生物学和免疫学杂志

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.

Advances in Klebsiella pneumoniae and Acinetobacter baumannii type Ⅵ secretion systems / 中华微生物学和免疫学杂志

Nosocomial infections caused by gram-negative opportunistic pathogens, including Acinetobacter baumannii ( A. baumannii) and Klebsiella pneumoniae ( K. pneumoniae), pose a great challenge to health care management and human health. New treatment strategies are urgently needed to tackle with the spread of multi-drug resistant strains and the increase in bacterial virulence. Type Ⅵ secretion system (T6SS), a conservative secretory apparatus encoded in a variety of gram-negative bacteria, can inject effectors into other prokaryotic or eukaryotic cells in a contact-dependent manner to achieve antibacterial and anti-host properties. It is closely related to the environmental adaptability, competitiveness and colonization ability of bacteria in hosts. The T6SS gene cluster is composed of core genes, effector genes and associated genes, and the effectors encoded by it are highly diverse and play an important role in pathogen infection. This review summarized the advances in A. baumannii and K. pneumoniae T6SS in terms of competition, host colonization, interaction between conjugative plasmids and expression regulation, aiming to provide reference for future study on T6SS-related antimicrobial activity, virulence and resistance.

Production and identification of monoclonal antibodies against Brucella melitensis VirB5 protein / 中华地方病学杂志

Objective To prepare monoclonal antibodies (mAb) against the type Ⅳ secretion system protein VirB5 of Brucella melitensis and to provide a basis for pathgenic diagnosis and research of brucellosis.Methods Four SPF female BALB/c mice were subcutaneously immunized with purified VirB5 protein at a dose of 60 μg/mice,and immunization was strengthened every 2 weeks at a dose of 30 μg/mice,three times in total.Two weeks later,the orbital venous blood of mice was taken to determine the antibody titer,and then intraperitoneally injected for the fourth time to strengthen immunization.Three days later,mouse spleen cells were fused with mouse myeloma SP2/O cells in a ratio of 5:1.After 3 times of cell screening and monoclonal cloning,the hybridoma cell lines with stable secretion of VirB5 antibody were established;one BALB/c mouse was intraperitoneally injected with hybridoma cells,and ascites were collected and antibody was purified when the mouse abdomen was significantly enlarged.The immunological characteristics of mAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting.Results A total of 6 monoclonal cell lines (2-2,2-12,2-19,2-25,2-31 and 2-40) capable of secreting VirB5 antibody were established.Among them,the cell line 2-19 can stably secrete an antibody that specifically recognized the VirB5 protein,and the VirB5 antibody secreted by the cell line was identified as an IgG1 subtype,a kappa light chain,a mAb affinity constant of 1.6 × 108.The titer of ascites antibody of mouse intraperitoneally injected with hybridoma cell 2-19 was 1:51 200.Conclusion The high-affinity mAb of type Ⅳ secretion system protein VirB5 is successfully prepared,and the antibody can rapidly bind specifically to pathogens,providing an alternative material for establishment of brucellosis pathogen diagnostic method.

Type VI secretion system and quorum sensing system involved in biofilm formation in Pseudomonas aeruginosa / 第二军医大学学报

[Abstract] Objective To explore the roles of type VI secretion system (T6SS) and quorum sensing (QS) system in the biofilm formation in Pseudomonas aeruginosa. Methods The PAO1 biofilm bacteria and planktonic bacteria were established. The expression levels of T6SS associated genes hemolysin co-regulated protein (Hcp) gene (Hcp1, Hcp2, Hcp3), QS associated gene LasR, exopolysaccharide associated genes polysaccharide synthesis locus A (PslA) and pellicle A (PelA), type pili gene pilus A (PilA) and flagellin C (FliC) were detected by quantitative real-time PCR, and the expression levels of those genes were compared between PAO1 biofilm bacteria and planktonic bacteria using independent t-test. Results The expression levels of PslA, PelA, PilA and FliC genes in PAO1 biofilm bacteria were respectively 714, 274, 604 and 42 folds of those in the planktonic bacteria (all P0.05), suggesting that PAO1 bacteria formed mature biofilm with flagellum and pili. The expression levels of Hcp1, Hcp2, Hcp3, and LasR genes in PAO1 biofilm bacteria were respectively 1 045, 11 268, 6 654 and 1 226 folds of those in planktonic bacteria (all P0.05), suggesting that T6SS and QS systems were related to the biofilm formation of PAO1 bacteria. Conclusion T6SS and QS systems might be involved in biofilm formation in Pseudomonas aeruginosa, and its regulatory mechanism needs further study.

Molecular cloning and characterization of gene virD[STHX]4 in Helicobacter pylori / 临床检验杂志

Objective@#To investigate the function of virD4 gene in Helicobacter pylori clinical strain SBK. @*Methods@#The virD4 gene segment was obtained through T-A cloning method. The prokaryotic expression vector pET-28a(＋)-virD4 was constructed and transformed into E. coli Rosetta for the expression by induction of IPTG. The recombinant proteins were obtained and purified by KCl dyeing with gel cutting method, and identified via SDS-PAGE analysis. The purified recombinant virD4 protein was used to immunize mice to produce polyclonal antibodies. The titer of the polyclonal antibodies was tested by ELISA and the antigenic specificity was identified by western blot. The purified recombinant virD4 proteins were co-cultured with GES-1 cells followed by detecting the expression of inflammatory cytokines secretion and the ability of cell proliferation. @*Results@#The full length of virD4 gene was 1 728 bp. The sequence shared quite high homology with the virD4 gene of isolate Shi470. The recombinant prokaryotic expression plasmid pET-28a(＋)-virD4 was successfully constructed. The recombinant virD4 proteins were obtained by IPTG induction and purified via KCl dyeing method. SDS-PAGE showed that the relative molecular weight of recombinant virD4 protein was 63 000. The purified proteins were used to immunize mice to obtain the anti-virD4 polyclonal antibodies with the titer 512 000. The reaction between anti-virD4 polyclonal antibodies and recombinant virD4 proteins was highly specific. The recombinant virD4 protein induced inflammatory cytokines secretion and promoted GES-1 cell proliferation. @*Conclusion@#The virD4 gene was successfully cloned and highly expressed in prokaryotic expression system, and its antibodies were prepared. The recombinant virD4 protein can induce cytokine secretion and cell proliferation.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Caracterização de dois pares efetor/inibidor associados ao sistema de secreção tipo IV de Xanthomonas citri / Characterization of the two effector/inhibitor pair associated with the type IV secretion system of Xanthomonas citri

O sistema de secreção tipo IV (T4SS) da família de bactérias Xanthomonadaceae transfere efetores (X-Tfes) com a capacidade de matar outras bactérias, conferindo uma vantagem em comunidades bacterianas mistas para colonizar diferentes nichos como o solo ou as superfícies das plantas. Os X-Tfes possuem diferentes domínios putativos com atividades hidrolíticas contra componentes do envelope celular bacteriano do tipo: glicohidrolases, transglicosilases, amidases e lipases. Os X-Tfes por sua atividade biológica inata podem ocasionar dano intracelular para a bactéria que os produz. Para se proteger contra estas atividades, também são produzidas lipoproteínas com função inibitoria (X-Tfis) localizadas no periplasma. Os genes que codificam os X-Tfes e os X-Tfis estão organizados em operons, o que permite gerar os pares efetor/inibidor simultaneamente. Entre os potenciais X-Tfes do fitopatógeno Xanthomonas citri estão Xac1918 e Xac0574. Xac1918 é uma proteína com um domínio da superfamília da lisozima e um domínio conhecido como RTX (Repeats in Toxin) de ligação ao cálcio, enquanto Xac0574 tem um domínio da superfamília da lipase 3. Os seus possíveis inibidores, Xac1917 e Xac0573 respectivamente, apresentam um peptídeo sinal no N-terminal contendo o lipobox representativo das lipoproteínas. As proteínas Xac0574 e Xac0573 são monômeros em solução que formam um complexo estável 1:1, favorecido termodinamicamente (ΔG°= -12 Kcal/mol) com uma constante de dissociação de 2,45 nM, garantindo que a bactéria fique protegida contra os efeitos nocivos de Xac0574 quando é produzida intracelularmente. Xac0574 é uma fosfolipase A1, sem atividade lisofosfolipase, com a capacidade de hidrolisar os três fosfolipídios majoritários que compõem a membrana celular bacteriana, fosfatidilglicerol (PG), cardiolipina e fosfatidiletanolamina (PE), mostrando uma aparente preferência pelo último. A atividade enzimática de Xac0574 explica a forte inibição do crescimento celular em E. coli após da sua indução heteróloga, já que gera uma diminuição de quase 10 vezes da população celular comparada com a cultura não induzida com a mesma construção. Poroutro lado, Xac0573 inibe efetivamente a atividade enzimática de Xac0574 ao formar o complexo, além de não ter atividade fosfolipase nem lisofosfolipase. Foram produzidos cristais da Xac1918 e Xac0573 que difrataram com uma resolução de 3,0 e 2,5 Å, respectivamente. Porém, só foi gerado um modelo de Xac0573. Xac0573 está composta por duas folhas ß antiparalelas com uma topologia característica de ß sanduíche Com uma pequena hélice e duas voltas. Um alinhamento de homólogos de Xac0573 identificou nas extremidades da proteína as regiões conservadas, constituindo duas possíveis interfaces de interação que podem ser as responsáveis por bloquear o acesso dos fosfolipídios ao sítio catalítico ou impedir os rearranjos estruturais de Xac0574 que são necessários para a sua atividade enzimática. Adicionalmente, a topologia da Xac0573 é semelhante do domínio C2, conhecido em eucariotos como domínio de ligação ao lipídio e ao cálcio, e está envolvido em processos de sinalização de segundos mensageiros lipídicos, proteínas de trafego de membranas e mecanismos de fusão de membranas. Nossos resultados apontam para uma nova função biológica do domínio C2 como um inibidor enzimático intracelular em bactérias

The type IV secretion system (T4SS) of the bacteria family Xanthomonadaceae transfers effectors (X-Tfes) with that can kill other bacterial cells, conferring an advantage to the bacterial community during colonization of different niches in the soil or on the plant surface. The X-Tfes possess different putative domains with hydrolytic activity against components of the bacterial cellular envelope, including glycohydrolase, transglycolase, amidase and lipase domain. The innate biological activity of X-Tfes can cause intracellular damage. Therefore, the bacteria that produce them also produce lipoproteins with inhibitor function (X-Tfis) located in the periplasm for their protection. The genes that code for X-Tfes and X-Tfis are organized in operons that allow for their simultaneous expression. Among the X-Tfes of the phytopathogen Xanthomonas citri are Xac1918 and Xac0574. Xac1918 is carries a lysozyme superfamily domain, as well as a domain known as RTX (Repeats in Toxic) predict to bind calcium, while, Xac0574 has a domain belonging to the lipase 3 superfamily. Their possible inhibitors, Xac1917 e Xac0573 respectively, carry an N-terminal signal peptide containing a lipobox found in bacterial lipoproteins. The Xac0574 and Xac0573 proteins are both monomers in solution, They can form a stable 1:1 complex, that is thermodynamically favored (ΔG°= -12 Kcal/mol) with a dissociation constant of 2,45 nM. This affinity ensure that the bacterium is protected against the harmful effects of Xac0574 when it is produced intracellularly. We show that Xac0574 is a phospholipase A1, without lisophospholipase activity, and is able to hydrolyze the three most common phospholipids found in the membranes of Gram negative bacteria, namely phosphatidylglycerol (PG), cardiolipin and phosphatidylethanolamine (PE), presenting an apparent preference for PE. The enzymatic activity of Xac0574 explains the strong inhibition of growth of E. coli cells after its heterologous induction: a nearly 10-fold decrease in the cell population is observed when compared to the non-induced culture with the same construct. On the other hand, Xac0573 effectively inhibits the enzymatic activity of Xac0574. Furthermore, Xac0573 does not possess when forming the complex, besides not having phospholipase nor lysophospholipase activity.Crystals of Xac1918 and Xac0573 were produced which diffracted with to resolution of 3.0 and 2.5 Å, respectively. However, we were able to resolve the structure of only Xac0573. Xac0573 is composed of two anti-parallel sheet that form a ß-sandwich with three small helices. An alignment to Xac0573 homologs identified conserved regions at the ends of the protein that constitute two possible interfaces of interaction that may be responsible for blocking the access of the phospholipids to the catalytic site or impede the structural rearrangements of Xac0574 that are necessary for its enzymatic activity. Additionally, the topology of Xac0573 is similar to that to C2 domains, known in eukaryotes to bind lipids and calcium and to be involved in signaling processes mediated by lipid second messengers, membrane trafficking and membrane fusion mechanisms. Our results point to a new biological function of the C2 domain as an intracellular enzyme inhibitor in bacteria

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Type Ⅶ secretion system in Mycobacteria / 中华微生物学和免疫学杂志

Type Ⅶ secretion system(T7SS) is a novel and specialized secretion system discovered in recent years. It was first found in Mycobacterium tuberculosis. Type Ⅶ secretion system is involved in the secretion of virulence-associated proteins, the interaction between pathogens and hosts and the balance of zinc/iron ions. Moreover,it plays a critical role in the growth and pathogenesis of Mycobacteria. This review summarizes the components,substrates and translocation mechanisms of the type Ⅶ secretion system and its relation to the virulence of Mycobacteria aiming to provide references for developing novel strategies for disea-ses diagnosis,treatment and prevention.

Complete plasmid sequence carrying type IV-like and type VII secretion systems from an atypical mycobacteria strain

The genus Mycobacterium is highly diverse and ubiquitous in nature, comprehending fast- and slow-growing species with distinct impact in public health. The plasmid-mediated horizontal gene transfer represents one of the major events in bacteria evolution. Here, we report the complete sequence of a 160,489 bp circular plasmid (pCBMA213_2) from an atypical and fast-growing environmental mycobacteria. This is a unique plasmid, in comparison with the characterised mycobacteria plasmids, harboring a type IV-like and ESX-P2 type VII secretion systems. pCBMA213_2 can be further explored for evolutionary and conjugation studies as well as a tool to manipulate DNA within this bacteria genus.

Comparative genomic analysis of Salmonella type Ⅲsecreted candidate gene yiiG / 基础医学与临床

Objective To study the distribution and evolution of yiiG, a Salmonella gene encoding a candidate type secretedsubstrate .Methods Salmonella genomes were comprehensively screened for yiiG distribution with se-quence alignment strategies .The evolutionary history of yiiG was traced .Comparative genomic analysis was per-formed to study the evolutionary mechanisms of yiiG gene acquisition , loss and duplication .RNA-seq data were combined to analyzing the correlation between yiiG and other virulence factors .A variety of bioinformatic tools were used for discovering the possible type Ⅲsecretion signals .Results yiiG distributed in S.enterica subsp.enterica but variable in other subspecies of S.enterica.No yiiG was found in S.bongori.Besides Salmonella, only a part of Shigella and E.coli strains were detected with yiiG homologs .The genomic locus of yiiG and its adjacency showed conservation among all Salmonella, E.coli and Shigella strains.In most of the serovars of S.enterica subsp.enteri-ca, there was a head-to-head tandem whole yiiG repeat sequence upstream the yiiG gene, which was renamed as yiiGRrc.RNA-seq analysis showed that yiiG gene expression level was highly correlated with T 3SS-related genes . Bioinformatic prediction also indicated the T 3SS effector signals in YiiG N-terminus.Conclusions yiiG represented an ancient genomic locus , which will be a hot spot where rearrangement events frequently happened .The function of yiiG could potentially be related with Salmonella virulence.Finally, a new protein-encoding gene (yiiGRrc) was newly identified that was closely related with yiiG, providing the target for further understanding the composition , function and function variation of yiiG gene family .

Detection of type three secretion system virulence gene expression difference of pseudomonas aeruginosa in Zhongshan area * / 国际检验医学杂志

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 .

Research progress on the type Ⅸ secretion system of Porphyromonas gingivalis / 华西口腔医学杂志

In recent years, the study found that Porphyromonas gingivalis type Ⅸ secretion system (T9SS) is a novel protein secretion system, also known as Por secretion system (PorSS). Unlike the eight protein secretion systems found in the past, the system is a polyprotein complex found only in Bacteroides. The secreted proteins have both N- and C-terminus, where the former includes Sec-dependent type Ⅰ signals peptide, and the latter contains conserved domains (C-terminal conserved domain, CTD). Porphyromonas gingivalis T9SS includes proteins such as intima, outer membrane, cytoplasm, and cell cycle, including at least 34 proteins containing CTD. Porphyromonas gingivalis T9SS is involved in regulating associated virulence factors including gingivin, fimbriae, lipopolysaccharide, HBP35, CPG70 protein and peptidyl-arginine deiminase. These CTD-containing virulence proteins are localized by T9SS and then released to the extracellular domain, thereby destroying periodontal tissue. Therefore, this review summarizes the research progress on the T9SS of Porphyromonas gingivalis.

Infección de callo embriogénico friable de yuca con Xanthomonas axonopodis pv. manihotis (Xam) / Cassava friable embryogenic calli infection with Xanthomonas axonopodis pv. manihotis (Xam)

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.

In silico dissection of Type VII Secretion System components across bacteria: New directions towards functional characterization.

Type VII Secretion System (T7SS) is one of the factors involved in virulence of Mycobacteriun tuberculosis H37Rv. Numerous research efforts have been made in the last decade towards characterizing the components of this secretion system. An extensive genome-wide analysis through compilation of isolated information is required to obtain a global view of diverse characteristics and pathogenicity-related aspects of this machinery. The present study suggests that differences in structural components (of T7SS) between Actinobacteria and Firmicutes, observed earlier in a few organisms, is indeed a global trend. A few hitherto uncharacterized T7SS-like clusters have been identified in the pathogenic bacteria Enterococcus faecalis, Saccharomonospora viridis, Streptococcus equi, Streptococcuss gordonii and Streptococcus sanguinis. Experimental verification of these clusters can shed lights on their role in bacterial pathogenesis. Similarly, verification of the identified variants of T7SS clusters consisting additional membrane components may help in unraveling new mechanism of protein translocation through T7SS. A database of various components of T7SS has been developed to facilitate easy access and interpretation of T7SS related data.

Construction and characterization of type III secretion system of attenuated Salmonella typhimurium / 生物工程学报

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.

Comparative proteomic analysis of Yersinia pestis 201 and 201△pCD1 strains / 军事医学

Objective To explore the potential pathogenesis of Yersinia pestis and provide new clues for vaccine development through comparative proteomic analysis of wild-type and pCD1 cured strain of Yersinia pestis 201.Methods Differentially expressed proteins at 26℃ and 37℃ were separated and identified using two-dimensional electrophoresis coupled with mass spectrometry .Results A total of 24 differently expressed proteins were successfully identified from the samples of bacteria grown at 26℃ and 25 proteins at 37℃.Among these, 7 proteins were encoded by pCD 1 plasmid. Conclusion Through comparative proteomic research, we have found that the abundance of several proteins can be dramatically changed when the large plasmid pCD 1 is missing,suggesting that the plasmid can regulate the expression of many genes located in the chromosome .

Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.