高致病性2型猪链球菌毒素-抗毒素系统SezAT的鉴定与活性研究

【目的】对我国高致病性2型猪链球菌05Z33基因组的89K毒力岛序列进行生物信息学分析,发现存在一对与化脓链球菌Epsilon-zeta(ε-ζ)同源的Ⅱ型毒素-抗毒素系统(Toxin-antitoxin system,TA)——SezAT,推测该系统具有稳定89K毒力岛使其不易丢失的作用。验证SezAT为有活性的TA系统。【方法】对SezAT进行了生物信息学分析;RT-PCR验证SezAT共转录特性;在大肠杆菌中选择性地诱导表达毒素蛋白SezT和抗毒素蛋白SezA;最后通过同源重组技术敲除SezAT系统。【结果】sezAT由同一操纵子控制,SezT可抑制细菌生长,SezA可中和SezT的毒性作用,同源重组成功获得sezT敲除突变株。【结论】证实SezAT为一对有活性的毒素-抗毒素(TA)系统,为进一步研究SezAT可能发挥稳定89K毒力岛的功能,同时获得89K毒力岛缺失突变株并深入认识89K在我国高致病性SS2中的作用奠定了基础。     

2型猪链球菌gene0969基因敲除突变体的构建及毒力分析

目的:构建2型猪链球菌强毒株05ZYH33毒力岛89K上的Ⅳ型分泌系统组分gene0969敲除突变体,初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以05ZYH33基因组为模板,PCR扩增gene0969基因的上下游片段;以穿梭质粒pSET1为模板,PCR扩增氯霉素抗性基因Cm;采用重叠PCR方法搭建3个片段,并克隆到自杀载体pSET4s上,构建基因敲除载体,通过同源重组构建gene0969突变体,再用小鼠感染模型对突变株和野生株的毒力进行比较。结果:获得了gene0969基因敲除突变体,并发现其毒力与野生型相比有下降趋势。结论:2型猪链球菌假想毒力因子gene0969可能与毒力有关,其作用和机制值得进一步分析。     

猪链球菌2型ABC转运蛋白gene0910敲除突变体的构建及活性分析

目的:构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH3389K毒力岛上的ABC转运蛋白gene0910敲除突变体,并初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以猪链球菌2型05ZYH33基因组为模板,扩增gene0910两侧各约500bp左右的片段为上下游同源臂,以pSET1质粒为模板,扩增氯霉素抗性基因Cm为中间片段,采用重叠PCR方法搭建三个片段,并克隆到自杀载体pSET4S上,构建基因敲除的载体。电转化05ZYH33感受态细胞,经30℃双交换和40℃质粒丢失,最后点板法筛选出基因敲除突变体△0910。对突变株和野生株的生物学活性及小鼠的致病性进行了初步比较。结果:PCR分析和测序结果均显示gene0910完全被氯霉素抗性基因Cm所替代,基因敲除突变体构建成功。结论:突变株的生物学活性和对小鼠的致病性与野生株相比差异不显著。     

猪链球菌2型89K毒力岛研究进展

高致病性猪链球菌2型的致病机制仍是未解之谜.毒力岛不仅赋予病原菌特殊的致病能力,而且在细菌的适应性进化过程中扮演重要角色.对猪链球菌2型89K毒力岛功能性基因的深入剖析有助于更全面地掌握病原菌的致病特性.综述了猪链球菌2型89K毒力岛的结构与进化过程,以及国内外对毒力岛中二元信号转导系统、Ⅳ型分泌系统、ABC转运蛋白、毒素-抗毒素系统等重要基因的研究进展,力图从基因水平为猪链球菌2型的致病机制寻找突破口.     

Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island

Enterococcus faecalis, a leading cause of nosocomial antibiotic resistant infections, frequently possesses a 150 kb pathogenicity island (PAI) that carries virulence determinants. The presence of excisionase and integrase genes, conjugative functions and multiple insertion sequence elements suggests that the PAI, or segments thereof, might be capable of horizontal transfer. In this report, the transfer of the E. faecalis PAI is demonstrated and a mechanism for transfer elucidated. In filter matings, chloramphenicol resistance was observed to transfer from strain MMH594b, a clinical isolate possessing the PAI tagged with a cat marker, to OG1RF (pCGC) with a frequency of 3.2 x 10(-10) per donor. Secondary transfer from primary transconjugant TCRFB1 to strain JH2SS in filter and broth matings occurred with a frequency of 1 and 2 x 10(-1) per donor respectively. Analysis of the transconjugants demonstrated that a 27,744 bp internal PAI segment was capable of excision and circularization in the donor, and is mobilized as a cointegrate with a pTEF1-like plasmid. High-frequency transfer also occurred from TCRFB1 to JH2SS during transient colonization of the mouse gastrointestinal tract. This is the first demonstration of the horizontal transfer of PAI-encoded virulence determinants in E. faecalis and has implications for genome evolution and diversity.     

Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.     

Horizontal transfer of large plasmid with type IV secretion system and mosquitocidal genomic island with excision and integration capabilities in Lysinibacillus sphaericus

We identified a ~30-kb genomic island (named GI8) carrying the binary toxin gene operon binA/binB on both the chromosome and large pBsph plasmid in the mosquitocidal Lysinibacillus sphaericus C3-41 strain. We found that GI8 is related to the occurrence of binA/binB within L. sphaericus and displays excision and integration capability by recognizing the attB region, which consists of a 2-nt target site (AT) flanked by an 11-nt imperfect inverted repeat. pBsph and two pBsph-like plasmids (p2362 and p1593) were found to carry a type IV secretion system (T4SS) and displayed transmissibility within a narrow host range specific to L. sphaericus. GI8 can be co-transferred with pBsph as a composite element by integration into its attB site, then excised from pBsph and re-integrated into the chromosomal attB site in the new host. The potential hosts of GI8, regardless of whether they are toxic or non-toxic to mosquito larvae, share good collinearity at the chromosomal level. Data indicated that the appearance of the mosquitocidal L. sphaericus lineage was driven by horizontal transfer of the T4SS-type conjugative plasmid and GI8 with excision and specific integration capability.     

Role of capsular sialic acid in virulence and resistance to phagocytosis of Streptococcus suis capsular type 2

Abstract Streptococcus suis capsular type 2 has a capsule rich in sialic acid (NANA). Sialic acid, known to be an antiphagocytic factor for many bacterial species, inhibits the activation of the alternative complement pathway. The role of capsular NANA in virulence, resistance to phagocytosis and intracellular survival of S. suis capsular type 2 was evaluated. In general, a low concentration of NANA was observed for all the S. suis strains tested. In addition, no difference could be found in NANA concentrations between strains of different virulence degrees. Sialic acid concentration increased in the virulent strain 89–1591 and the avirulent strain 90–1330 after in vivo growth with an increased capsular material thickness compared to growth in vitro. No significant difference could be found in the phagocytosis rate by porcine blood monocytes of either strain and strain 89–1591 treated with sialidase or the sialic acid-binding lectin from Sambucus nigra (SNA I). Intracellular survival of strain 89–1591 decreased after treatments with sialidase or lectin, becoming comparable to that of strain 90–1330. Finally, no difference could be seen in virulence using a murine model, even if strain 89–1591 was treated with the enzyme or the lectin. Thus, NANA does not seem to be a critical virulence factor for S. suis capsular type 2.     

猪链球菌2型05Z33强毒株转录调控因子Rgg基因敲除突变体的构建及毒力分析

目的:构建猪链球菌2型强毒株05ZYH33转录调控因子Rgg的基因敲除突变体,观察其生物学性状,并在动物感染实验中比较敲除株与野生株的毒力差异,为进一步研究猪链球菌转录调控因子在致病中的作用提供实验基础。方法:分别以猪链球菌2型05ZYH33基因组和pSET1质粒为模板,扩增基因SSU05_1997两侧各约500 bp的片段为上下游同源臂,氯霉素(Cm)抗性基因为中间片段,采用重叠PCR方法连接3个片段;连接产物先克隆到T载体上,再经过酶切克隆到温度敏感自杀载体pSET4S上;将构建的基因敲除载体pSET4S-1997电转化入05ZYH33感受态细胞,通过改变培养温度筛选出基因敲除突变体05Z33△rgg;对敲除株和野生株的生物学性状及小鼠和猪的致病性进行了初步比较。结果:PCR分析和测序结果均显示基因SSU05_1997完全被Cm抗性基因所替代,基因敲除突变体构建成功;05ZYH33△rgg对小鼠和猪的致病性与野生株相比无明显差异。结论:转录调控因子Rgg可能和猪链球菌2型的毒力无关。     

Identification of candidate susceptibility and resistance genes of mice infected with Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is an important swine pathogen and zoonosis agent. A/J mice are significantly more susceptible than C57BL/6 (B6) mice to SS2 infection, but the genetic basis is largely unknown. Here, alterations in gene expression in SS2 (strain HA9801)-infected mice were identified using Illumina mouse BeadChips. Microarray analysis revealed 3,692 genes differentially expressed in peritoneal macrophages between A/J and B6 mice due to SS2 infection. Between SS2-infected A/J and control A/J mice, 2646 genes were differentially expressed (1469 upregulated; 1177 downregulated). Between SS2-infected B6 and control B6 mice, 1449 genes were differentially expressed (778 upregulated; 671 downregulated). These genes were analyzed for significant Gene Ontology (GO) categories and signaling pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database to generate a signaling network. Upregulated genes in A/J and B6 mice were related to response to bacteria, immune response, positive regulation of B cell receptor signaling pathway, type I interferon biosynthesis, defense and inflammatory responses. Additionally, upregulated genes in SS2-infected B6 mice were involved in antigen processing and presentation of exogenous peptides, peptide antigen stabilization, lymphocyte differentiation regulation, positive regulation of monocyte differentiation, antigen receptor-mediated signaling pathway and positive regulation of phagocytosis. Downregulated genes in SS2-infected B6 mice played roles in glycolysis, carbohydrate metabolic process, amino acid metabolism, behavior and muscle regulation. Microarray results were verified by quantitative real-time PCR (qRT-PCR) of 14 representative deregulated genes. Four genes differentially expressed between SS2-infected A/J and B6 mice, toll-like receptor 2 (Tlr2), tumor necrosis factor (Tnf), matrix metalloproteinase 9 (Mmp9) and pentraxin 3 (Ptx3), were previously implicated in the response to S. suis infection. This study identified candidate genes that may influence susceptibility or resistance to SS2 infection in A/J and B6 mice, providing further validation of these models and contributing to understanding of S. suis pathogenic mechanisms.     

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.     

Immunoproteomic assay of surface proteins of Streptococcus suis serotype 9

Streptococcus suis is an important swine pathogen responsible for a diverse group of diseases. Studies on vaccines have focused on S. suis serotype 2 strains, which are the most invasive isolates worldwide. However, in China S. suis serotype 9 (SS9) is also a prevalent serotype, which is frequently isolated from diseased pigs. Little is known about immunogenic proteins for SS9. Therefore, an immunoproteomic-based approach was developed to identify immunogenic proteins of SS9. Cell wall proteins extracted from SS9 strain GZ0565 isolated from a diseased pig with meningitis were screened by two-dimensional Western blotting using anti-SS9 sera pooled from specific pathogen-free mice. Protein spots were excised from preparative gels and identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) or MALDI-TOF-TOF-MS, which led to the identification of eight immunogenic proteins (arginine deiminase, extracellular solute-binding protein, translation elongation factor Ts, neprilysin, peptide ATP-binding cassette transporter peptide-binding protein, pyruvate kinase, phosphate acetyltransferase, and fructose-bisphosphate aldolase). These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, could be developed as vaccine candidates.     

Pathogenicity island integrase cross-talk: a potential new tool for virulence modulation

Instability and excision of pathogenicity islands (PAIs) have already been described in Escherichia coli 536. In this edition of Molecular Microbiology, Bianca Hochhut and colleagues from the University of Würzburg in Germany have shown that the instability of four of the E. coli 536 PAIs is mediated by a P4-type integrase encoded within the specific PAI by a site-specific recombination mechanism. The integrase encoded on PAI II(536) is able to mediate excision and integration of both PAI II(536), and also PAI V(536). The att sites of both these PAIs have a region of sequence similarity, which is also found in several other PAIs and in tRNA genes in several bacterial species. The cross-PAI activity of this integrase (Int(PAI II)) suggests that it plays an important role in both genome evolution and horizontal transfer of pathogenicity elements, possibly even across species barriers. Deletion of PAIs that carry genes for adhesins and other traits might lead to a phase variation-like phenomenon. Differential regulation of integrase activity or production might add a further level of fine-tuning during bacterial infection.     

Infection with Streptococcus Suis Serotypes 1 and 2 in the Same Diseased Pig

Streptococcus suis is an important pig pathogen which is associated with respiratory problems, meningitis and less fre-quently with a variety of other conditions(Hommez et al. 1986). S. suis type 1 causes disease mainly in 1–2 week old pigs while serotype 2 is found commoaily in 2–22 week old pigs, S. suis type 2 is a zoonosis. It can cause meningitis and septicaemia in man (Christensen & Kronvall 1985). Several other serotypes of S. suis have also been identified on the basis of the capsular poly-saccharide (Perch et al. 1983, Hommez et al. 1986). We present a case where we isolated S. suis types 1 and 2 from the brain and lungs respectively of the same diseased suckling piglet. This i/s the first reported case of S. suis types 1 and 2 in Finland.     

猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940的比较转录谱分析

目的:比较猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940毒力相关基因转录水平的差异,为进一步研究强毒株S.suis 05ZY毒力增强的原因提供实验基础。方法:分别提取S.suis 05ZY和S.suis 1940的RNA,反转录成cDNA并纯化,用Cy5或Cy3标记,与猪链球菌全基因组DNA芯片进行杂交,扫描芯片进行数据分析,比较二者在转录水平上的差异基因。结果:编码溶血素、精氨酸氨基肽酶的基因分别上调4.4和6.0倍,参与荚膜多糖合成的相关基因cps2H、cps2I、cps2J和一些可能的毒力相关基因ofs、dpr、SSU05₀196、SSU05₀272、SSU05₁408-1409均发生转录水平的上调。结论:溶血素、荚膜多糖、精氨酸氨基肽酶及一些可能的毒力因子在转录水平的上调很可能与S.suis 05ZY的毒力增强有关。     

Analysis of the type IV secretion system-dependent cell motility of Helicobacter pylori-infected epithelial cells

The pathogenesis of Helicobacter pylori-associated disorders is strongly dependent on a specialized type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). Cytotoxin-associated gene A (CagA) is the only known H. pylori protein translocated into the host cell followed by tyrosine phosphorylation through host protein kinases. H. pylori induces cellular processes which are either PAI- or CagA-dependent (e.g., cell motility), PAI-dependent, but CagA-independent (e.g., interleukin-8 release), or PAI- and CagA-independent (e.g., cyclooxygenase-2 release). Here, we investigated H. pylori strains mutated in single PAI genes of the wild type strain Hp26695 and their effects on cell motility. We found 17 gene products out of 27 PAI genes playing a superordinated role and five PAI-encoded proteins exhibiting a clearly critical role in motogenic host cell responses, whereas the remaining five PAI gene products had no significant influence on the motogenic response in reaction to H. pylori infection. This study clearly demonstrated that H. pylori-induced cell motility and invasive growth involve type IV secretion system-dependent signalling as well as translocated and phosphorylated CagA. These findings reveal a deeper insight in to the meaning of the T4SS of H. pylori for host cell motility.