弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的研究Ⅱ:生物学特性的研究

进一步对FS54—2、—3、—4、—5、—6、—7及—7b等双价减毒株的侵袭力、毒力、免疫原性与免疫保护性及遗传稳定性等进行了研究,证明双价减毒株仍保持了入侵肠上皮细胞的能力;小动物实验安全低毒;有很好的免疫原性,对小鼠有很好的双重免疫保护性。     

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.     

鉴定痢疾杆菌毒力基因的SW480细胞模型构建

信号标签诱变技术（STM）是一种在体内高通量筛选病原体毒力基因的新方法,在应用时的一个先决条件是要建立合适的体内筛选系统。为将该技术应用于福氏痢疾杆菌,我们使用三个福氏痢疾杆菌菌株进行了预试验：通过同源重组构建而成的带有氯霉素抗性且aroA和virG基因失活的突变株RC426;因在侵袭质粒上自发缺失3个基因座(ipaBCDA, invA 和 virG)的另一减毒突变株T32,其曾被用作福氏痢疾杆菌的口服疫苗;还有具侵袭宿主细胞能力的野生性菌株2457T。将RC426、T32和2457T混合后侵袭结肠细胞系SW480,不同时间回收经侵袭后细胞裂解液中的菌体并统计。结果显示在侵袭12h内回收到减毒突变株的量与野生有毒株存在显著性差异,表明SW480 细胞系可用于痢疾杆菌的STM研究。Abstract: Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo. An appropriate animal or cell line model is one of prerequisites by exploiting this technique. In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; Another attenuated strain T32 was used as an oral S. flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid. The wild type strain 2457T had the invasion ability into host cells. The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time. The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Triphenyltin salicylate-antimicrobial effect and resistance--the pyrophosphatase connection

The effect of Triphenyltin salicylate (TPS) was tested against six bacteria, Escherichia coli, Staphylococcus aureus, Shigella flexneri, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi and five fungi, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhodotorula spp. and Saccharomyces spp. Sensitivity tests were determined with 5-500 microg/ml of TPS. All organisms were sensitive to the compound except Klebsiella pneumoniae, Pseudomonas aeruginosa, Rhodotorula spp. and Saccharomyces spp. The minimum dose of TPS that can kill 50% of the susceptible microorganisms is in the range 5-50 microg/ml. Membrane bound pyrophosphatase(s) from the organisms was non-competitively inhibited by 5 microM TPS with Ki values of 7.6, 18, 8.8 and 6.9 microM for Escherichia coli, Shigella flexneri, Aspergillus niger, and Aspergillus fumigatus, respectively. The physiological index of efficiency of the enzyme (Vmax/KM) for TPS susceptible organisms was reduced by 17-68% in the presence of 5-10 microM of the compound. In contrast the index for the non-susceptible organisms was unaffected. The mode of action of TPS is discussed.     

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.     

肠毒素大肠杆菌定居因子抗原基因CFA-I在痢疾杆菌中的表达

通过体外重组的方法 ,构建了包含asd基因的重组表达质粒 pZHY2 1,与福氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统 ,Western印迹结果表明 ,在没有抗生素条件选择的情况下 ,稳定表达肠毒素大肠杆菌定居因子抗原CFA I。电镜结果显示 ,在重组菌株的菌体表面 ,表达产物能够装配成菌毛。重组菌通过口服和鼻饲免疫小鼠后 ,可以诱生CFA I的抗体 ;同时可以检测到分泌型IgA产生 ,表明重组菌可以诱导相应的粘膜免疫反应     

The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo.

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.     

稳定、无抗药的痢疾福氏2a和宋内双价菌苗候选株的构建

通过体内外基因重组,将大肠杆菌粘附因子cs3基因定位整合到痢疾杆菌福氏2a疫苗株T32菌染色体的asd基因内,使asd基因灭活;将来内O抗原基因克隆至无抗药性表达载体pXL378,获得重组质粒pXL390,将其转化asd-的T32受体菌,构建成福氏2a和宋内双价苗苗株FS01。实验表明:重组质粒pXL390在不带任何抗菌素基因的情况下,在asd-的T32受体菌内是稳定的。FS01株遗传稳定,能表达两种痢疾菌的PLS-O抗原,无明显毒性作用。动物试验表明,以FS01株皮下免疫的小鼠对福氏2a和宋内有毒株的腹腔攻击有100％的保护。     

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.     

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

志贺菌福氏2a信号标签诱变突变体文库的构建

以合成的单链序列特异性标签为模板,通过PCR得到双链DNA标签并将其克隆到自杀质粒pUT-Tn5 Km2的转座子中,转化大肠杆菌S17-1λpir;然后用经转化的S17-1λpir与福氏志贺菌2a 2457T交配,挑出对氨苄青霉素敏感,对卡那霉素和萘啶酮酸抗性的菌落,结果表明构建了包含4376个福氏志贺菌突变体信号标签诱变库,为进一步鉴定该病原体的毒力基因打下了基础。     

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.     

Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis

The nucleotide sequence of a small plasmid, designated pSFD10, is isolated from the vaccine strain Salmonella choleraesuis C500 in China, has been determined. This plasmid is 4091 bp long with a total G+C content of 51.4%, which is in the range of Salmonella genomic DNA. Analysis of the complete nucleotide sequence reveals that pSFD10 has a high degree of similarity to ColE1-type plasmid, having the possible cer and rom genes, and a putative mobilization origin of ColE1-type. Plasmid pSFD10 possesses six main open reading frames (ORFs), five of which have a very high degree of amino acid identity to ColE1-type plasmid gene products involved in mobilization and copy number control. The other ORF (ORF6) encodes a putative protein, which has 49% homology to the invasion plasmid antigen J protein (IpaJ) secreted by the type III secretion apparatus of Shigella flexneri. In addition, pSFD10 belongs to a different incompatibility group than ColE1-type and pMB1-type to which it is related. Plasmid pSFD10 can be mobilized by the plasmid RP4 in E. coli.     

Electron microscopic study of the interaction of virulent and avirulent Shigella flexneri strains with the macrophages of murine peritoneal exudate

The electron microscopic dynamic study of the interaction of S. flexneri virulent strain 2a and S. flexneri avirulent strain, genetically related to strain 2a and having the same antigenic structure, with peritoneal macrophages of CBA mice at an early period showed differences in the cytotoxic action of these strains. Even at the early period of the interaction between macrophages and virulent shigellae structural changes were observed in macrophages: ruptures in the membranes of parasitophore phagosomes, the vacuolization of the cytoplasm, the destruction of mitochondria. These changes were indicative of the early cytotoxic action of shigellae belonging to the virulent strain. In macrophages infected with avirulent shigellae the manifestations of this cytotoxicity were less pronounced and appeared later than in macrophages infected with S. flexneri virulent strain.     

Analysis of merodiploid strains of Sh. flexneri that have lost virulence]

Episome F'13 introduced into the genome of a virulent Sh. flexneri strain brought about changes in a number of properties of the recipient strain. The expression of these properties was not connected with the chromosome area allelic to the plasmid genome. These changes seem to be induced by the mobilization of the chromosome genes of E. coli. The loss of virulence in Sh. flexneri strains carrying episome F'13 seemed to be the consequence of two reasons: the overlapping of kcpA gene by its dominant avirulent allele and abnormal synthesis of cell wall lipopolysaccharide due to the transfer of the mobilized genes from the donor strain F'13. When the preliminary mapping of genes on the chromomome was made with the use of plasmids, it was found necessary to use F-episomes which had no influence on the changes occurring in the phenotypic characteristics of the recipient.     

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.