The Periplasmic Enzyme,AnsB, of Shigella flexneri Modulates Bacterial Adherence to Host Epithelial Cells

S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.     

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

Background

On most common microarray platforms many genes are represented by multiple probes. Although this is quite common no one has systematically explored the concordance between probes mapped to the same gene.

Results

Here we present an analysis of all the cases of multiple probe sets measuring the same gene on the Affymetrix U133a GeneChip and found that although in the majority of cases both measurements tend to agree there are a significant number of cases in which the two measurements differ from each other. In these cases the measurements can not be simply averaged but rather should be handled individually.

Conclusion

Our analysis allows us to provide a comprehensive list of the correlation between all pairs of probe sets that are mapped to the same gene and thus allows microarray users to sort out the cases that deserve further analysis. Comparison between the set of highly correlated pairs and the set of pairs that tend to differ from each other reveals potential factors that may affect it.     

Behavior of Coliphage Lambda in Shigella flexneri 2a

The insensitivity of wild-type Shigella flexneri 2a to coliphage lambda is a consequence of its native genetic defect in the malA gene cluster. The "smooth" S. flexneri 2a lipopolysaccharide layer affects the efficient adsorption of lambda. Derivatives, capable of serving as functional hosts for lambda, were obtained by repairing the malA lesion, enabling the expression of the malB-lambdarcp region of S. flexneri. Introduction of a mutation into S. flexneri causing a "rough" lipopolysaccharide character resulted in more efficient adsorption of lambda. Such S. flexneri hosts can be stably lysogenized and upon induction yield gal(+)-transducing lysates. Lambda propagated on a malA(+) rough S. flexneri host was restricted by Escherichia coli K-12 and E. coli B, but not by E. coli C. This S. flexneri host did not restrict lambda grown on these E. coli strains.     

痢疾福氏2a asd基因的克隆及其序列分析

本文根据大肠杆菌（Ｅ．ｃｏｌｉ）Ｋ１２ａｓｄ基因两侧序列设计了一对引物,用全菌ＰＣＲ扩增了福氏２ａ Ｔ３２株的ａｓｄ基因及其两侧序列。对ＰＣＲ产物的初步结果表明,在ａｓｄ基因两端存在ＢａｍＨ Ｉ位点。为了防止由ＰＣＲ扩增带来的差错,我们又从福氏２ａ Ｔ３２株染色体中克隆了全长的ａｓｄ基因。序列分析了结果表明,福氏２ａＴ３２株ａｓｄ基因的序列与Ｅ．ｃｏｌｉ Ｋ１２的完全一致,全长１６８０ｂｐ,其两侧     

福氏志贺氏2a及Y变种保护性抗原的研究

福氏志贺菌Y变种曾经作为一种痢疾疫苗的候选株,其特有的抗原结构在疫苗的有效性抗原研究中起主要作用。以Y变种毒株与无毒株、野生型F2a株与T32株及失去Ⅱ型抗原结构的T32-1株之间分别进行了各种毒力表型的检测、四种外膜侵袭蛋白表达、菌株的外膜蛋白提取物(OMPs)分析、质粒DNA图谱和小鼠主动免疫、被动保护试验的对比分析,了解其抗原特性。结果显示：细菌外膜蛋白抗原和具有完整型特异性抗原结构的福氏菌LPS在动物机体免疫中都发挥着重要的作用。这些抗原物质的共同存在似乎能达到更好的免疫效果。     

Contribution of the lipopolysaccharide to resistance of Shigella flexneri 2a to extreme acidity

Shigella flexneri is endemic in most underdeveloped countries, causing diarrheal disease and dysentery among young children. In order to reach its target site, the colon, Shigella must overcome the acid environment of the stomach. Shigella is able to persist in this stressful environment and, because of this ability it can initiate infection following the ingestion of very small inocula. Thus, acid resistance is considered an important virulence trait of this bacterium. It has been reported that moderate acid conditions regulate the expression of numerous components of the bacterial envelope. Because the lipopolysaccharide (LPS) is the major component of the bacterial surface, here we have addressed the role of LPS in acid resistance of S. flexneri 2a. Defined deletion mutants in genes encoding proteins involved in the synthesis, assembly and length regulation of the LPS O antigen were constructed and assayed for resistance to pH 2.5 after adaptation to pH 5.5. The results showed that a mutant lacking O antigen was significantly more sensitive to extreme acid conditions than the wild type. Not only the presence of polymerized O antigen, but also a particular polymer length (S-OAg) was required for acid resistance. Glucosylation of the O antigen also contributed to this property. In addition, a moderate acidic pH induced changes in the composition of the lipid A domain of LPS. The main modification was the addition of phosphoethanolamine to the 1' phosphate of lipid A. This modification increased resistance of S. flexneri to extreme acid conditions, provide that O antigen was produced. Overall, the results of this work point out to an important role of LPS in resistance of Shigella flexneri to acid stress.     

Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.     

Comprehensive proteomic analysis of Shigella flexneri 2a membrane proteins

Shigella flexneri is the causative agent of most shigellosis cases in developing countries. We used different proteolytic enzymes to selectively shave the protruding proteins on the surface of purified bacterial membrane sheets or vesicles, and recovered peptides were subsequently identified using 2-D LC-MS/MS. As a result, a total of 666 proteins were unambiguously assigned, including 159 integral membrane proteins, 35 outer membrane proteins and 114 proteins previously annotated as hypothetical. The former had an average grand average hydrophobicity score of 0.362 and were predicted to separate within a pH range of 4.1-10.6 with molecular mass 8-148 kDa, which represents the largest validated set of integral membrane proteins in this organism to date. A functional classification revealed that a large proportion of the identified proteins were involved in cell envelope biogenesis and energy production and conversion. For the first time, this work provides a global view of the S. flexneri 2a membrane subproteome.     

The Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both Ply^WT and the mutant Ply molecules (Ply^A370G, Ply^A370E, Ply^A406G, Ply^A406E, Ply^W433G, Ply^W433E, Ply^W433F, Ply^L460G, and Ply^L460E) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, Ply^WT localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (Ply^A370G) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (Ply^A370E, Ply^W433G, Ply^W433E, and Ply^L460E) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except Ply^A370G, exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.     

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。     

痢疾杆菌体内诱导基因筛选方法的建立

分别用氯霉素乙酰转移酶基因(cat)和天冬氨酸半醛脱氢酶基因(asd)作为报告基因,用小鼠肺感染模型和HeLa细胞侵袭模型来试验筛选痢疾杆菌体内诱导基因的可行性。结果表明,以asd为报告基因,用HeLa细胞侵袭试验作为筛选模型,可成功地用于筛选痢疾杆菌的体内诱导基因。     

Gene expression profiling of the pH response in Shigella flexneri 2a

The pH response of Shigella flexneri 2a 301 was identified by gene expression profiling. Gene expression profiles of cells grown in pH 4.5 or 8.6 were compared with the profiles of cells grown at pH 7.0. Differential expression was observed for 307 genes: 97 were acid up-regulated, 102 were acid down-regulated, 91 were base up-regulated, and 86 were base down-regulated. Twenty-seven genes were found to be both acid and base up-regulated, and 29 genes were both acid and base down-regulated. This study showed that (1) the most pH-dependent genes regulate energy metabolism; (2) the RpoS-dependent acid-resistance system is induced, while the glutamate-dependent acid resistance system is not; (3) high pH up-regulates some virulence genes, while low pH down-regulates them, consistent with Shigella infection of the low gut; and (4) several cross-stress response genes are induced by pH changes. These results also illustrate that many unknown genes are significantly regulated under acid or basic conditions, providing researchers with important information to characterize their function.     

肠毒素大肠杆菌CS3定居因子抗原和融合肠毒素基因在减毒痢疾杆菌中的共表达

肠毒素和定居因子抗原 (CFAs)是肠毒素大肠杆菌 (ETEC)两种主要的致病因素。有效的ETEC疫苗应包括这两种成分。采用基因重组技术 ,将定居因子CFA/II的共有抗原成分CS3菌毛抗原和LT B/ST融合肠毒素基因克隆至以asd基因为选择标记的重组质粒上 ,与asd基因剔除的弗氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统。结果表明 ,在无抗生素条件选择的情况下 ,该重组菌可稳定表达CS3菌毛抗原和LT B/ST融合肠毒素抗原。通过口服和鼻饲方式免疫小鼠 ,可诱生相应的血清IgG抗体 ,同时能够检测到分泌型IgA的产生 ,表明该重组菌可以有效的诱导产生粘膜免疫。     

Syntaxin 16 Binds to Cystic Fibrosis Transmembrane Conductance Regulator and Regulates Its Membrane Trafficking in Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a key membrane protein in the complex network of epithelial ion transporters regulating epithelial permeability. Syntaxins are one of the major determinants in the intracellular trafficking and membrane targeting of secretory proteins. In the present study we demonstrate the biochemical and functional association between CFTR and syntaxin 16 (STX16) that mediates vesicle transport within the early/late endosomes and trans-Golgi network. Immunoprecipitation experiments in rat colon and T84 human colonic epithelial cells indicate that STX16 associates with CFTR. Further analyses using the domain-specific pulldown assay reveal that the helix domain of STX16 directly interacts with the N-terminal region of CFTR. Immunostainings in rat colon and T84 cells show that CFTR and STX16 highly co-localize at the apical and subapical regions of epithelial cells. Interestingly, CFTR-associated chloride current was reduced by the knockdown of STX16 expression in T84 cells. Surface biotinylation and recycling assays indicate that the reduction in CFTR chloride current is due to decreased CFTR expression on the plasma membrane. These results suggest that STX16 mediates recycling of CFTR and constitutes an important component of CFTR trafficking machinery in intestinal epithelial cells.