EAggEC中小肠结肠炎耶尔森菌强毒力岛结构和功能研究

目的了解小肠结肠炎耶尔森菌（Yersinia enterocolitica,Yen）强毒力岛在肠聚集性大肠杆菌（EAggEC）中的结构和摄铁功能，为进一步研究细菌毒力的进化奠定理论基础。方法PCR扩增、原位杂交及SDS-PAGE。结果在检测的6株EAggEC有5株检出了毒力岛，而且这些阳性菌株中的毒力岛都位于asntRNA（天门冬氨酸tRNA）位点；在缺铁条件下，EAggRC能够表达和耶尔森菌相同的摄铁蛋白HMWP1和HMWP2。结论小肠结肠炎耶尔森菌强毒力岛在肠聚集性大肠杆菌中有阳性率较高的分布，并具有相同的摄铁功能，很可能EAggRC和耶尔森菌中的强毒力岛有相同的转移机制。     

致泻性大肠杆菌中发现小肠结肠炎耶尔森菌毒力岛

目的　研究肠产志贺样毒素且具侵袭力的大肠杆菌 (ESIEC)菌株是否含有耶尔森菌的HPI(毒力岛 )基因。方法　采用PCR扩增和Southern杂交的方法。结果　从 35 %的ESIEC菌的染色体上同时扩出irp1、irp2和fyuA 3个片段 ,片段大小分别与小肠结肠炎耶尔森菌WA菌株的相应片段一致 ,鼠疫耶尔森菌HPI上的 6对引物均未扩出目的片段。 6 5 %的ESIEC使用以上 9对引物未扩出目的片段。ESIEC菌的染色体EcoRI酶切产物电泳后与小肠结肠炎耶尔森菌WA菌株的fyuA探针杂交出大小一致的条带 ,与irp1和irp2探针杂交出不同的带型。结论　ESIEC菌含有小肠结肠炎耶尔森菌的HPI,且有变异现象。ESIEC和EAEC的关系还有待于进一步的研究     

我国stx-大肠埃希菌eae基因分型的研究

目的：了解我国大肠埃希菌LEE毒力岛eae基因的分布流行病学特征。方法：对从浙江省分离的20株携LEE毒力岛大肠埃希菌株的eae基因3ˊ端部分以PCR法和限制性酶切法进行分型，未能分型的进行核酸序列测定，确定型别；对LEE毒力岛在染色体上的插入位点进行鉴定，对菌株进行ERIC-PCR分型。结果：20株大肠埃希菌的LEE毒力岛的eae基因以β型为主（占45.00%），用限制酶切β型可进一步分为二个亚型，γ型eae与EHEC O157：H7 933株的γ型eae酶切图谱差异较大。对未能用PCR法分型的3株eae阳性大肠埃希菌菌株，其推定的intimin C端氨基酸序列分析结果表明，96-1株为γ2型，C130-1株为ε型的新亚型（C端序列与ε型最相近，一致性为83.39%），97-3株C端序列与α型最相近（一致性为58.06%），将其定为新的λ型。另外发现97-3株中，1个IS200变种插入到eae和escD基因间隔区。9株菌的LEE插入位点为selC，其中2株同时还存在完整的selC位点；另11株可能存在着除selC和pheU以外的插入位点。几乎所有20株菌间的ERIC-PCR指纹呈现不同程度的差异，同一型的eae可出现在指纹相差很大的菌株中，但同时也存在着少量的携同型eae的同一克隆群菌株。结论：我国分离的stx^-大肠埃希菌eae基因在3ˊ端有较高的多态性，eae基因型别并不能反映菌株间亲缘关系，可能存在有新的LEE毒力岛插入位点。     

志贺菌毒力岛的结构和功能

志贺菌毒力岛对了解细菌的致病性具有重要意义。痢疾岛的序列和开放读码框架、多数痢疾岛的一侧或两侧均伴有插入序列元件、转座子或者tRNAs。许多毒力岛都与短DRs侧链相连,志贺菌PAI依赖int基因通过位点特异性重组、切除和整合。不同的是志贺氏菌毒力岛没有连接传递功能的基因或抗菌素的外膜蛋白。     

大肠杆菌O157：H7 OI115岛的多态性分析

目的研究大肠杆菌0157：H7测序菌株EDL933的OI115岛在肠杆菌科细菌中的分布。方法用PCR和杂交方法检测OI115岛的21个基因，同时进行该岛序列测定。结果OI115岛和SPI-1毒力岛的分布特点相似，具有种属局限性。仅在大肠杆菌中存在，在EPEC、CTEC、EAggEC均可检测到该岛的缺失形式，在沙门菌、志贺菌、耶尔森、摩根菌等其他肠杆菌科细菌中未发现该岛的存在。在不同类别的致泻性大肠杆菌中，Oi115岛也各有特点，共检测到该岛的3种存在形式。产生志贺毒素的大肠杆菌O157：H7菌株、2株EAggEC和2株不典型大肠杆菌均具有完整的OI115岛。在不产生志贺毒素的大肠杆菌O157中具有H5、H12、H29、H42、H11、H19、H26、H10、H21鞭毛型的菌株缺失了第3基因簇，具有H16鞭毛型的菌株第2、第3基因簇同时缺失。结论本研究发现，OI115岛在致泻性大肠杆菌中分布广泛，可能和病原菌的进化有关。     

霍乱弧菌毒力表达调控基因toxR缺失株的构建及其功能的研究

目的　通过构建霍乱弧菌toxR基因缺失株来研究toxR基因对霍乱弧菌减毒菌株IEM101和高产毒株569B毒力表达的调控作用。方法　采用自杀性质粒和接合转移技术，将2个中间含有四环素基因的toxR基因分别与霍乱弧菌减毒株IEM101和高产毒株569B染色体toxR基因重组，从而获得toxR基因缺失株IEM101-4和569B-43，并对2个toxR基因缺失株和其原出发菌株的霍乱肠毒素的产率和主要外膜蛋白图谱进行比较。结果　采用GM1-ELISA检测受测菌CT基因表达，toxR基因缺失株569B-43的P/N值为1.82，而其原出发菌株569B的P/N为4.52，而IEM101和其toxR基因缺失株的P/N值均低于2。采用SDS-PAGE对受试菌外膜蛋白进行分析，toxR基因缺失株569B和IEM101的外膜蛋白图谱相比，均多出2条相对分子质量（Mr）为40×103和43×103外膜蛋白区带。结论　toxR蛋白是霍乱肠毒素基因ctx表达的正调控因子，是霍乱弧菌主要外膜蛋白（Mr为40×103和43×103)编码基因的负调控因子。     

携耶尔森菌HPI大肠埃希菌的致泻性调查

ＨＰＩ(ｈｉｇｈ ｐａｔｈｏｇｅｎｉｃｉｔｙｉｓｌａｎｄ)为高致病力群耶尔森菌 (包括鼠疫耶尔森菌、假结核耶尔森菌和小肠结肠炎耶尔森菌生物群 1Ｂ)中发现的一种毒力岛 ,介导铁载体耶尔森杆菌素 (ｓｉｄｅｒｏｐｈｏｒｅｙｅｒｓｉｎｉａｂａｃｔｉｎ)的生物合成和摄取 ,为菌株致小鼠死亡所必需。近年来国内外学者在许多大肠埃希菌分离菌株中先后也发现了此ＨＰＩ〔1,2〕,然而 ,目前尚不明了大肠埃希菌中检出的ＨＰＩ是否与菌株的致病性有关。我们对分离自杭州各类人群的大肠埃希菌菌株 ,应用菌落原位杂交技术和ＰＣＲ技术检测ＨＰＩ的…     

大肠杆菌O157∶H7 OI115岛的多态性分析

目的研究大肠杆菌O157∶H7测序菌株EDL933的OI115岛在肠杆菌科细菌中的分布。方法用PCR和杂交方法检测OI115岛的21个基因,同时进行该岛序列测定。结果OI115岛和SPI-1毒力岛的分布特点相似,具有种属局限性。仅在大肠杆菌中存在,在EPEC、ETEC、EAggEC均可检测到该岛的缺失形式,在沙门菌、志贺菌、耶尔森、摩根菌等其他肠杆菌科细菌中未发现该岛的存在。在不同类别的致泻性大肠杆菌中,OI115岛也各有特点,共检测到该岛的3种存在形式。产生志贺毒素的大肠杆菌O157∶H7菌株、2株EAggEC和2株不典型大肠杆菌均具有完整的OI115岛。在不产生志贺毒素的大肠杆菌O157中具有H5、H12、H29、H42、H11、H19、H26、H10、H21鞭毛型的菌株缺失了第3基因簇,具有H16鞭毛型的菌株第2、第3基因簇同时缺失。结论本研究发现,OI115岛在致泻性大肠杆菌中分布广泛,可能和病原菌的进化有关。     

毒力岛和细菌毒力的进化

近年来,在医学细菌学领域出现了一个新名词——毒力岛（Ｐａｔｈｏｇｅｎｉｃｉｔｙｉｓｌａｎｄ）〔１〕。毒力岛的发现和研究对了解细菌致病性和毒力因子具有重要的意义。毒力岛最早是用来描述泌尿道致病性大肠杆菌的两个相对分子质量很大的、编码许多毒力相关基因的、...     

Transfer of the core region genes of the Yersinia enterocolitica WA-C serotype O:8 high-pathogenicity island to Y. enterocolitica MRS40, a strain with low levels of pathogenicity, confers a yersiniabactin biosynthesis phenotype and enhanced mouse virulence

The high-pathogenicity island (HPI) of yersiniae encodes an iron uptake system represented by its siderophore yersiniabactin (Ybt). The HPI is present in yersiniae with high levels of pathogenicity--i.e., Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica biogroup (BG) 1B--but absent in Y. enterocolitica strains with low (BG 2 to 5) and no (BG 1A) levels of pathogenicity and has been shown to be an important virulence factor. Comparison of the HPI in Y. enterocolitica (Yen-HPI) and that in Y. pestis and Y. pseudotuberculosis revealed that, in contrast to genes of the variable region, genes of the core region (genes irp9 to fyuA) are highly homologous. In the present work the Yen-HPI core genes were rescued from the chromosome of Y. enterocolitica WA-C (BG 1B, serotype O:8) using the FRT-FLP recombinase system. Transfer of the resulting plasmid pCP1 into the siderophore-deficient strain Y. enterocolitica NF-O (BG 1A) led to no halo on siderophore indicator chrome azurol S (CAS) agar. Transfer of pCP1 into the Y. enterocolitica strain MRS40 (serotype O:9, BG 2; phenotype, CAS negative) led to a CAS halo larger than that of parental strain WA-C, indicating high Ybt production. pCP1 was highly unstable in iron-deficient medium, and no enhanced mouse virulence conferred by MRS40 carrying pCP1 could be detected. To overcome the problem of instability, pCP1 was integrated into the chromosome of MRS40, leading to the formation of a CAS halo comparable to that seen with WA-C and correspondingly to increased mouse virulence. Thus, the core genes of Yen-HPI are sufficient to confer a positive CAS phenotype and mouse virulence to Y. enterocolitica MRS40, BG 2, but are insufficient to confer this phenotype to Y. enterocolitica NF-O, BG 1A.     

Yersiniabactin production requires the thioesterase domain of HMWP2 and YbtD,a putative phosphopantetheinylate transferase

One requirement for the pathogenesis of Yersinia pestis, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the pgm locus. We have identified a gene, ybtD, encoded outside the HPI and pgm locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of Escherichia coli. A deletion within ybtD yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from Ybt-producing strains of Y. pestis. The promoter region of ybtD was fused to lacZ; beta-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the ybt system. The ybtD mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other ybt biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any ybt regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.     

Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis.

We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.     

The Yersinia high-pathogenicity island (HPI): evolutionary and functional aspects

The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.     

The Yersinia high pathogenicity island is present in Salmonella enterica Subspecies I isolated from turkeys

The Yersinia high pathogenicity island (HPI) encodes a yersiniabactin-mediated iron acquisition system present in highly pathogenic strains of Yersinia and several members of the Enterobacteriaceae. In this study, 420 salmonellae representing multiple serovars recovered from diverse hosts were investigated for the presence of the HPI. The isolates were initially screened via PCR with primers specific for irp2, a conserved gene involved in yersiniabactin biosynthesis. Seventeen isolates produced an amplicon of the expected size. These isolates were further investigated using PCR primers spanning the HPI core region and all isolates produced identical results. HPI-positive isolates were recovered only from turkeys (feces) or production samples (feed and water) from a single flock and identified as Salmonella enterica serovar Senftenberg, a Subspecies I. Southern hybridization and genome sequencing of isolate 3-70-31 revealed that the island is plasmid-borne and is 92-97% identical to the core region in Yersinia species. To our knowledge, this is the first report of the HPI in Salmonella Subspecies I. This study illustrates the presence of the HPI in a new species, and the continued importance of poultry as a reservoir and vehicle for the dissemination of zoonotic pathogens.     

Yersinia high-pathogenicity island contributes to virulence in Escherichia coli causing extraintestinal infections

The Yersinia high-pathogenicity island (HPI) encodes an iron uptake system mediated by the siderophore yersiniabactin (Ybt) and confers the virulence of highly pathogenic Yersinia species. This HPI is also widely distributed in human pathogenic members of the family of Enterobacteriaceae, above all in extraintestinal pathogenic Escherichia coli (ExPEC). In the present study we demonstrate a highly significant correlation of a functional HPI and extraintestinal virulence in E. coli. Moreover, using a mouse infection model, we show for the first time that the HPI contributes to the virulence of ExPEC.     

Identification of an orphan response regulator required for the virulence of Francisella spp. and transcription of pathogenicity island genes

Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 10(8) CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10(6) CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.     

The high-pathogenicity island of Yersinia enterocolitica Ye8081 undergoes low-frequency deletion but not precise excision, suggesting recent stabilization in the genome.

Highly pathogenic strains of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are characterized by the possession of a pathogenicity island designated the high-pathogenicity island (HPI). This 35- to 45-kb island carries an iron uptake system named the yersiniabactin locus. While the HPIs of Y. pestis and Y. pseudotuberculosis are subject to high-frequency spontaneous deletion from the chromosome, we were initially unable to obtain HPI-deleted Y. enterocolitica 1B isolates. In the present study, using a positive selection strategy, we identified three HPI-deleted mutants of Y. enterocolitica strain Ye8081. In these three independent clones, the chromosomal deletion was not limited to the HPI but encompassed a larger DNA fragment of approximately 140 kb. Loss of this fragment, which occurred at a frequency of approximately 5 x 10(-7), resulted in the disappearance of several phenotypic traits, such as growth in a minimal medium, hydrolysis of o-nitrophenyl-beta-D-thiogalactopyranoside, Tween esterase activity, and motility, and in a decreased virulence for mice. However, no precise excision of the Ye8081 HPI was observed. To gain more insight into the molecular basis for this phenomenon, the putative machinery of HPI excision in Y. enterocolitica was analyzed and compared to that in Y. pseudotuberculosis. We show that the probable reasons for failure of precise excision of the HPI of Y. enterocolitica Ye8081 are (i) the interruption of the P4-like integrase gene located close to its right-hand boundary by a premature stop codon and (ii) lack of conservation of 17-bp att-like sequences at both extremities of the HPI. These mutations may represent a process of HPI stabilization in the species Y. enterocolitica.