致肾盂肾炎大肠杆菌的毒力因子和调控

致肾盂肾炎大肠杆菌引起人的尿路感染,它的毒力因子包括表面毒力因子和分泌毒力因子两大类。表面毒力因子包括菌毛、鞭毛、黏附素和多糖类物质,主要在细菌的侵染过程中起作用。分泌毒力因子主要是溶血素、细胞毒性坏死因子等毒素蛋白,主要对宿主细胞产生毒力作用。本文简要综述致肾盂肾炎大肠杆菌毒力因子分泌所需要的5种分泌机制,并论及毒力因子的宏观调控和影响毒力调控的因素。     

Assessment of the Significance of Virulence Factors of Uropathogenic Escherichia coli in Experimental Urinary Tract Infection in Mice

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype O2:H^? and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD₅₀ and ID₅₀ of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.     

Adhesins of escherichia coli

E. coli has got increasing importance as a causative agent of intestinal and extra-intestinal diseases. In both these infections adhesion of the bacteria to mucous surface cells are initial events for coionization and development of infection. Adhesins are bacterial recognition proteins which specifically interact with carbohydrate moieties of glycoproteins or glycolipids on mammalian cells. The adhesiveness of bacteria is associated with filamentous surface appendages, designated as fimbriae or pili, as well as with non-fimbrial components. Some recent data on the nomenclature, classification, disease association, receptor specificity, and topographic arrangement are presented. The correlation between E. coli O : K : H serovar and fimbrial antigens is demonstrated on the basis of E. coli isolated from patients with urinary tract infections. Hitherto unknown non-fimbrial adhesins are briefly described.     

Heterobinary Adhesins Based on the Escherichia coli FimH Fimbrial Protein

The FimH adhesin of Escherichia coli type 1 fimbriae confers the ability to bind to d-mannosides by virtue of a receptor-binding domain located in its N-terminal region. This protein was engineered into a heterobifunctional adhesin by introducing a secondary binding site in the C-terminal region. The insertion of histidine clusters into this site resulted in coordination of various metal ions by recombinant cells expressing chimeric FimH proteins. In addition, libraries consisting of random peptide sequences inserted into the FimH display system and screened by a “panning” technique were used to identify specific sequences conferring the ability to adhere to Ni²⁺ and Cu²⁺. Recombinant cells expressing heterobifunctional FimH adhesins could adhere simultaneously to both metals and saccharides. Finally, combining the metal-binding modifications with alterations in the natural receptor-binding region demonstrated the ability to independently modulate the binding of FimH to two ligands simultaneously.

Expression systems for the display of heterologous protein segments facilitate the presentation of both defined and random peptide sequences at exposed regions of surface proteins of filamentous bacteriophage virions, bacteria, and yeasts (2, 4). We are particularly interested in the display of heterologous peptides in type 1 fimbriae. Such surface organelles are found on the majority of Escherichia coli strains and confer the ability to bind to specific surfaces. A single type 1 fimbria is a heteropolymer that is 7 nm wide and approximately 1 μm long. It consists of approximately 1,000 subunits of the major element, FimA, that are polymerized in a right-handed helical structure that also contains low levels of the minor components FimF, FimG, and FimH (9). The FimH protein has been shown to be the actual receptor-binding molecule which recognizes α-d-mannose-containing structures (10). Because of this, type 1 fimbriated bacteria readily agglutinate yeast cells (a rich source of mannan).The FimH adhesin is located at the tip of each fimbria and also is interspersed along the fimbrial shaft (6, 10). The results of linker insertion mutagenesis (16) and analyses of naturally occurring variants (17–19) and hybrid proteins constructed by fusing FimH to FocH (8) and MalE (21) suggest that the FimH protein consists of two major domains, each constituting roughly one-half of the molecule; the N-terminal domain seems to contain the receptor-binding site, while the C-terminal domain seems to contain the recognition sequences for export and bioassembly.In previous studies we investigated the ability of FimH to display heterologous peptides in connection with the development of vaccine systems. Various heterologous sequences, representing immune-relevant sectors of foreign proteins, were authentically displayed on the bacterial surface in FimH (12). These observations led us to believe that the FimH protein is an ideal candidate for display of random peptide sequences and for construction of designer adhesins (i.e., proteins manipulated to bind to targets of choice). Here we describe simultaneous heterobifunctional binding of recombinant cells expressing chimeric FimH proteins to metal and d-mannose targets.     

Distribution of Virulence Factors in Escherichia coli Isolated from Urine of Cystitis Patients

The distribution of 7 urovirulence factors, such as type 1 pilus (pil), pilus associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin I (afaI), hemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnf1) was examined by a DNA colony hybridization test among 194 Escherichia coli strains isolated from the urine of cystitis patients and in 80 strains isolated from the stool specimens of healthy adults. All virulence factors examined, except pil, were significantly more frequently detected among the cystitis isolates than among the fecal isolates. When individual virulence factors were analyzed against the others, an association was discernible which was not apparent when all 7 virulence factors were considered collectively. There was an apparent correlation between the genotypes and serotypes of the E. coli strains from the cystitis patients. From the data presented, it was proposed that genetic detection of virulence factors would be useful for rapid diagnosis of cystitis, especially in patients without severe pyuria or bacteriuria.     

华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子

从江苏、江西、安徽等7个省疑似黄、白痢直肠棉拭及病死猪的十二指肠和肠系膜淋巴结中分离鉴定出339株病原性大肠杆菌。经O血清型鉴定,除77株未能定型、41株自凝外,测定出221个分离株的O血清型,这些分离株覆盖了64个血清型,以O107、O101、O20、O93、O11和O149为主,共99株,占定型菌株的44.80%。这些血清型与已报道的常见血清型间存在一定差异。运用黏附素单抗对以上菌株进行F4、F5、F6、F18、F41　5种黏附素检测,共97个分离株表达黏附素(28061%),而表达两种和3种黏附素的菌株分别有22株和8株,它们分别占表达黏附素菌株的22.68%和8.25%,其中单独表达F4、F6、F5+F41黏附素菌株分别有18、30、15株,分别占表达黏附素菌株的18.56%、30.93%和15.46%;同时运用多重PCR对其中145个分离株进行毒素基因(Sta、STb、LT、SLT2e)的检测,拥有Sta和STb毒素基因的菌株分别占检测菌株的51.72%和3724%。F6、F4、F5+F41和Sta、STb为该地区致初生仔猪腹泻大肠杆菌常见的毒力因子。     

Virulence of Escherichia coli Serotypes for Mice

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.     

Regulation of Virulence Factors of Enterohemorrhagic Escherichia coli O157:H7 by Self-Produced Extracellular Factors

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans. The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase. Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase. Inhibition of EspD and intimin expression was also induced by medium conditioned by E. coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in Gram-negative bacteria. These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.     

Pathogenomics of the Virulence Plasmids of Escherichia coli

Summary: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. coli virulence plasmids exist, including those essential for the virulence of enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and extraintestinal pathogenic E. coli. Despite their diversity, these plasmids belong to a few plasmid backbones that present themselves in a conserved and syntenic manner. Thanks to some recent research, including sequence analysis of several representative plasmid genomes and molecular pathogenesis studies, the evolution of these virulence plasmids and the implications of their acquisition by E. coli are now better understood and appreciated. Here, work involving each of the E. coli virulence plasmid types is summarized, with the available plasmid genomic sequences for several E. coli pathotypes being compared in an effort to understand the evolution of these plasmid types and define their core and accessory components.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

Tir细胞骨架偶联蛋白:大肠杆菌O157：H7的一种新的毒力因子

肠出血性大肠杆菌O157:H7是一种重要的传染性病原菌,可引起多种致死性疾病的爆发流行。Tir细胞骨架偶联蛋白(TccP)是近年来发现的O157:H7的一种新的重要的毒力因子,在O157:H7黏附宿主细胞造成黏附擦拭(A/E)损伤过程中起重要作用,TccP相关研究对阐明O157:H7致病机制具有重要意义。     

Sensitivity of an Immunomagnetic-Separation-Based Test for Detecting Escherichia coli O26 in Bovine Feces

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED₈₀) was 1.0 × 10⁴ CFU g⁻¹ feces (95% confidence interval, 4.7 × 10³ to 2.4 × 10⁴). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED₈₀ for each strain and fecal pat combination ranged from 4.2 × 10² to 4.8 × 10⁵ CFU g⁻¹. These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

Genomic Comparative Study of Bovine Mastitis Escherichia coli

Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.