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1.
An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus. 相似文献
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IuM Romanova MIu Kirillov AA Terekhov AL Gintsburg 《Canadian Metallurgical Quarterly》1996,32(9):1184-1190
Mutants of Salmonella typhimurium with an impaired process of transition to the nonculturable state were tainted. Mutants were divided into four phenotypic groups. In four mutants (representatives of each phenotypic group), genes with TnPhoA transposon insertions were cloned; these insertions caused a disturbance in the process of mutant cell transition to the nonculturable state. Nucleotide sequences of mutant gene fragments were determined. Comparison of nucleotide sequences obtained with a data bank on DNA nucleotide sequences of enterobacterial genomes allowed the identification of four genes involved in the control of nonculturable form generation in salmonellae. 相似文献
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A Polissi A Pontiggia G Feger M Altieri H Mottl L Ferrari D Simon 《Canadian Metallurgical Quarterly》1998,66(12):5620-5629
Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia. 相似文献
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Salmonella enterica serovar Typhi (hereafter referred to as S. typhi) is a host-restricted pathogen that adheres to and invades the distal ileum and subsequently disseminates to cause typhoid fever in humans. However, S. typhi appears to be avirulent in small animals. In contrast, other pathogenic salmonellae, such as S. enterica serovars Typhimurium and Dublin (S. typhimurium and S. dublin, respectively), typically cause localized gastroenteritis in humans but have been used as models for typhoid fever because these organisms cause a disease in susceptible rodents that resembles human typhoid. In vivo, S. typhi has been demonstrated to attach to and invade murine M cells but is rapidly cleared from the Peyer's patches without destruction of the M cells. In contrast, invasion of M cells by S. typhimurium is accompanied by destruction of these M cells and subsequently sloughing of the epithelium. These data have furthered our view that the early steps in the pathogenesis of typhoidal and nontyphoidal Salmonella serovars are distinct. To extend this concept, we have utilized an in vitro model to evaluate three parameters of initial host-pathogen interactions: adherence of three Salmonella serovars to human and murine small intestinal epithelial cell (IEC) lines, the capacity of these salmonellae to invade IECs, and the ability of the bacteria to induce interleukin-6 (IL-6) in these cell lines as a measure of host cell activation and the host acute-phase response. The results demonstrate that S. typhi adheres to and invades human small IECs better than either S. typhimurium or S. dublin. Interestingly, invA and invE null mutants of S. typhi are able neither to adhere to nor to invade IECs, unlike S. typhimurium invA and invE mutants, which adhere to but cannot invade IECs. S. typhi also induces significantly greater quantities of IL-6 in human small IEC lines than either of the other two Salmonella serovars. These findings suggest that differential host cytokine responses to bacterial pathogens may play an important role in the pathological sequelae that follow infection. Importantly, S. typhimurium did not induce IL-6 in murine IECs. Since S. typhimurium infection in mice is often used as a model of typhoid fever, these findings suggest that, at least in this case, the mouse model does not reflect the human disease. Taken together, our studies indicate that (i) marked differences occur in the initial steps of S. typhi, S. typhimurium, and S. dublin pathogenesis, and (ii) conclusions about S. typhi pathogenesis that have been drawn from the mouse model of typhoid fever should be interpreted conservatively. 相似文献
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Salmonella enterica poxA mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivity to the herbicide sulfometuron methyl, alpha-ketobutyrate, and amino acid analogs. These mutants also failed to grow in the presence of the host antimicrobial peptide, protamine. In this study, PoxA- mutants of S. enterica serovar Typhimurium (S. typhimurium) were found to be 10,000-fold attenuated in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperitoneally inoculated BALB/c mice, compared to wild-type S. typhimurium UK-1. In addition, poxA mutants were found to be capable of colonizing the spleen, mesenteric lymph nodes, and Peyer's patches; to induce strong humoral immune responses; and to protect mice against a lethal wild-type Salmonella challenge. A 2-kb DNA fragment was isolated from wild-type S. typhimurium UK-1 based on its ability to complement an isogenic poxA mutant. The nucleotide sequence of this DNA fragment revealed an open reading frame of 325 amino acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated sequence to the available databases indicated high homology to a family of lysyl-tRNA synthetases. Our results indicate that a mutation of poxA has an attenuating effect on Salmonella virulence. Further, poxA mutants are immunogenic and could be useful in designing live vaccines with a variety of bacterial species. To our knowledge, this is the first report on the effect of poxA mutation on bacterial virulence. 相似文献
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AW van der Velden AJ B?umler RM Tsolis F Heffron 《Canadian Metallurgical Quarterly》1998,66(6):2803-2808
Adhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the Salmonella typhimurium fimbrial operons lpf, pef, or fim only moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or S. typhimurium may encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of S. typhimurium: fim, lpf, pef, and agf. A mutation in the agfB gene resulted in a threefold increase in the oral 50% lethal dose (LD50) of S. typhimurium for mice. In contrast, an S. typhimurium strain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD50. The quadruple mutant, but not the agfB mutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever. 相似文献
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Salmonella typhimurium zwf mutants lacking glucose 6-phosphate dehydrogenase (G6PD) activity have increased susceptibility to reactive oxygen and nitrogen intermediates as well as attenuated virulence in mice. Abrogation of the phagocyte respiratory burst oxidase during experimental infection with zwf mutant Salmonella causes a prompt restoration of virulence, while inhibition of inducible nitric oxide synthase results in delayed lethality. These observations suggest that G6PD-dependent bacterial antioxidant defenses play an important pathogenic role during early salmonellosis and additionally may help to antagonize NO-dependent antimicrobial mechanisms later in the course of infection. 相似文献
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S Sukupolvi A Edelstein M Rhen SJ Normark JD Pfeifer 《Canadian Metallurgical Quarterly》1997,65(2):838-842
The invasive disease caused by Salmonella typhimurium in mice resembles the acute phase of human typhoid fever caused by Salmonella typhi, and experimental murine salmonellosis is a widely used experimental model for systemic salmonellosis. In this paper we demonstrate that murine S. typhimurium infection can also be used to model the development of the chronic carrier state that develops in humans after infection with S. typhi. We describe a virulent variant of S. typhimurium that has decreased expression of AgfA fibers under all environmental conditions studied and that causes a chronic carrier state in BALB/c mice after peroral inoculation. The chronic carrier state is associated with persistence of bacteria in the small intestine, spleen, and liver, and chronic infection continues despite the development of protective immunity to challenge with virulent Salmonella. 相似文献
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It was previously reported that Salmonella typhimurium LT2 cob mutants defective in the biosynthesis of vitamin B12 (cobalamin) are more virulent than the wild type in mice. Here we show that the strains used previously are non-isogenic and that the proposed increase in virulence of the cob mutant strain results from an uncharacterized mutation in the "wild type" which attenuates virulence, most likely by decreasing expression of the spv genes on the virulence plasmid. As a result the cob mutant will appear as hyper-virulent. Examination of the virulence of reconstructed wild-type and cob mutant strains showed that their growth rates were similar in mice, and we conclude that vitamin B12 does not affect the virulence of S. typhimurium LT2. 相似文献
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S Pinker 《Canadian Metallurgical Quarterly》1998,66(3):199-213
Salmonella abortusovis strain Rv6 (Sao Rv6) is a live attenuated vaccine used for a few years to protect ewes against abortive salmonellosis. As Salmonellae, particularly Salmonella aro mutants, have considerable potential as vehicles for the presentation of heterologous vaccine antigens, Sao Rv6 was tested in order to develop a vaccinal vehicle for small ruminants. Five vector plasmids were tested in Sao Rv6; these plasmids, which carry Maltose Binding Protein (MBP) expressed as protein, but differ in their promotors, had been previously tested in S. typhimurium strain SL3261, and were transferred into Sao Rv6. The five plasmids were stable in vitro, and the recombinant Sao Rv6 expressed MBP at various levels. Intraperitoneal infection of OF1 mice with the recombinant bacteria did not modify the characteristics of Sao Rv6; dissemination and infection levels were similar in all groups and all mice developed antibodies to Salmonella antigens as measured by ELISA. In contrast, only animals immunized with Sao Rv6 carrying the pNTE plasmid developed a serum antibody response to MBP. This plasmid was then tested in sheep; following subcutaneous immunization with Sao Rv6-pNTE, dissemination and infection levels were not modified in comparison with sheep immunized with Sao Rv6 lacking plasmid. Antibodies specific to MBP were detected in sera of sheep immunized with Sao Rv6-pNTE, purified MBP, and with S. typhimurium SL3261-pNTE as positive controls. These results demonstrate that Sao Rv6 can be used as a vehicle for heterologous antigens in sheep with pNTE as plasmid vector. 相似文献
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A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences. 相似文献
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FJ Lacroix A Cloeckaert O Grépinet C Pinault MY Popoff H Waxin P Pardon 《Canadian Metallurgical Quarterly》1996,135(2-3):161-167
Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium. 相似文献
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The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies. 相似文献
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In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S. typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S. typhimurium. Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S. typhimurium. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems. 相似文献
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The starvation-stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C-source. A subset of SSR genes, known as core SSR genes, are required for the long-term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimurium: rpoS, stiA, stiB, and stiC. Here we report that in S. typhimurium C-starvation induced a greater and more sustainable cross-resistance to oxidative challenge (15 mM hydrogen peroxide (H2O2) for 40 min) than either N- or P-starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C-starvation-inducible cross-resistance to H2O2 challenge. Interestingly, (unadapted) log-phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H2O2 resistance developed during a 60 min adaptive exposure to 60 microM H2O2 (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H2O2 challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H2O2 challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild-type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C-starvation-inducible cross-resistance to oxidative challenge, and (ii) the rpoS, stiA, and, in a delayed effect, stiB loci are needed for H2O2-inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 microM H2O2 exposure, but only stiA was regulated (repressed) by (reduced form) OxyR. 相似文献