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1.
Infect Immun ; 69(9): 5619-25, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11500437

ABSTRACT

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.


Subject(s)
DNA-Binding Proteins/genetics , Flagella/metabolism , Mutation , Salmonella Infections, Animal/microbiology , Salmonella typhimurium/pathogenicity , Trans-Activators/genetics , Animals , Cattle , Cell Line , DNA-Binding Proteins/metabolism , Disease Models, Animal , Enterocolitis/microbiology , Escherichia coli Proteins , Female , Flagella/genetics , Flagellin/genetics , Flagellin/metabolism , Gene Expression Regulation, Bacterial/genetics , Humans , Ileum/immunology , Intestines/cytology , Macrophages, Peritoneal/microbiology , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Mice, Inbred C57BL , Salmonella typhimurium/genetics , Salmonella typhimurium/growth & development , Trans-Activators/metabolism , Virulence/genetics
2.
Infect Immun ; 69(5): 3021-30, 2001 May.
Article in English | MEDLINE | ID: mdl-11292720

ABSTRACT

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.


Subject(s)
Bacterial Proteins , Flagellin/genetics , Gastroenteritis/etiology , Salmonella typhimurium/pathogenicity , Typhoid Fever/etiology , Animals , Bacterial Adhesion , Cattle , Disease Models, Animal , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Virulence
3.
J Clin Invest ; 107(1): 99-109, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11134185

ABSTRACT

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.


Subject(s)
Flagellin/metabolism , Inflammation/etiology , Intestinal Mucosa/microbiology , Salmonella typhimurium/pathogenicity , Cell Line , Epithelium/immunology , Epithelium/microbiology , Flagellin/genetics , Humans , Inflammation/immunology , Inflammation/microbiology , Inflammation Mediators/metabolism , Interleukin-8/metabolism , Intestinal Mucosa/immunology , Models, Biological , Mutation , Salmonella typhimurium/genetics , Salmonella typhimurium/physiology
4.
Emerg Infect Dis ; 5(2): 224-34, 1999.
Article in English | MEDLINE | ID: mdl-10221874

ABSTRACT

Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize the beneficial uses of toxins and toxoids. Our intent is to illustrate the importance of the analysis of bacterial toxins to both basic and applied sciences.


Subject(s)
Bacterial Toxins/toxicity , Escherichia coli Proteins , Animals , Bacterial Toxins/immunology , Bacterial Toxins/pharmacology , Bacterial Vaccines/immunology , Botulinum Toxins/toxicity , Cytotoxins/toxicity , Enterotoxins/toxicity , Humans , Superantigens/pharmacology
6.
J Bacteriol ; 178(10): 2911-5, 1996 May.
Article in English | MEDLINE | ID: mdl-8631681

ABSTRACT

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.


Subject(s)
Bacterial Proteins/genetics , Flagellin/biosynthesis , Mutation , Repressor Proteins/genetics , Salmonella typhimurium/pathogenicity , Animals , Antigens, Bacterial/biosynthesis , Lethal Dose 50 , Mice , Phenotype , Salmonella typhimurium/genetics , Salmonella typhimurium/growth & development , Sigma Factor/genetics , Typhoid Fever/microbiology , Virulence/genetics
7.
FEMS Microbiol Lett ; 135(2-3): 281-5, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8595870

ABSTRACT

FlgM inhibits the flagella-specific sigma factor FliA and is involved in the mouse-virulence of Salmonella typhimurium. In recent experiments, we observed that: (i) a flgM gene that could function to negatively regulate flagella synthesis was present in a variety of salmonellae; and (ii) the flgM gene derived from Salmonella species that are not normally virulent in mice could complement the S. typhimurium flgM mutant for virulence. Our results suggest that a functional flgM has been retained in most, and perhaps all, Salmonella species, regardless of the motility or virulence phenotype of the strain.


Subject(s)
Bacterial Proteins/genetics , Flagella/genetics , Genes, Bacterial , Salmonella typhimurium/genetics , Salmonella/genetics , Animals , Cloning, Molecular , Female , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Mice , Mice, Inbred C57BL , Plasmids/genetics , Recombinant Fusion Proteins/biosynthesis , Salmonella typhimurium/pathogenicity , Sequence Analysis, DNA , Virulence
8.
Infect Immun ; 62(2): 623-31, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8300218

ABSTRACT

Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-like toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytotoxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related toxins have been described. Because EHEC O91:H21 strain B2F1, which produces two SLT-II-related toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD50], < 10 organisms), we asked whether the pathogenicity of strain B2F1 was a consequence of SLT-II-related toxin production. For this purpose, we compared the lethality of orally administered E. coli DH5 alpha (Strr) strains that produced different cytotoxic levels of SLT-II, SLT-IIvha (cloned from B2F1), SLT-IIvhb (also cloned from B2F1), or SLT-IIc (cloned from EHEC O157:H7 strain E32511) on Vero cells. We also calculated the specific activities of purified SLT-IIvhb and SLT-II in intraperitoneally injected mice and on Vero cells. The two purified toxins were equally toxic for mice, but SLT-IIvhb was approximately 100-fold less active than SLT-II on Vero cells and bound to the glycolipid receptor Gb3 with lower affinity than did SLT-II. In addition, characterization of SLT-II-related toxin-binding (B) subunit mutants generated in this study revealed that the reduced in vitro cytotoxic levels of the SLT-II-related toxins were due to Asn-16 in the B subunit. Taken together, these findings do not support the idea that B2F1 is uniquely virulent because of the in vivo toxicity of SLT-II-related toxins but do demonstrate differences in in vitro cytotoxic activity among the SLT-II group produced by human EHEC isolates.


Subject(s)
Bacterial Toxins/toxicity , Enterotoxins/toxicity , Escherichia coli/pathogenicity , Animals , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Base Sequence , Chlorocebus aethiops , DNA, Bacterial/genetics , Enterotoxins/genetics , Enterotoxins/metabolism , Escherichia coli/genetics , Escherichia coli Infections/etiology , Escherichia coli Infections/pathology , Gastrointestinal Hemorrhage/etiology , Glycolipids/metabolism , Kidney Cortex/pathology , Lethal Dose 50 , Male , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, Cell Surface/metabolism , Shiga Toxin 2 , Vero Cells
9.
J Bacteriol ; 176(2): 368-77, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8288531

ABSTRACT

Salmonella typhimurium ST39 exhibits reduced virulence in mice and decreased survival in mouse macrophages compared with the parent strain SL3201. Strain ST39 is nonmotile, carries an indeterminate deletion in and near the flgB operon, and is defective in the mviS (mouse virulence Salmonella) locus. In flagellum-defective strains, the flgM gene product of S. typhimurium negatively regulates flagellar genes by inhibiting the activity of FliA, the flagellin-specific sigma factor. In this study, flgM of wild-type S. typhimurium LT2 was found to complement the mviS defect in ST39 for virulence in mice and for enhanced survival in macrophages. Transduction of flgM::Tn10dCm into the parent strain SL3201 resulted in attenuation of mouse virulence and decreased survival in macrophages. However, a flgM-fliA double mutant was fully virulent in mice and survived in macrophages at wild-type levels. Thus, the absolute level of FliA activity appears to affect the virulence of S. typhimurium SL3201 in mice. DNA hybridization studies showed that flgM-related sequences were present in species other than Salmonella typhimurium and that sequences related to that of fliA were common among members of the family Enterobacteriaceae. Our results demonstrate that flgM and fliA, two genes previously shown to regulate flagellar operons, are also involved in the regulation of expression of virulence of S. typhimurium and that this system may not be unique to the genus Salmonella.


Subject(s)
Bacterial Proteins/genetics , Flagella/physiology , Gene Expression Regulation, Bacterial , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicity , Sigma Factor/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/biosynthesis , Bacteriophage T7/genetics , Base Sequence , Cell Survival , Genetic Complementation Test , Macrophages, Peritoneal/microbiology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Mutagenesis, Insertional , Phenotype , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/biosynthesis , Salmonella Infections, Animal/genetics , Sequence Homology, Amino Acid , Species Specificity , Virulence/genetics
10.
J Clin Microbiol ; 31(10): 2799-801, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8253989

ABSTRACT

We analyzed Escherichia coli O157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-like toxin profile, adherence and plasmid traits, mouse virulence, capsule, and enterohemolysin production. The profiles of the five isolates were indistinguishable from each other and similar to that of E. coli O157:H7 strain EDL933, an organism responsible for a similar hamburger-associated food poisoning episode in 1982.


Subject(s)
Disease Outbreaks , Escherichia coli/pathogenicity , Food Microbiology , Gastrointestinal Hemorrhage/epidemiology , Hemolytic-Uremic Syndrome/epidemiology , Meat/poisoning , Adolescent , Adult , Animals , Bacterial Toxins/analysis , Child , Female , Gastrointestinal Hemorrhage/etiology , Hemolytic-Uremic Syndrome/etiology , Humans , Male , Mice , Shiga Toxin 2 , Virulence , Washington
11.
J Bacteriol ; 173(20): 6507-14, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1917875

ABSTRACT

Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.


Subject(s)
Cell Membrane Permeability/genetics , Escherichia coli/metabolism , F Factor/genetics , Genes, Viral/genetics , T-Phages/genetics , Escherichia coli/genetics , Gene Expression/physiology , Nucleotides/analysis , Operon/genetics
12.
Infect Immun ; 59(3): 1065-73, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1997410

ABSTRACT

Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia coli O157:H- strain E32511. One copy of the operon was found to be essentially identical to slt-II; it differed from slt-II by only one nucleotide base. This single nucleotide difference did not affect the predicted amino acid sequence. The predicted amino acid sequence of the A subunit of the second operon was identical to that of SLT-II, but the predicted amino acid sequence of the B subunit was identical to that of the B2F1 toxin VT2ha. We designated this second operon slt-IIc. Neutralization assays using several monoclonal antibodies and polyclonal antiserum prepared against SLT-II showed that SLT-IIc was antigenically related to but distinct from SLT-II.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Toxins/genetics , Escherichia coli/genetics , Genes, Bacterial , Amino Acid Sequence , Animals , Bacterial Toxins/toxicity , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA, Bacterial , Escherichia coli/immunology , Escherichia coli/pathogenicity , HeLa Cells , Humans , Molecular Sequence Data , Multigene Family , Nucleic Acid Hybridization , Sequence Homology, Nucleic Acid , Shiga Toxin 2 , Species Specificity , Vero Cells
13.
J Bacteriol ; 173(4): 1536-43, 1991 Feb.
Article in English | MEDLINE | ID: mdl-1995595

ABSTRACT

Plasmids expressing bacteriophage T7 gene 1.2 or gene 10 DNA transform F plasmid-containing strains of Escherichia coli only at low efficiency, though they transform plasmid-free strains normally. The gene products T7 gp1.2 and T7 gp10 appear to be the toxic agents, and their effects are directed towards the product of the F pifA gene, PifA. T7 gp1.2 and gp10 are also the two targets of the pif exclusion system of F, and their synthesis normally triggers the abortive infection of T7 in pifA+ hosts. The properties of plasmids containing T7 gene 1.2 or 10 suggest that they can be used to study the molecular mechanisms of phage exclusion in model systems that avoid the pleiotropic dysfunctions associated with an abortive infection.


Subject(s)
Genes, Lethal , T-Phages/genetics , Viral Proteins/biosynthesis , Escherichia coli/genetics , Gene Expression , Plasmids , Transformation, Bacterial
14.
J Mol Biol ; 207(3): 563-74, 1989 Jun 05.
Article in English | MEDLINE | ID: mdl-2668536

ABSTRACT

Mutants of bacteriophage T7 that escape F restriction have been isolated. Two mutations in gene 10, which codes for the capsid protein, and one mutation in gene 1.2 are required for these phages to grow on F-containing strains. The products of these two genes are the two targets of the exclusion system; the presence of either wild-type product results in an abortive infection. Phages that grow normally in male hosts still lead to membrane dysfunction and nucleotide efflux from the infected cell. This type of membrane damage and the abortive infection are therefore separable phenomena.


Subject(s)
F Factor , T-Phages , DNA, Viral/genetics , DNA-Directed RNA Polymerases , Escherichia coli , Genes, Viral , Membranes , Mutation
15.
J Oral Surg ; 35(5): 351, 1977 May.
Article in English | MEDLINE | ID: mdl-265373
16.
J Oral Surg ; 34(5): 391, 1976 May.
Article in English | MEDLINE | ID: mdl-1063818
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