Biomedical subjects

S Falkow

Publications and source records attributed to S Falkow.

At least 145 records · Page 8Linked to original sources

PubMed1990

Invasion and replication of Salmonella typhimurium in animal cells.

A total of 81 avirulent Tn10 insertion mutants of Salmonella typhimurium have previously been described. These mutants were selected for the inability to survive in murine macrophages. We have characterized the abilities of the most avirulent of these mutants to adhere to, invade, and replicate in both macrophages and nonphagocytic epithelial cells. The results suggest that most mutants contain a defect that is specific to survival within professional phagocytes. These mutants invaded and replicated normally within nonphagocytic human colon adenocarcinoma cells (Caco-2) but did not survive in the macrophage cell line J774. One mutant invaded both macrophages and epithelial cells much less efficiently than the parental strain. The defect associated with this mutant appears to be a result of decreased adherence to animal cells.

Animals↗

PubMed1990

Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv-homologous sequences.

Previous studies have demonstrated a correlation between the ability of isolates of Yersinia enterocolitica to cause disease and to invade tissue culture cells in vitro. Two genes, inv and ail, isolated from a pathogenic strain of Y. enterocolitica have each been shown to confer this invasive phenotype upon Escherichia coli. Eighty pathogenic, invasive isolates studied by Miller et al. (Infect. Immun. 57:121-131, 1989) contained sequences homologous to both of these genes. Thirty-five nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. We investigated inv-homologous sequences from four nonpathogenic isolates. Recombinant clones of these inv-homologous sequences did not confer the invasive phenotype upon E. coli. No RNA transcripts capable of encoding a full-length Inv protein were detected in the four noninvasive Yersinia strains. When the inv gene from a pathogenic isolate was introduced into two of these strains, the resulting transformants invaded tissue culture cells in vitro. The inv gene was transcribed in a pathogenic Yersinia isolate grown at 30 degrees C but not at all in these cells grown at 37 degrees C. The production of RNA transcripts homologous to inv in transformants was not regulated by temperature to the same degree as was seen for pathogenic isolates. We conclude that the inv gene in nonpathogenic strains of Y. enterocolitica is nonfunctional. Y. enterocolitica isolates epidemiologically linked to disease contain both a functional inv gene and a functional ail gene. Environmental isolates not associated with disease have a nonfunctional inv gene and no ail gene.

Gene Expression Regulation, Bacterial↗

PubMed1990

Genetic analysis of Escherichia coli urease genes: evidence for two distinct loci.

Studies with two uropathogenic urease-producing Escherichia coli strains, 1021 and 1440, indicated that the urease genes of each are distinct. Recombinant plasmids encoding urease activity from E. coli 1021 and 1440 differed in their restriction endonuclease cleavage sites and showed minimal DNA hybridization under stringent conditions. The polypeptides encoded by the DNA fragments containing the 1021 and 1440 urease loci differed in electrophoretic mobility under reducing conditions. Regulation of urease gene expression differed in the two ureolytic E. coli. The E. coli 1021 locus is probably chromosomally encoded and has DNA homology to Klebsiella, Citrobacter, Enterobacter, and Serratia species and to about one-half of the urease-producing E. coli tested. The E. coli 1440 locus is plasmid encoded; plasmids with DNA homology to the 1440 locus probe were found in urease-producing Salmonella spp., Providencia stuartii, and two E. coli isolates. In addition, the 1440 urease probe was homologous to Proteus mirabilis DNA.

Blotting, Southern↗

PubMed1990

Nucleotide sequence of the Yersinia enterocolitica ail gene and characterization of the Ail protein product.

The ability to enter (invade) eucaryotic cells is a property common to many pathogenic bacteria. Yersinia enterocolitica is a facultative intracellular pathogen whose primary site of multiplication is the reticuloendothelial system. In an effort to understand how Y. enterocolitica crosses the intestinal epithelial cell layer, we previously reported the cloning of two loci from Y. enterocolitica that individually conferred an invasive phenotype to the normally noninvasive Escherichia coli HB101. One of these loci, ail, is encoded by a region of DNA that is less than 650 base pairs. We have identified the ail gene product in maxicells as a 17-kilodalton membrane-associated protein. The Ail protein has been purified, and its N-terminal sequence has been determined. The nucleotide sequence of the ail gene revealed a single unique open reading frame of 178 amino acids. Comparison of amino acid sequences deduced from the gene and obtained by analysis of the purified protein identified the first 23 amino acids as a signal sequence. The site(s) at which transcription initiates on the ail gene was identified by primer extension analysis and shown to be identical in E. coli and Y. enterocolitica. Two small open reading frames downstream of ail were found and shown to exhibit considerable identity to the proposed IS3 transposase.

Amino Acid Sequence↗

PubMed1989

Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system.

Bordetella pertussis, the aetiological agent of whooping cough, coordinately regulates the expression of many virulence-associated determinants, including filamentous haemagglutinin, pertussis toxin, adenylyl cyclase toxin, dermonecrotic toxin and haemolysin. The coordinate regulation is apparent in the repression of synthesis of these determinants in response to environmental stimuli; a phenomenon known as antigenic or phenotypic modulation. B. pertussis also varies between metastable genetic states, or phases. There is a virulent phase in which virulence-associated determinants are synthesized, and an avirulent phase in which they are not. Previous studies have shown that a genetic locus, vir, is required for expression from many virulence-associated loci, and that replacing the cloned vir locus in trans can restore the virulent phase phenotype to spontaneously occurring avirulent phase strains. Here, we show that phase variation in one series of strains is due to a frameshift mutation within an open reading frame that is predicted to code for a Vir protein product. The deduced protein sequence is similar to both components of the 'two-component' regulatory system which control gene expression in response to environmental stimuli in a range of bacterial species.

Base Sequence↗

PubMed1989

Coordinate regulation and sensory transduction in the control of bacterial virulence.

Genes and operons that encode bacterial virulence factors are often subject to coordinate regulation. These regulatory systems are capable of responding to various environmental signals that may be encountered during the infectious cycle. For some pathogens, proteins that mediate sensory transduction and virulence control are similar to components of other bacterial information processing systems. Understanding the molecular mechanisms governing global regulation of pathogenicity is essential for understanding bacterial infectious diseases.

Bacteria↗

PubMed1989

Epithelial cell surfaces induce Salmonella proteins required for bacterial adherence and invasion.

Salmonella bacteria are capable of entering (invading) and multiplying within eukaryotic cells. Stable adherence to and invasion of epithelial cells by S. choleraesuis and S. typhimurium were found to require de novo synthesis of several new bacterial proteins. This inducible event appears to be a coordinately regulated system dependent on trypsin- and neuraminidase-sensitive structures present on the epithelial cell surface. Mutants of S. choleraesuis and S. typhimurium were unable to synthesize these proteins and did not stably adhere to nor invade eukaryotic cells. Two such S. typhimurium mutants were avirulent in mice, an indication that these proteins are required for Salmonella virulence.

Animals↗

PubMed1989

Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins.

The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence. The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid. The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present. Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype. The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems. We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm. Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.

Amino Acid Sequence↗

PubMed1989

Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence.

Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.

Amino Acid Sequence↗

PubMed1989

Expression of pertussis toxin correlates with pathogenesis in Bordetella species.

Pertussis toxin is a principal determinant of virulence produced by Bordetella pertussis in the disease whooping cough and is the primary toxinogenic component of the pertussis vaccine. This toxin is not produced by the closely related species Bordetella parapertussis. Toxinogenic strains of B. parapertussis were constructed by the conjugative introduction of cloned pertussis toxin genes. These and analogous toxinogenic and nontoxinogenic strains of B. pertussis were assayed for their toxicity and reactogenic activities. Expression of active toxin by either Bordetella sp. correlated with the induction of leukocytosis, anaphylaxis, and histamine sensitivity.

Anaphylaxis↗

PubMed1989

Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus.

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.

Bordetella↗

PubMed1989

Salmonella as an intracellular parasite.

Salmonella species are facultative intracellular parasites, capable of penetrating (invading), surviving, and often multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. These processes are essential for virulence, and involve both bacterial and host cell products. The use of cultured eukaryotic cells and other model systems has facilitated the study of bacterial-host cell interactions, and has led to a better understanding of the genetic and molecular basis of Salmonella pathogenicity.

Animals↗

PubMed1989

Expression of gonococcal protein II in Escherichia coli by translational fusion.

A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.

Amino Acid Sequence↗

PubMed1989

A Yersinia pestis-specific DNA fragment encodes temperature-dependent coagulase and fibrinolysin-associated phenotypes.

The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30 degrees C but fibrinolytic activity increases with higher temperatures (greater than 30 degrees C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to 'flea blockage' and subsequent transmission of the plague bacillus to animals.

Amino Acid Sequence↗

PubMed1989

The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease.

Yersinia enterocolitica is a heterogeneous group of organisms with more than 50 serotypes and several biotypes. Only a few of these serotypes cause gastrointestinal disease in otherwise healthy hosts; these serotypes are the pathogenic serotypes. Although Y. enterocolitica requires a high-molecular-weight plasmid to cause disease, chromosome-encoded determinants are required for the full expression of virulence. The ability of Yersinia spp. to invade eucaryotic cells is thought to be a virulence factor, because nonpathogenic serotypes are noninvasive in animals and in tissue culture cell models. Current evidence indicates that invasion ability is chromosome encoded. We recently reported cloning two loci, inv and ail, from Y. enterocolitica O8 strain 8081c that allow Escherichia coli to invade tissue culture cells. We investigated the link between invasion in an in vitro tissue culture invasion (TCI) model and hybridization to probes derived from the two invasion loci, inv and ail. We examined 177 Yersinia strains. Strains of serotypes and species associated with disease were TCI+, whereas strains of serotypes and species not associated with disease were TCI-. Only TCI+ strains had DNA homologous to probes derived from ail. All strains (TCI+ and TCI-) had DNA homologous to probes derived from inv, but there were certain restriction fragment-linked polymorphisms that were associated primarily with TCI+ strains. These observations held true for strains epidemiologically associated with disease. Both the inv and ail loci were found to be clearly located on the chromosome. No other genera, including other invasive organisms, had DNA homologous to inv or ail. These data support the hypothesis that the ail locus encodes a Y. enterocolitica invasion factor that may be involved in pathogenesis.

Bacteriological Techniques↗

PubMed1989

The bvgA gene of Bordetella pertussis encodes a transcriptional activator required for coordinate regulation of several virulence genes.

The bvg region of the respiratory pathogen Bordetella pertussis coordinately regulates the expression of several unlinked virulence determinants in response to environmental signals. The DNA sequence of the bvg region contains three genes (bvgA, bvgB, and bvgC). Transcription of a single-copy fusion consisting of the upstream region of a bvg-activated B. pertussis gene (fhaB) attached to the promoterless lac operon in Escherichia coli requires the entire bvgABC region in trans. Activation of the fhaB::lacZYA fusion is sensitive to the same environmental stimuli in E. coli that modulate the expression of bvg-activated genes in B. pertussis. Our data show that overexpression of the bvgA gene from a strong heterologous promoter results in transcriptional activation of the fhaB::lacZYA fusion even in the absence of the bvgB and bvgC products. Activation of fhaB transcription by bvgA overexpression in E. coli is no longer repressed by environmental conditions. The bvgA product has been identified by maxicell analysis as a 23-kilodalton protein. A B. pertussis mutant containing an in-frame deletion in bvgA was constructed. This mutant was nonhemolytic and no longer produced filamentous hemagglutinin and pertussis toxin. The mutation in this strain was complemented by returning the bvgA gene in trans. Transcriptional chloramphenicol acetyltransferase fusions to the fhaB and ptx promoter regions were returned to both the B. pertussis bvgA deletion mutant and its parental wild-type strain. Analysis of these strains indicated that the deletion mutant was defective in transcription of both ptx and fhaB. We conclude from these data that bvgA, bvgB, and bvgC comprise an operon encoding the components essential for coordinate regulation and sensory transduction. The BvgA protein is a transcriptional regulatory factor. The bvgB and bvgC products may be important in regulating the activity of BvgA in response to the changing environmental stimuli that B. pertussis encounters during the diseases whooping cough.

Bordetella pertussis↗

PubMed1989

Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli.

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

Bordetella pertussis↗

PubMed1989

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.

Amidohydrolases↗