Search PubMed⌕ Search

Biomedical subjects

S Falkow

Publications and source records attributed to S Falkow.

At least 127 records · Page 7Linked to original sources
PubMed1991

Characterization of interactions of enteropathogenic Escherichia coli O127:H6 with mammalian cells in vitro.

Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

Adhesins, Escherichia coli↗
PubMed1991

Structural and genetic analysis of the bvg locus in Bordetella species.

The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.

Amino Acid Sequence↗
PubMed1991

Surface structures and adherence properties of diverse strains of Haemophilus influenzae biogroup aegyptius.

Haemophilus influenzae biogroup aegyptius is an important cause of conjunctivitis and has recently been associated with Brazilian purpuric fever (BPF), a fulminant systemic disease of children. To gain insight into the bacterial factors involved in the pathogenesis of this disease, we investigated the surface structures and adherence properties of eight different strains of H. influenzae biogroup aegyptius, including both BPF and non-BPF isolates. All eight strains were able to express long peritrichous pili similar to those observed in H. influenzae. As in H. influenzae, piliation correlated with colony binding of human erythrocytes. However, two strains were capable of hemagglutination in the absence of pili; in these strains, hemagglutination was insensitive to protease treatment, suggesting a nonproteinaceous hemagglutinin. All strains possessed short, thin, surface fibers distinct from long pili and demonstrated efficient attachment to cultured human conjunctival cells. Attachment to conjunctival cells occurred independently of long pili or a capacity for hemagglutination.

Bacterial Adhesion↗
PubMed1991

Loss of capsule expression by Haemophilus influenzae type b results in enhanced adherence to and invasion of human cells.

Haemophilus influenzae type b is a common cause of systemic bacterial disease in children, and the serotype b capsule is a major determinant of virulence. Nevertheless, as a consequence of the genetic configuration of the capb locus, type b strains become capsule deficient at a high frequency. To investigate the potential biological relevance of the predisposition to capsule loss, we compared the adherent and invasive abilities of several strains of H. influenzae type b and their isogenic capsule-deficient mutants by using cultured human epithelial cells. In all cases the capsule-deficient mutant demonstrated significantly greater adherence and invasion than the encapsulated parent. Transformation of one capsule-deficient mutant to restore encapsulation resulted in a marked decrease in adherence and invasion. All strains were capable of adherence and invasion by a pilus-independent mechanism. We conclude that capsule loss by H. influenzae type b results in enhanced in vitro adherence and invasion, properties that may be relevant to colonization of the nasopharynx and persistence within the respiratory tract. These observations suggest an explanation for the evolution of the capb locus as directly repeated segments of DNA with a consequent predisposition to recombination resulting in capsule loss.

Bacterial Adhesion↗
PubMed1991

Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon.

Transcription of numerous virulence genes in Bordetella pertussis is positively regulated by the products of the bvgAS genes. In this study a series of lacZYA fusions containing deletions in either the fhaB or bvgA promoter regions was used to identify cis-acting regulatory regions required for bvg activation of these two genes. Gel retardation and DNase I protection analyses have shown that specific protein-DNA interactions occur at these regulatory regions and that these interactions require the transcriptional activator protein BvgA. The regulatory regions found upstream of fhaB and bvgA which are involved in protein binding both contain the sequence TTTCCTA. This sequence is part of an inverted repeat upstream of fhaB and a direct repeat upstream of bvgA. Homologous repeats are not apparent upstream of other bvg-activated genes, such as ptx and cyaA. These data suggest that the mechanism for transcriptional regulation of the other bvg-activated genes is complex and may require regulatory factors in addition to the bvgAS gene products.

Base Sequence↗
PubMed1991

Characterization of a Salmonella choleraesuis mutant that cannot multiply within epithelial cells.

A mutant of Salmonella choleraesuis was identified that could invade (enter) and penetrate through polarized monolayers of Caco-2 and MDCK epithelial cells at normal levels but was defective for intracellular multiplication within these cells. It was also able to survive inside cultured J774 macrophage cells. These bacteria remained inside membrane-bound vacuoles, which coalesced at later times in the perinuclear region of the epithelial cell. This mutant exhibited slightly slower growth rates in rich or minimal media than the parental strain but was normal for iron usage, phosphate usage, and anaerobic growth and was a prototroph. The mutant was completely avirulent when administered orally or intravenously to susceptible mice. These results suggest that the ability to multiply within eukaryotic cells may contribute to S. choleraesuis virulence.

Animals↗
PubMed1990

The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.

BACKGROUND: Bacillary angiomatosis is an infectious disease causing proliferation of small blood vessels in the skin and visceral organs of patients with human immunodeficiency virus infection and other immunocompromised hosts. The agent is often visualized in tissue sections of lesions with Warthin-Starry staining, but the bacillus has not been successfully cultured or identified. This bacillus may also cause cat scratch disease. METHODS: In attempting to identify this organism, we used the polymerase chain reaction. We used oligonucleotide primers complementary to the 16S ribosomal RNA genes of eubacteria to amplify 16S ribosomal gene fragments directly from tissue samples of bacillary angiomatosis. The DNA sequence of these fragments was determined and analyzed for phylogenetic relatedness to other known organisms. Normal tissues were studied in parallel. RESULTS: Tissue from three unrelated patients with bacillary angiomatosis yielded a unique 16S gene sequence. A sequence obtained from a fourth patient with bacillary angiomatosis differed from the sequence found in the other three patients at only 4 of 241 base positions. No related 16S gene fragment was detected in the normal tissues. These 16S sequences associated with bacillary angiomatosis belong to a previously uncharacterized microorganism, most closely related to Rochalimaea quintana. CONCLUSIONS: The cause of bacillary angiomatosis is a previously uncharacterized rickettsia-like organism, closely related to R. quintana. This method for the identification of an uncultured pathogen may be applicable to other infectious diseases of unknown cause.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis.

During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 (alpha M beta 2, CD11b/CD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097-1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.

Amino Acid Sequence↗
PubMed1990

Autogenous regulation of the Bordetella pertussis bvgABC operon.

The bvgABC operon of the bacterial pathogen Bordetella pertussis encodes a sensory transduction system that regulates the expression of several virulence genes in response to environmental stimuli. In this study we have examined the transcriptional regulation of the bvgABC operon. Transcriptional bvg::lacZYA fusions in Escherichia coli show that the bvgABC operon is autogenously activated. Autoactivation is inhibited by the same environmental stimuli that result in the lack of expression of bvg-activated genes in B. pertussis. These observations were confirmed in B. pertussis using a chromosomal chloramphenicol acetyltransferase transcriptional fusion in bvgC. Transcriptional initiation sites upstream of bvgA were mapped by primer extension analysis in E. coli and B. pertussis. Two differentially regulated bvg promoters were identified. The bvgP1 promoter is a positively autoregulated promoter located 90 base pairs upstream of bvgA. The bvgP2 promoter is located 141 base pairs upstream of bvgA and does not appear to require any positive regulatory factors for activity. These results suggest a model describing the regulatory events that take place upstream of the bvgABC operon.

Base Sequence↗
PubMed1990

The ability of Salmonella to enter mammalian cells is affected by bacterial growth state.

We have examined the effect of different growth conditions on the ability of Salmonella to interact with Madin-Darby canine kidney cells. Two growth conditions that affect the expression of Salmonella adherence and invasiveness have been identified. First, bacteria lose their invasiveness in the stationary phase of growth. Second, bacteria growing in oxygen-limited growth conditions are induced for adherence and invasiveness, whereas those growing aerobically are relatively nonadherent and noninvasive. Salmonella from cultures aerated with gas mixtures containing 0% or 1% oxygen were 6- to 70-fold more adherent and invasive than those from cultures aerated with a gas mixture containing 20% oxygen. The Salmonella typhimurium oxrA gene that is required for the anaerobic induction of many proteins is not involved in the regulation of Salmonella invasiveness. We speculate that oxygen limitation might be an environmental cue that triggers the expression of Salmonella invasiveness within the intestinal lumen and other tissues.

Anaerobiosis↗
PubMed1990

Detection of Shigella in feces using DNA amplification.

A rapid diagnostic method employing a polymerase chain reaction procedure (PCR) was used to identify Shigella and enteroinvasive Escherichia coli. This procedure amplified a region of the invasive-associated locus (ial) from a crude DNA extract of feces. A synthetic 21-base oligonucleotide corresponding to the ial gene sequence was shown to specifically hybridize only with enteroinvasive E. coli (EIEC) strains and Shigella species. Upon PCR amplification, a 320-base pair fragment was generated in DNA extracted from feces reconstituted with EIEC or Shigella flexneri but not in DNA from 70 normal stools lacking these organisms and could be readily detected by the ial probe. For identifying Shigella and EIEC, the PCR assay was 10(5)- and 10(2)-fold more sensitive than standard biochemical tests and the macrocolony hybridization assay, respectively. These findings demonstrate a novel methodology for rapid, sensitive, and culture-independent diagnosis of diarrhea caused by these pathogens and underscores the utility of PCR in the diagnostic laboratory.

Base Sequence↗
PubMed1990

Salmonella interactions with polarized human intestinal Caco-2 epithelial cells.

Polarized monolayers of the human intestinal epithelial Caco-2 cell line were grown on permeable filters and infected apically with either Salmonella choleraesuis or Salmonella typhimurium. Both Salmonella species penetrated through the monolayer, requiring 2 h before appearing in the basolateral medium. Both species caused a loss in transepithelial resistance by 3-4 h, and the monolayer's integrity was completely disrupted by 6 h. Scanning and transmission electron microscopy revealed that the bacteria interacted with well-defined apical microvilli and caused disruptions in the brush border, including elongation and denuding of the microvilli. The cytoplasm was also disrupted locally, with blebs protruding from the apical surface. The bacteria entered (invaded) these cells and were enclosed in membrane-bound vacuoles within the cytoplasm. By 6 h there were many bacteria within most Caco-2 cells, and these organisms caused serious cytopathic consequences. These morphologic observations correlated well with animal infection models, indicating that this in vitro system will be useful to study pathogens that interact with human intestinal epithelia.

Cell Line↗
PubMed1990

A soluble 60 kiloDalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL.

Two major 60 kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl-soluble 60 kD protein is (i) structurally and antigenically distinct from the previously characterized 60 kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD mycobacterial antigen and the GroEL heat-shock protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl-GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide-rich cell wall late in the growth cycle. Chl-GroEL is identical to the Triton X-100-soluble, ocular delayed-type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl-GroEL homology to host proteins is hypothesized.

Antigens, Bacterial↗
PubMed1990

Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor.

The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella pertussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220 kD biologically active, mature polypeptide product, suggesting a role for co- or post-translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220 kD product is encoded by the 5' portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N-terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.

Adhesins, Bacterial↗
PubMed1990

Sequence, localization and function of the invasin protein of Yersinia enterocolitica.

The inv locus of Yersinia enterocolitica is sufficient to convert a non-invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835-amino-acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C-terminal 88 amino acids of this open reading frame detected a unique 100 kD protein in cell lysates of Y. enterocolitica strain 8081 c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp-2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.

Adhesins, Bacterial↗
PubMed1990

Bordetella pertussis filamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model.

Filamentous hemagglutinin (FHA) is a cell surface protein of Bordetella pertussis which functions as an adhesin for this organism. It is a component of many new acellular pertussis vaccines. The proposed role of FHA in immunity to pertussis is based on animal studies which have produced some conflicting results. To clarify this situation, we reexamined the protective activity of FHA in an adult mouse respiratory infection model. Four-week-old BALB/c mice were immunized with one or two doses of 4 or 8 micrograms of FHA and then aerosol challenged with B. pertussis Tohama I. In control mice receiving tetanus toxoid, the CFU in the lungs increased from 10(5) immediately following infection to greater than 10(6) by days 5 and 9 after challenge. Mice immunized with FHA by the intraperitoneal or intramuscular route had significantly reduced bacterial colonization in the lungs. A decrease in colonization of the trachea was also observed in FHA-immunized mice. Evaluation of antibody responses in these mice revealed high titers of immunoglobulin G (IgG) and IgM to FHA in sera and of IgG to FHA in lung lavage fluids. No IgA to FHA was detected. BALB/c mice were also passively immunized intravenously with either goat or rat antibodies to FHA and then aerosol challenged 24 h later, when anti-FHA antibodies were detected in the respiratory tract. Lung and tracheal colonization was markedly reduced in mice immunized with FHA-specific antibodies compared with those receiving control antibodies. In additional studies, the role of FHA in the colonization of the mouse respiratory tract was evaluated by using strain BP101, an FHA mutant of B. pertussis. FHA was important in the initial colonization of the mouse trachea, but was not required for colonization of the trachea later in the infection. FHA was not a factor in colonization of the lungs. Collectively, these experiments demonstrate (i) that systemic immunization with FHA can provide significant protection against B. pertussis infection in both the lower and upper respiratory tract of mice as defined by the lungs and trachea, respectively; (ii) that this protection is mediated primarily by serum antibodies to FHA, which transudate into respiratory secretions; and (iii) that FHA is an important upper respiratory tract colonization factor. These studies provide further evidence for the role of FHA in pertussis pathogenesis and immunity.

Animals↗
PubMed1990

Haemophilus influenzae adheres to and enters cultured human epithelial cells.

Haemophilus influenzae is a common commensal organism of the human respiratory tract that initiates infection by colonizing the nasopharyngeal epithelium. In some individuals, colonization is followed by localized respiratory tract or systemic disease. To gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx, we examined the interactions between a nonpiliated clinical isolate of H. influenzae and human epithelial cells. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. Comparison of profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent and nonadherent bacteria identified several candidate adhesin molecules. In addition, a small number of adherent bacteria were capable of entering epithelial cells in a process that was inhibited by cytochalasin D and colchicine. The suggestion from our studies is that one or more of several newly synthesized nonpilus bacterial proteins are required for maximal in vitro adherence and invasion. We speculate that H. influenzae entry into epithelial cells may provide a mechanism for evasion of host defenses, thereby allowing persistence in the nasopharynx.

Bacterial Adhesion↗