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S Falkow

Publications and source records attributed to S Falkow.

At least 91 records · Page 5Linked to original sources
PubMed1994

Mycobacterium marinum persists in cultured mammalian cells in a temperature-restricted fashion.

We have explored the relatively rapidly growing animal and human pathogen Mycobacterium marinum as an experimental model for mycobacterial pathogenesis. M. marinum, which has a lower temperature for optimal growth than does Mycobacterium tuberculosis, has a much shorter generation time and can be safely studied in ordinary laboratory facilities and examined in multiple animal infection models. We have established an in vitro assay for its interaction with eukaryotic cells and shown that it persists in these cells in a temperature-specific fashion that correlates with its ability to cause disease in vivo at lower temperatures. Additionally, preliminary evidence that M. marinum causes a chronic disease with some features resembling tuberculosis in frogs of the species Rana pipiens is presented.

Animals↗
PubMed1994

Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.

Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.

Acanthamoeba↗
PubMed1994

Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

Amino Acid Sequence↗
PubMed1993

Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism.

Invasive Salmonella typhimurium induces dramatic actin rearrangements on the membrane surface of mammalian cells as part of its entry mechanism. These changes, which are best characterized as membranous ruffles, closely resemble the membrane changes that occur when a growth factor binds to its receptor. Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated ruffling and stress-fiber formation, respectively. In addition, actin changes induced by the oncogene ras were also shown to be regulated by rac and rho. Because Salmonella-induced actin rearrangements resemble those caused by growth factors, we investigated whether ras, rho, or rac regulates the membrane ruffling elicited by S. typhimurium. Surprisingly, inhibition of the functions of these GTPases had no effect on the ability of invasive S. typhimurium to induce membrane ruffles on a variety of tissue culture cells including Madin-Darby canine kidney cells, Swiss 3T3 fibroblasts, and Hep-2 cells. These results led us to examine the interactions of S. typhimurium with Henle-407 intestinal cells, which lack epidermal growth factor receptor on their membrane surface. We found no difference in the ability of invasive S. typhimurium to induce membrane ruffling and to enter Henle-407 cells with or without the epidermal growth factor receptor on the membrane surface. We, therefore, conclude that invasive S. typhimurium induces membrane ruffling and its own internalization by a rac-independent, growth factor-receptor-independent signaling pathway.

3T3 Cells↗
PubMed1993

Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria.

Ruffles are specialized plasma membrane ultrastructures of mammalian cells though to be integral to growth, development and locomotion. Induced by growth factors, mitogens or oncogene expression, ruffles are sites of filamentous actin rearrangement and are temporally associated with enhanced pinocytosis. But the function of ruffles, their mechanism of induction and their role in pinocytosis are not understood. We have observed formation of structures resembling ruffles associated with the site of entry of invasive Salmonella typhimurium. Here we report that ruffles elicited by invasive Salmonella directly mediate internalization of non-invasive bacteria in a macropinocytotic fashion, a phenomenon we term 'passive entry'. Furthermore, ruffles induced in the absence of Salmonella also facilitate passive entry. We present evidence that ruffles, common to many signalling events, comprise the macropinocytotic machinery mediating pinocytosis and are subverted by Salmonella so as to enter mammalian cells.

Animals↗
PubMed1993

High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells.

Nontypable Haemophilus influenzae are Gram-negative bacilli that represent a common cause of human disease. These organisms initiate infection by colonizing the upper respiratory tract. Despite the essential role of colonization, the bacterial determinants of this process remain poorly defined. We recently identified a family of surface-exposed high-molecular-weight proteins of nontypable H. influenzae that are related to filamentous hemagglutinin, a critical adherence factor of Bordetella pertussis. The genes encoding the two such high-molecular-weight proteins (HMW-1 and HMW-2) expressed by a prototypic nontypable H. influenzae strain have now been cloned and sequenced. In this study we examined the role of the HMWs in adherence to human epithelial cells. We found that loss of expression of HMW-1 by the prototypic strain and a HMW-1-like protein in a heterologous nontypable H. influenzae strain markedly decreased the capacity to adhere. The absence of expression of both HMW-1 and HMW-2 in the prototypic strain or their homologs in the second strain was associated with a further decrease in adherence. Expression of either HMW-1 or HMW-2 in nonadherent laboratory strains of Escherichia coli resulted in acquisition of the adherence phenotype. These results indicate that both HMW-1 and HMW-2 and the homologous proteins from a second strain can mediate attachment. We speculate that these proteins and the related proteins in other nontypable H. influenzae isolates are important colonization factors.

Bacterial Adhesion↗
PubMed1993

The role of host tyrosine phosphorylation in bacterial pathogenesis.

Recent studies on the pathogenic mechanisms of several bacterial genera, including the Yersinia, Salmonella and Escherichia, have revealed novel strategies of infection that involve the signal transduction processes of eukaryotic cells. These model systems are reviewed here, with emphasis on the role of tyrosine phosphorylation in these bacterial-host cell interactions.

Animals↗
PubMed1993

Mutation in the pla gene of Yersinia pestis alters the course of the plague bacillus-flea (Siphonaptera: Ceratophyllidae) interaction.

Yersinia pestis possesses a unique gene (pla) encoding coagulase and fibrinolysin which is implicated in the transmission of plague by fleas. This gene is encoded on the highly conserved but poorly characterized 'pesticin' plasmid pKYP1. The role of the pKYP1-encoded gene, pla, in plague transmission was addressed by feeding fleas on blood containing avirulent Y. pestis strain EV76-6 and three derivatives of this strain (K10-2, K10-3, and K10-5) carrying Tn801 insertions in pKYP1. One of these mutant strains, K10-5, contains an insertion within the pla gene that eliminates both coagulase and fibrinolysin activities, whereas strains K10-3 and K10-2 retain both pla-associated phenotypes. After feeding, it was found that flea mortality at 4 d after infection associated with strain K10-5 (26%) was significantly lower than the mortality observed with other strains (53-64%). These results suggest that expression of the pla gene product may contribute to the deleterious effects of plague bacilli on fleas that have been associated with flea blockage and plague transmission. This increased mortality is not caused simply by an increased bacterial load in fleas containing pla+ bacteria because fleas ingesting pla+ strains contained no more bacteria by flea blot hybridization analysis than did those that ingested the pla- strain K10-5. It is anticipated that further work in this area will clarify the mechanism by which pla acts and will reveal additional genetic loci in the plague bacillus which are required for transmission by fleas.

Animals↗
PubMed1993

Phylogenetic relationship of Chlamydia pneumoniae to Chlamydia psittaci and Chlamydia trachomatis as determined by analysis of 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Chlamydia pneumoniae was determined and compared with the corresponding gene sequences of Chlamydia psittaci and Chlamydia trachomatis. C. pneumoniae has been reported to exhibit little chromosomal DNA homology with the other chlamydial species, and its phylogenetic relationships within the genus Chlamydia have not been described. A polymerase chain reaction was employed to determine the 16S rRNA gene sequence of C. pneumoniae. Ten primers from the C. psittaci sequences were used to amplify a C. pneumoniae template in overlapping segments of the gene. Sequence data for 1,554 bases indicated that the levels of homology of C. pneumoniae with C. psittaci and C. trachomatis were 96.19 and 94.07%, respectively. These data support the results of previous biochemical and developmental studies indicating that C. pneumoniae is more closely related to C. psittaci than to C. trachomatis.

Base Sequence↗
PubMed1993

Cloning of Bordetella bronchiseptica urease genes and analysis of colonization by a urease-negative mutant strain in a guinea-pig model.

The genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription-translation system. At least four polypeptides with predicted molecular weights of 69,000, 26,000, 12,200 and 11,000 were found. Partial DNA sequence of the gene encoding the 69,000 Da polypeptide revealed high amino acid identity to the alpha-subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease-negative mutant of B. bronchiseptica. To assess colonization in a guinea-pig model, the urease-negative strain was inoculated with the urease-positive parental strain in a mixed infection. The urease-negative strain out competed the urease-positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea-pig respiratory and digestive tracts.

Amino Acid Sequence↗
PubMed1993

The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia.

Transmissible murine colonic hyperplasia is characterized by proliferation of anchored stem cells in the mucosa of the descending colon of laboratory mice and is caused by Citrobacter freundii biotype 4280. This bacterium produces attaching and effacing lesions in the descending colon prior to the onset of gross hyperplasia. By mutational analysis, the chromosomal eae gene of C. freundii biotype 4280 was shown to be necessary for colonic colonization. Conversely, bacteria cured of a 65-kb plasmid, which was identified in C. freundii biotype 4280, were not attenuated for colonic colonization or for the induction of colonic hyperplasia.

Adhesins, Bacterial↗
PubMed1993

The ail gene of Yersinia enterocolitica has a role in the ability of the organism to survive serum killing.

Two Yersinia enterocolitica genes, inv and ail, play a major role in the ability of this microorganism to enter cultured mammalian cells. ail-homologous sequences are present only in pathogenic species and strains of Yersinia. We previously demonstrated (D. E. Pierson and S. Falkow, Infect. Immun. 58:1059-1064, 1990) that four different nonpathogenic isolates of Y. enterocolitica are not able to invade tissue culture cells because they contain functionally inactive variants of the inv gene. When a functional version was introduced into these strains, they became invasive. In this study, we introduced a functional ail gene into the same strains and found that the ail gene was expressed but that these strains neither adhere to nor invade cultured animal cells. However, these recombinant strains became resistant to killing by human serum, whereas their parental strains were not. Using an ail mutant, we also demonstrate that the ail gene has a role in both invasion/adherence and serum resistance in a pathogenic isolate of Y. enterocolitica. These results support a role for Ail in the pathogenesis of Y. enterocolitica infection and disease.

Animals↗
PubMed1993

Isolation, expression, and nucleotide sequencing of the pilin structural gene of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

In this study we isolated the pilin gene from the Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius, expressed the gene in Escherichia coli, and determined its nucleotide sequence. Comparison of the nucleotide sequence of the BPF pilin gene with the sequences of pilin genes from strains of H. influenzae sensu stricto demonstrated a high degree of identity. Consistent with this observation, hemagglutination inhibition studies performed with a series of glycoconjugates indicated that BPF pili and H. influenzae type b pili possess the same erythrocyte receptor specificity.

Amino Acid Sequence↗
PubMed1993

Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia.

Citrobacter freundii biotype 4280 produces attaching and effacing (AE) lesions in the large intestine of laboratory mice and is the causative agent of transmissible murine colonic hyperplasia. AE lesions are also produced by enteropathogenic Escherichia coli in humans. Southern analysis revealed that biotype 4280, but not 20 other strains of C. freundii, contained DNA homologous to the eae (E. coli attaching and effacing) gene which is necessary for AE activity by enteropathogenic E. coli in vitro. We have cloned and determined the nucleotide sequence of the C. freundii eae homolog. Our findings suggest that the eae locus of C. freundii biotype 4280 is necessary for AE activity and has a role in the pathogenesis of transmissible murine colonic hyperplasia.

Adhesins, Bacterial↗
PubMed1993

The Yersinia pseudotuberculosis adhesin YadA mediates intimate bacterial attachment to and entry into HEp-2 cells.

We characterized a bacterium-host cell interaction that is mediated by the Yersinia adhesin YadA. Derivatives of the virulence plasmid pIB1 harboring mutations in yadA, yopE, or yopH or in a low-calcium-response regulatory locus were introduced into a Yersinia pseudotuberculosis YPIII strain defective for Inv. The mutant strains were tested for the capacity to attach to and enter HEp-2 cells and express the cytotoxic activities of YopE and YopH. As previously shown, expression of YadA was necessary for bacterial attachment and Yop activity in the absence of Inv (R. Rosqvist, A. Forsberg, M. Rimpilainen, T. Bergman, and H. Wolf-Watz, Mol. Microbiol. 4:657-667, 1990). In addition, bacterial entry into HEp-2 cells occurred efficiently when YadA was expressed in the absence of YopE and YopH. These results demonstrated that YadA mediates intimate attachment of Y. pseudotuberculosis to HEp-2 cells and that phagocytic uptake of bacteria by this pathway is inhibited by the synergistic activities of YopH and YopE. A role for beta 1 integrins as host cell receptors for this bacterial attachment and entry mechanism was supported by HEp-2 cell adhesion and monoclonal antibody neutralization studies.

Adhesins, Bacterial↗
PubMed1993

Isolation and characterization of "Flexispira rappini" from laboratory mice.

A bacterium with an unusual ultrastructure and possessing a fusiform protoplasmic cylinder, spiral periplasmic fibers, and bipolar tufts of sheathed flagella was identified in the intestinal mucosae of laboratory mice. The organism was cultured under microaerophilic conditions and was found to rapidly hydrolyze urea. On the basis of 16S rRNA gene sequence analysis, the organism was shown to be "Flexispira rappini." "F. rappini" is closely related to members of the genus Helicobacter and has been reported to be associated with human gastroenteritis and ovine abortion. "F. rappini" has not previously been observed in the gastrointestinal tracts of mice.

Animals↗