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B B Finlay

Publications and source records attributed to B B Finlay.

At least 109 records · Page 6Linked to original sources
PubMed1996

A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.

Actins↗
PubMed1996

M cells and the pathogenesis of mucosal and systemic infections.

M cells are specialized epithelial cells of mucosal surfaces lining the respiratory and intestinal tracts. They participate in generating mucosal immune protection by sampling and delivering antigens to the underlying lymphoid tissue. A variety of viral, bacterial and protozoal pathogens exploit the M cell transport pathway to breach mucosal barriers and establish local or systemic infections.

Animals↗
PubMed1996

Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells.

Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (lgp)-containing vacuoles. Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable lgp-containing filamentous structures that connect with the Salmonella-containing vacuoles. Salmonella typhimurium SL1344::Tn l0dCm mutant strains unable to induce these structures were isolated. All contained insertions within a novel Salmonella induced filament gene A (sifA). sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38 kDa and an apparent molecular mass of 35 kDa. sifA is flanked by 300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases. sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines. Fourteen-base-pair direct repeats mark the probable site of integration of sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52-54%). Deletion mutant strains in sifA or in the downstream potC were constructed. Delta sifA does not produce Salmonella-induced filaments in epithelial cells, and is attenuated in mice. Delta potC produces Salmonella-induced filaments in epithelial cells, and was fully virulent. Collectively, these results suggest that sifA arose by horizontal gene transfer into Salmonella and its product is involved in a virulence-associated intracellular phenotype related to Salmonella-induced filament formation.

Animals↗
PubMed1996

EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea. EPEC mediates several effects on host epithelial cells, including activation of signal-transduction pathways, cytoskeletal rearrangement along with pedestal and attaching/effacing lesion formation. It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria. DNA-sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino-terminal sequence of a 25 kDa EPEC secreted protein. A mutant with a non-polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. These phenotypes were complemented by a cloned espA gene. The espA mutant is also defective for invasion. It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.

Adhesins, Bacterial↗
PubMed1996

Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells.

It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium. These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes. This may be relevant to the etiology of diarrhea caused by EPEC infection.

Caco-2 Cells↗
PubMed1996

Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells.

Enteropathogenic Escherichia coli (EPEC) induces tyrosine phosphorylation of a 90-kDa protein (Hp90) in infected epithelial cells. This in turn facilitates intimate binding of EPEC via the outer membrane protein intimin, effacement of host cell microvilli, cytoskeletal rearrangement, and bacterial uptake. This phenotype has been commonly referred to as attaching/effacing (A/E). The ability of EPEC to induce A/E lesions was dependent on bacterial growth phase and temperature. Early-logarithmic-phase EPEC grown at 37 degrees C elicits strong A/E activity within minutes after infection of HeLa epithelial cells. EPEC de novo protein syntheses during the first minutes of interaction with the host cell was required to elicit A/E lesions. However, once formed, bacterial viability was not needed to maintain A/E lesions. The type of growth media and partial O2 pressure level do not seem to affect the ability of EPEC to cause A/E lesions. These results indicates that the A/E activity of EPEC is tightly regulated by environmental and host factors.

Adhesins, Bacterial↗
PubMed1996

Effect of cell polarization and differentiation on entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line.

The entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line was studied as a function of cell polarization and differentiation. L. monocytogenes entered through the entire surface of nonpolarized cells and, predominantly, through the basolateral surface of polarized cells based on the following observations: (i) sites of L. monocytogenes invasion paralleled the distribution of the transferrin receptor, a well-known basolateral marker of polarization; (ii) numbers of internalized bacteria decreased dramatically when Caco-2 monolayers cultured beyond confluency were used (about 0.1% of the inoculated bacteria versus 1 to 2% with nonconfluent monolayers); and (iii) L. monocytogenes entry into postconfluent monolayers was greatly enhanced by treating cells with Ca(2)+ -free medium, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N' -tetraacetic acid (EGTA) had contradictory effects on L. monocytogenes entry as this reagent opened intercellular junctions but inhibited binding and internalization of bacteria. Finally, the role of the inlAB locus in L. monocytogenes entry was confirmed because and inlAB mutant was 50- to 100-fold less invasive than the parental strain regardless of the monolayer's age. However, the inlAB mutant was still able to enter cells and to induce intracellular actin polymerization. Entry of inlAB bacteria into Caco-2 cells was not inhibited by EGTA.

Bacterial Adhesion↗
PubMed1996

Listeriolysin O activates mitogen-activated protein kinase in eucaryotic cells.

Infection with Listeria monocytogenes induces the activation of mitogen-activated protein (MAP) kinase in several tissue culture cell lines (P.Tang, I. Rosenshine, and B. B. Finlay, Mol. Biol. Cell 5:455-464, 1994). After various mutants were examined, the bacterial factor responsible for MAP kinase activation was identified as listeriolysin O (LLO). Growth supernatant containing LLO or purified LLO alone can induce MAP kinase tyrosine phosphorylation in HeLa cells. Single-amino-acid mutations in LLO that do not affect its membrane binding capacity but reduce its cytolytic activity also reduced its ability to induce MAP kinase activity in HeLa cells. Streptolysin O, another sulfhydryl-activated hemolysin, and the detergent saponin are also able to activate MAP kinase in target cells. Thus, the increased MAP kinase activity observed in L. monocytogenes-infected cells is most likely a result of the permeabilization of the host cell membrane by LLO and may not be linked with invasion.

Bacterial Toxins↗
PubMed1996

Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins.

Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.

Amino Acid Sequence↗
PubMed1995

Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells.

Enteropathogenic Escherichia coli (EPEC), a major cause of pediatric diarrhea, adheres to epithelial cells and activates host cell signal transduction pathways. We have identified five proteins that are secreted by EPEC and show that this secretion process is critical for triggering signal transduction events in epithelial cells. Protein secretion occurs via two pathways: one secretes a 110-kDa protein and the other mediates export of the four remaining proteins. Secretion of all five proteins was regulated by temperature and the perA locus, two factors which regulate expression of other known EPEC virulence factors. Amino-terminal sequence analysis of the secreted polypeptides identified one protein (37 kDa) as the product of the eaeB gene, a genetic locus previously shown to be necessary for signal transduction. A second protein (39 kDa) showed significant homology with glyceraldehyde-3-phosphate dehydrogenase, while the other three proteins (110, 40, and 25 kDa) were unique. The secreted proteins associated with epithelial cells, and EaeB became resistant to protease digestion upon association, suggesting that intimate interactions are required for transducing signals.

Amino Acid Sequence↗
PubMed1995

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells.

Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pylori-infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or alpha-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pylori-infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.

Cell Line↗
PubMed1995

The varied lifestyles of intracellular pathogens within eukaryotic vacuolar compartments.

Many bacterial pathogens and eukaryotic parasites can enter mammalian cells and live intracellularly inside membrane-bound vacuoles. The intravacuolar lifestyle of these pathogens plays a key role in pathogenesis. Understanding the molecular basis of the development of these specialized intracellular compartments is critical to understanding how these organisms cause disease.

Animals↗
PubMed1995

Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors.

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.

Acid Phosphatase↗
PubMed1995

Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.

Animals↗
PubMed1995

Interactions between Salmonella typhimurium, enteropathogenic Escherichia coli (EPEC), and host epithelial cells.

The interactions that occur between pathogenic micro-organisms and their host cells are complex and intimate. We have used two enteric pathogens, Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC), to examine the interactions that occur between these organisms and epithelial cells. Although these are enteric pathogens, the knowledge and techniques developed from these systems may be applied to the study of dental pathogens. Both S. typhimurium and EPEC disrupt epithelial monolayer integrity, although by different mechanisms. Both pathogens cause loss of microvilli and re-arrangement of the underlying host cytoskeleton. Despite these similarities, both organisms send different signals into the host cell. EPEC signal transduction involves generation of intracellular calcium and inositol phosphate fluxes, and activation of host tyrosine kinases that results in tyrosine phosphorylation of a 90-kDa host protein. Bacterial mutants have been identified that are deficient in signaling to the host. We propose a sequence of events that occur when EPEC interacts with epithelial cells. Once inside a host cell, S. typhimurium remains within a vacuole. To define some of the parameters of the intracellular environment, we constructed genetic fusions of known genes with lacZ, and used these fusions as reporter probes of the intracellular vacuolar environment. We have also begun to examine the bacterial and host cell factors necessary for S. typhimurium to multiply within epithelial cells. We found that this organism triggers the formation of novel tubular lysosomes, and these structures are linked with intracellular replication.

Cells, Cultured↗