Cell adhesion and invasion mechanisms in microbial pathogenesis.
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Amino Acid Sequence↗
Biomedical subjects
B B Finlay
Publications and source records attributed to B B Finlay.
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↗
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↗
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↗
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↗
Common themes in microbial pathogenicity.
A bacterial pathogen is a highly adapted microorganism which has the capacity to cause disease. The mechanisms used by pathogenic bacteria to cause infection and disease usually include an interactive group of virulence determinants, sometimes coregulated, which are suited for the interaction of a particular microorganism with a specific host. Because pathogens must overcome similar host barriers, common themes in microbial pathogenesis have evolved. However, these mechanisms are diverse between species and not necessarily conserved; instead, convergent evolution has developed several different mechanisms to overcome host barriers. The success of a bacterial pathogen can be measured by the degree with which it replicates after entering the host and reaching its specific niche. Successful microbial infection reflects persistence within a host and avoidance or neutralization of the specific and nonspecific defense mechanisms of the host. The degree of success of a pathogen is dependent upon the status of the host. As pathogens pass through a host, they are exposed to new environments. Highly adapted pathogenic organisms have developed biochemical sensors exquisitely designed to measure and respond to such environmental stimuli and accordingly to regulate a cascade of virulence determinants essential for life within the host. The pathogenic state is the product of dynamic selective pressures on microbial populations.
Animals↗
Passage of Salmonella through polarized epithelial cells: role of the host and bacterium.
Salmonella are intracellular parasites which enter their hosts by penetrating the intestinal epithelial barrier. We examined the interaction of S. choleraesuis and S. typhimurium with Madin Darby canine kidney (MDCK) and human larynx (HEp-2) epithelial cells to characterize bacterial adherence, invasion and penetration through epithelial monolayers. Epithelial cell microfilaments were required for bacterial internalization and surrounded the bacteria as they were internalized. The bacteria entered membrane-bound vacuoles inside epithelial cells where they replicated. When polarized MDCK cell monolayers were infected, we found that Salmonella could pass through this barrier and enter medium bathing the opposite surface, although most bacteria remained within the monolayer. Synthesis of several Salmonella proteins was induced by the presence of epithelial cell surfaces, and these proteins were required for bacterial adherence and invasion. This induction was stimulated by trypsin- and neuraminidase-sensitive structures on epithelial cells.
Actin Cytoskeleton↗
Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication.
Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.
Animals↗
Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer.
Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.
Animals↗
The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli.
Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.
Amino Acid Sequence↗
Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer.
Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.
Alkaline Phosphatase↗
Two unusual pilin sequences from different isolates of Pseudomonas aeruginosa.
The pilin genes of two Pseudomonas aeruginosa strains isolated from two different patients with cystic fibrosis were cloned and sequenced. The predicted protein sequences of these two pilins had several unusual features compared with other published P. aeruginosa pilin sequences.
Amino Acid Sequence↗
Factors essential for the penetration of mammalian cells by Yersinia.
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Animals↗
Virulence factors associated with Salmonella species.
Salmonella species can cause diseases ranging in severity from acute gastroenteritis to typhoid fever. These bacteria are considered as intracellular pathogens and produce several products which are required for entry and survival in the intracellular environment, in addition to factors necessary for existence in the gastrointestinal tract and the outside environment. The virulence factors used by these bacteria to cause these diseases are complex, and only recently have we begun to characterize these factors and determine the contribution they make to Salmonella virulence.
Bacterial Adhesion↗
Novel serine proteases encoded by two cytotoxic T lymphocyte-specific genes.
Genes that are expressed exclusively in cytotoxic T cells should encode proteins that are essential for target cell lysis in cell-mediated immune responses. The sequences of two cytotoxic T lymphocyte-specific complementary DNA's (cDNA's) suggest that the two genes encode serine proteases. A full-length cDNA corresponding to one of the genes was isolated and sequenced. The predicted protein resembles serine proteases in that it includes all the residues that form the catalytic triad of the active site of serine proteases. Moreover, it has sequence characteristics thought to occur only in rat mast cell protease type II. These results are in accord with the view that a protease cascade plays a key role in cytotoxic T-cell activation.
Animals↗
Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.
Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.
Bacterial Outer Membrane Proteins↗
Nucleotide sequences of the R1-19 plasmid transfer genes traM, finP, traJ, and traY and the traYZ promoter.
The complete nucleotide sequences of the R1 drd-19 (R1-19) plasmid transfer genes traM, finP, traJ, and traY and the region encoding the traYZ promoter were determined. The traM protein from R1-19 was similar to the 127-amino-acid traM product from the conjugative plasmid F; only 28 residues were not identical. finP, a negative regulatory element of the traJ gene, contained a 12-base-pair inverted repeat identical to that found in the F plasmid, but differed in the 7 base pairs found between the repeats. The traJ gene and the traYZ promoter (the site of transcriptional stimulation by the traJ product) were completely different from the equivalent sequences in plasmid F. Galactokinase fusion studies of the traYZ promoter indicated that the R1-19 and F plasmids have analogous but not homologous traYZ promoter strengths and regulation. The traY protein from R1-19 was 44 residues shorter than the traY product from plasmid F, but there was some homology within the C-terminal halves of the traY gene products. The predicted translational start codon for the traY gene is GUG.
Alleles↗
Nucleotide sequence of the surface exclusion genes traS and traT from the IncF0 lac plasmid pED208.
pED208 is a 90-kilobase conjugative plasmid belonging to the incompatibility group IncF0 lac. The surface exclusion system from this plasmid was cloned and sequenced, and two genes demonstrated exclusion ability. traS encoded a 186-amino-acid hydrophobic protein which, when transcribed from a vector promoter, caused exclusion of pED208. The product of traT (TraTp) was a 245-residue protein which was highly expressed independently of a vector promoter in Escherichia coli minicells. The TraTp from pED208 was homologous with traT products from the IncF plasmids R-100 and F (80% homology), but recombinants containing the pED208 surface exclusion system excluded F poorly.
Base Sequence↗