Enteropathogenic E. coli acts through WASP and Arp2/3 complex to form actin pedestals.
Explore the source record for details and available documents.
Actin-Related Protein 2↗
Biomedical subjects
B B Finlay
Publications and source records attributed to B B Finlay.
High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements.
A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.
Actins↗
Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion.
Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-secreted proteins) that are required for full virulence. Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure. In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir-CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifically binding to its amino-terminus, forming a multimeric stabilized complex.
Amino Acid Sequence↗
Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells via a bacterial outer membrane adhesion molecule, intimin, and its receptor in the host cell membrane, Tir. Tir is a bacterial protein translocated into the host cell membrane and tyrosine phosphorylated after insertion. Tir-intimin binding induces organized actin polymerization beneath the adherent bacteria, resulting in the formation of pedestal-like structures. A series of Tir deletion derivatives were constructed to analyse which Tir domains are involved in intimin binding. We have localized the intimin-binding domain (IBD) of Tir using a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino acids that is predicted to be exposed on the surface of the plasma membrane. A truncated Tir protein lacking this domain was translocated to the host cell membrane and tyrosine phosphorylated, but failed to bind intimin or to induce either actin polymerization or Tir accumulation beneath the bacteria. These results indicate that only a small region of Tir is needed to bind intimin and support the predicted topology for Tir, with both N- and C-terminal regions in the mammalian cell cytosol. They also confirm that Tir-intimin interactions are needed for cytoskeletal organization. We have also identified N-terminal regions involved in Tir stability and Tir secretion to the media.
Actins↗
Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway.
In epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella-containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (Igps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immunofluorescence microscopy and epitope-specific flow cytometry. We show that proteins specific to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when Igp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the Igp is acquired, and that these processes occur progressively and not as the result of a single fusion/fission event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of Igp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.
Adenosine Triphosphatases↗
Type III secretion-dependent hemolytic activity of enteropathogenic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and EspD display homology to pore-forming molecules. Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells.
Amino Acid Sequence↗
Salmonella typhimurium virulence genes are induced upon bacterial invasion into phagocytic and nonphagocytic cells.
Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.
Animals↗
Enteropathogenic Escherichia coli inhibits phagocytosis.
Enteropathogenic Escherichia coli (EPEC) interacts with intestinal epithelial cells, activating host signaling pathways leading to cytoskeletal rearrangements and ultimately diarrhea. In this study, we demonstrate that EPEC interacts with the macrophage-like cell line J774A.1 to inhibit phagocytosis by these cells. Antiphagocytic activity was also observed in cultured RAW macrophage-like cells upon EPEC infection. The EPEC antiphagocytic phenotype was dependent on the type III secretion pathway of EPEC and its secreted proteins, including EspA, EspB, and EspD. Intimin and Tir mutants displayed intermediate antiphagocytic activity, suggesting that intimate attachment mediated by intimin-Tir binding may also play a role in antiphagocytosis. Tyrosine dephosphorylation of several host proteins was observed following infection with secretion-competent EPEC but not with secretion-deficient mutants. Dephosphorylation was detectable 120 min after infection with EPEC, directly correlating with the onset of the antiphagocytic phenotype. Inhibition of protein tyrosine phosphatases by pervanadate treatment increased the number of intracellular wild-type EPEC organisms to levels seen with secretion-deficient mutants, suggesting that dephosphorylation events are linked to the antiphagocytic phenotype. No tyrosine phosphatase activity was detected with the EPEC-secreted proteins, suggesting that EPEC induces antiphagocytosis via a different mechanism than Yersinia species. Taken together, the present findings demonstrate a novel function for EPEC-secreted proteins in triggering macrophage protein tyrosine dephosphorylation and inhibition of phagocytosis.
Animals↗
Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7.
Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.
Alleles↗
Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated.
Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.
Adhesins, Bacterial↗
Protein translocation: delivering virulence into the host cell.
A wide variety of plant and human bacterial pathogens use a specialized 'type III' protein secretion system to deliver virulence factors into host cells. Appendage-like surface structures have recently been identified on several bacterial pathogens and there are indications that these may be conduits for virulence factor delivery.
Animals↗
Lipopolysaccharide induces apoptosis in a bovine endothelial cell line via a soluble CD14 dependent pathway.
We investigated the mode of bovine endothelial cell death induced by lipopolysaccharide (LPS). Inter-nucleosomal DNA fragmentation was observed indicating apoptotic cell death. Apoptosis was confirmed by examining cell morphology using confocal laser scanning and transmission electron microscopy, which revealed characteristic nuclear condensation in LPS-treated endothelial cells. Apoptosis was blocked by a monoclonal antibody against CD14, suggesting that LPS triggers apoptosis via a soluble CD14 (sCD14) dependent mechanism.
Animals↗
Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.
The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms. After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles. In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells. Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells. Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells. We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization. We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L. Further, we report on differential processing of lysosomal hydrolases (such as cathepsin D and cathepsin L) associated with the isolated BCVs. Although there was some contamination of the S. typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y. pseudotuberculosis-containing vacuoles were predominately free of ER contamination. The Y. pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time. These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y. pseudotuberculosis traffics to lysosomes where they are degraded. The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y. pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.
Animals↗
Typhoid fever--important issues still remain.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
Intracellular delivery and antibacterial activity of gentamicin encapsulated in pH-sensitive liposomes.
Cell membranes are relatively impermeable to the antibiotic gentamicin, a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections. Liposomal encapsulation of this drug was used in order to achieve intracellular antibiotic delivery and therefore increase the drug's therapeutic activity against intracellular pathogens. Gentamicin encapsulation in several dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoylphosphatidylethanolamine (DOPE)-based carrier systems was characterized. To systematically test the antibacterial efficacies of these formulations, a tissue culture assay system was developed wherein murine macrophage-like J774A.1 cells were infected with bacteria and were then treated with encapsulated drug. Of these formulations, DOPE-N-succinyl-DOPE and DOPE-N-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activities, killing greater than 75% of intracellular vacuole-resident wild-type Salmonella typhimurium compared to the level of killing of the control formulations. These formulations also efficiently eliminated intracellular infections caused by a recombinant hemolysin-expressing S. typhimurium strain and a Listeria monocytogenes strain, both of which escape the vacuole and reside in the cytoplasm. Control non-pH-sensitive liposomal formulations of gentamicin had poor antibacterial activities. A fluorescence resonance energy transfer assay indicated that the efficacious formulations undergo a pH-dependent lipid mixing and fusion event. Intracellular delivery of the fluorescent molecules encapsulated in these formulations was confirmed by confocal fluorescence microscopy and was shown to be dependent on endosomal acidification. This work shows that encapsulation of membrane-impermeative antibiotics in appropriately designed lipid-based delivery systems can enable their use in treating intracellular infections and details the development of a general assay for testing the intracellular delivery of encapsulated drug formulations.
Animals↗
Antigenic analysis of Bordetella pertussis filamentous hemagglutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies.
Although substantial advancements have been made in the development of efficacious acellular vaccines against Bordetella pertussis, continued progress requires better understanding of the antigenic makeup of B. pertussis virulence factors, including filamentous hemagglutinin (FHA). To identify antigenic regions of FHA, phage display libraries constructed by using random fragments of the 10-kbp EcoRI fragment of B. pertussis fhaB were affinity selected with rabbit anti-FHA polyclonal antibodies. Characterization of antibody-reactive clones displaying FHA-derived peptides identified 14 antigenic regions, each containing one or more epitopes. A number of clones mapped within regions containing known or putative FHA adhesin domains and may be relevant for the generation of protective antibodies. The immunogenic potential of the phage-displayed peptides was assessed indirectly by comparing their recognition by antibodies elicited by sodium dodecyl sulfate (SDS)-denatured and native FHA and by measuring the inhibition of this recognition by purified FHA. FHA residues 1929 to 2019 may contain the most dominant linear epitope of FHA. Clones mapping to this region accounted for ca. 20% of clones recovered from the initial library selection and screening procedures. They are strongly recognized by sera against both SDS-denatured and native FHA, and this recognition is readily inhibited by purified FHA. Given also that this region includes a factor X homolog (J. Sandros and E. Tuomanen, Trends Microbiol. 1:192-196, 1993) and that the single FHA epitope (residues 2001 to 2015) was unequivocally defined in a comparable study by E. Leininger et al. (J. Infect. Dis. 175:1423-1431, 1997), peptides derived from residues of 1929 to 2019 of FHA are strong candidates for future protection studies.
Adhesins, Bacterial↗
Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway.
PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.
Androstadienes↗
Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments.
Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.
Bacterial Outer Membrane Proteins↗