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3.
Mol Microbiol ; 42(3): 689-703, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11722735

ABSTRACT

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high-affinity binding of invasin to beta1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPgammaS, but was inhibited in mammalian cells transfected with the interfering Rac1-N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3-binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N-WASP, uptake was analysed in a cell line lacking expression of WASP and N-WASP. Uptake was unaffected by the absence of these proteins, indicating that beta1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N-WASP function.


Subject(s)
Adhesins, Bacterial , Cytoskeletal Proteins , Integrin beta1/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Yersinia pseudotuberculosis/pathogenicity , rac1 GTP-Binding Protein/metabolism , 3T3 Cells , Actin-Related Protein 2 , Actin-Related Protein 3 , Actins/metabolism , Animals , Bacterial Proteins/metabolism , COS Cells , Cell Line , Cell Membrane Permeability , Chlorocebus aethiops , Drug Combinations , Humans , Mice , Oils , Phagosomes/microbiology , Phenols , Wiskott-Aldrich Syndrome Protein, Neuronal , Yersinia pseudotuberculosis Infections/microbiology
4.
J Cell Sci ; 114(Pt 18): 3333-43, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11591821

ABSTRACT

We show that the interaction of the Yersinia surface protein, invasin, with rabbit synovial fibroblasts mediates bead phagocytosis and induces expression of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha) and MMP-1/collagenase-1 (CL-1). Presentation of invasin as a ligand on the surface of 4.5 microm beads induced phagocytosis and increased CL-1 expression 20-fold after 24 hours. By contrast, presentation of invasin as a spreading substrate did not induce CL-1 expression. CL-1 induction following phagocytosis of invasin-coated beads was mediated by a mechanism dependent on high-affinity binding to beta1 integrins and the function of the small GTPase RhoA. Expression of a function-perturbing mutant, RhoAN19, abrogated bead-induced CL-1 expression. RhoA activation coupled bead phagocytosis with signal transduction because expression of constitutively active mutant RhoV14 was sufficient to trigger CL-1 expression. The signal-transduction cascade elicited by bead phagocytosis triggered NFkappaB activation, stimulating a proinflammatory cellular response with transient increases in TNF-alpha production that peaked at 2 hours and induction of IL-1alpha that was sustained for at least 10 hours. Inhibition of IL-1alpha function by blocking antibodies or IL-1 receptor antagonist showed that IL-1alpha is the autocrine intermediary for subsequent CL-1 induction.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/metabolism , Collagenases/metabolism , Fibroblasts/metabolism , Interleukin-1/metabolism , Phagocytosis/physiology , Synovial Membrane/metabolism , Yersinia , Animals , Bacterial Proteins/chemistry , Fibroblasts/cytology , Integrin beta1/metabolism , NF-kappa B/metabolism , Rabbits , Signal Transduction/physiology , Synovial Membrane/cytology , Tumor Necrosis Factor-alpha/metabolism , Yersinia/chemistry , rhoA GTP-Binding Protein/metabolism
5.
J Exp Med ; 194(8): 1081-96, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11602638

ABSTRACT

The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.


Subject(s)
Genes, Bacterial/physiology , Legionella pneumophila/immunology , Macrophages/immunology , Pinocytosis/immunology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cell Membrane/microbiology , Cell Membrane/physiology , Cells, Cultured , Female , Macrophages/cytology , Macrophages/microbiology , Mice , Mice, Inbred A , Mice, Inbred C57BL
6.
Mol Microbiol ; 40(5): 1113-27, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11401716

ABSTRACT

A key event in legionellosis is the ability of Legionella pneumophila to survive and proliferate inside alveolar macrophages. The dot/icm genes, which are necessary for intracellular growth, show sequence similarity to genes encoding conjugative transfer systems, and it is believed that they are responsible for the formation of a secretion apparatus. Evidence is provided here that the IcmR and IcmQ proteins participate in a chaperone-substrate relationship similar to that observed for translocated proteins in type III and type IV secretion apparatuses. Immobilized IcmQ was found to bind IcmR from crude bacterial extracts efficiently. Furthermore, purified IcmR and IcmQ bind with high affinity. This interaction was also observed in vivo by co-immunoprecipitation. The presence of IcmR was found to affect the physical state of IcmQ directly. In the absence of IcmR, IcmQ formed high-molecular-weight complexes both in vivo and in vitro, whereas IcmR prevented and reversed the formation of these complexes.


Subject(s)
Bacterial Proteins/metabolism , Legionella pneumophila , Molecular Chaperones/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Chromatography, Affinity , Macromolecular Substances , Molecular Chaperones/genetics , Molecular Sequence Data , Molecular Weight
7.
Mol Microbiol ; 39(2): 313-29, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11136453

ABSTRACT

Legionella pneumophila grows in human alveolar macrophages and resides within a phagosome that initially lacks proteins associated with the endocytic pathway. Required for targeting to this unique location is the Dot/Icm complex, which is highly similar to conjugative DNA transfer apparatuses. Here, we show that exposure to three distinct inducing conditions resulted in the formation of a fibrous structure on the bacterial cell surface that contained the DotH and DotO proteins. These conditions included: (i) incubation for 2 h with mouse bone marrow-derived macrophages; (ii) incubation for 2 h in macrophage-conditioned media; or (iii) replication of bacteria for 22 h within macrophages. Introduction of bacteria harbouring the surface-exposed DotH and DotO onto a fresh monolayer resulted in loss of the surface localization of DotH and DotO shortly after uptake. Treatments that resulted in the production of the fibrous structure enhanced the rate at which the bacteria were internalized, but there was no corresponding increase in the efficiency of intracellular growth compared with bacteria that had been cultured in broth using conditions that resulted in maximal intracellular growth. These data indicate that the surface-exposed DotH and DotO on L. pneumophila may act either just before lysis from the macrophage or at the earliest stages of infection, transiently relocating in a fibrous structure on the bacterial cell surface.


Subject(s)
Bacterial Proteins/metabolism , Legionella pneumophila/growth & development , Legionella pneumophila/pathogenicity , Legionnaires' Disease/microbiology , Macrophages/microbiology , Animals , Bacterial Proteins/genetics , Cell Membrane Permeability , Cells, Cultured , Culture Media , Endocytosis , Fluorescent Antibody Technique , Gene Deletion , Humans , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Mice , Microscopy, Electron
8.
J Cell Sci ; 114(Pt 1): 21-28, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11112686

ABSTRACT

Enteropathogenic Yersinia are gram-negative bacterial species that translocate from the lumen of the intestine and are able to grow within deep tissue sites. During the earliest stages of disease, the organism is able to bind integrin receptors that are presented on the apical surface of M cells in the intestine, which allows its internalization and subsequent translocation into regional lymph nodes. The primary integrin substrate is the outer-membrane protein invasin, which binds with extraordinarily high affinity to at least five different integrins that have the (beta)(1) chain. Bacterial uptake into host cells is modulated by the affinity of receptor-substrate interaction, receptor concentration and the ability of the substrate to aggregate target receptors.


Subject(s)
Adhesins, Bacterial , Integrins/metabolism , Yersinia/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/metabolism , Humans , Molecular Sequence Data , Signal Transduction , Yersinia/physiology , Yersinia Infections/microbiology
9.
Trends Microbiol ; 8(10): 478-80, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11044684

ABSTRACT

Legionella pneumophila, the Gram-negative bacterium that causes Legionnaires' disease, can be cultured in the laboratory in a variety of fresh-water amoebae and macrophage-like cell lines. None of these hosts, however, is amenable to genetic analysis, which has limited the ability of researchers to analyse the host factors essential for L. pneumophila growth. In this article, we describe a novel method in which L. pneumophila is grown within the soil amoeba Dictyostelium discoideum and how D. discoideum genetics is being used to analyse the host cell factors involved in L. pneumophila pathogenesis.


Subject(s)
Dictyostelium/microbiology , Legionella pneumophila/growth & development , Animals , Bacteriological Techniques , Dictyostelium/genetics , Legionella pneumophila/genetics , Legionnaires' Disease/microbiology , Mutation
10.
Microbes Infect ; 2(7): 793-801, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10955960

ABSTRACT

The invasin protein encoded by enteropathogenic Yersinia allows entry of bacteria into intestinal M cells by binding to integrin receptors. In cultured cells, invasin-mediated uptake requires proteins involved in endocytosis and signaling to the cell cytoskeleton. At least four different factors have been demonstrated to play a role in regulating the efficiency of invasin-promoted uptake. These include receptor-ligand affinity, receptor clustering, signaling through focal adhesion kinase, and stimulation of cytoskeletal rearrangements by small GTP binding proteins.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/metabolism , Integrins/metabolism , Yersinia/pathogenicity , Animals , Bacterial Adhesion , Bacterial Proteins/chemistry , Cytoskeleton/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , GTP-Binding Proteins/metabolism , Humans , Integrins/chemistry , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Yersinia/chemistry , Yersinia/physiology
12.
Infect Immun ; 68(5): 2930-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10768991

ABSTRACT

The binding of the Yersinia pseudotuberculosis and Yersinia enterocolitica invasin proteins to beta(1) integrin receptors allows internalization of these organisms by cultured cells. The C-terminal 192-residue superdomain of the Y. pseudotuberculosis invasin is necessary and sufficient for integrin recognition, while a region located outside, and N-terminal to, this superdomain strongly enhances the efficiency of bacterial uptake. Within the enhancer region is a domain called D2 that allows invasin-invasin interaction. To investigate the role of the enhancer region, bacterial cell binding and entry mediated by the Y. pseudotuberculosis invasin protein (invasin(pstb)) was compared to that of Y. enterocolitica invasin (invasin(ent)), which lacks the D2 self-association domain. Invasin(ent) was shown to be unable to promote self-interaction, using the DNA binding domain of lambda repressor as a reporter. Furthermore, two genetically engineered in-frame deletion mutations that removed D2 from invasin(pstb) were significantly less proficient than wild-type invasin(pstb) at promoting uptake, although the amount of surface-exposed invasin as well as the cell binding capacity of the recombinant Escherichia coli strains remained similar. Competitive uptake assays showed that E. coli cells expressing invasin(pstb) had a significant advantage in the internalization process versus either E. coli cells expressing invasin(ent) or the invasin(pstb) derivatives deleted for D2, further demonstrating the importance of invasin self-interaction for the efficiency of invasin-mediated uptake.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/immunology , Endocytosis/immunology , Immunoglobulins/immunology , Integrin beta1/immunology , Yersinia pseudotuberculosis/immunology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Humans , Immunoglobulins/chemistry , Immunoglobulins/genetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Tumor Cells, Cultured , Two-Hybrid System Techniques
13.
Infect Immun ; 68(5): 2939-47, 2000 May.
Article in English | MEDLINE | ID: mdl-10768992

ABSTRACT

Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible to L. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some known D. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and the myoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a G(beta) mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.


Subject(s)
Dictyostelium/microbiology , Legionella pneumophila/growth & development , Animals , Culture Media , Dictyostelium/genetics , Genes, Bacterial , Intracellular Fluid/microbiology , Intracellular Membranes , Legionella pneumophila/genetics , Lysosomes , Membrane Proteins
14.
Cell Microbiol ; 2(3): 219-30, 2000 Jun.
Article in English | MEDLINE | ID: mdl-11207578

ABSTRACT

A highly conserved 14-amino-acid region of the chicken beta1 integrin chain (beta1chk residues 151-168) hypothesized to be involved in divalent cation coordination was analysed to determine whether invasin uses the same structural determinants as fibronectin (Fn) to recognize receptors. For the most part, both proteins required similar beta1 chain residues for integrin recognition, although the relative preference of the integrin for the two substrates could be inverted mutationally. Substitution mutations in the amino terminal residues of this region resulted in defective binding to both substrates by the receptor, while substitutions at the carboxyl-terminal end of this region were better tolerated. A derivative carrying the double substitution (KDD160RDV) had the unique phenotype of maintaining Fn binding while abolishing invasin binding, indicating that this region of the receptor may determine substrate specificity. These data indicate that the integrin beta1 chain possesses a ligand binding site shared by invasin and Fn that can be altered by mutation to allow greater preference for the normally lower affinity substrate Fn than for invasin. It was also established that the region analysed has the ability to bind divalent cations.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/metabolism , Integrin beta1/chemistry , Integrin beta1/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cations, Divalent/metabolism , Chickens , Fibronectins/chemistry , Fibronectins/metabolism , Humans , Integrin beta1/genetics , Maltose-Binding Proteins , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Transfection
15.
Science ; 286(5438): 291-5, 1999 Oct 08.
Article in English | MEDLINE | ID: mdl-10514372

ABSTRACT

The Yersinia pseudotuberculosis invasin protein promotes bacterial entry by binding to host cell integrins with higher affinity than natural substrates such as fibronectin. The 2.3 angstrom crystal structure of the invasin extracellular region reveals five domains that form a 180 angstrom rod with structural similarities to tandem fibronectin type III domains. The integrin-binding surfaces of invasin and fibronectin include similarly located key residues, but in the context of different folds and surface shapes. The structures of invasin and fibronectin provide an example of convergent evolution, in which invasin presents an optimized surface for integrin binding, in comparison with host substrates.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/chemistry , Integrins/metabolism , Yersinia pseudotuberculosis/chemistry , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Evolution, Molecular , Fibronectins/chemistry , Fibronectins/metabolism , Hydrogen Bonding , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Secondary , Yersinia pseudotuberculosis/metabolism
16.
EMBO J ; 18(5): 1199-213, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10064587

ABSTRACT

Invasin allows efficient entry into mammalian cells by Yersinia pseudotuberculosis. It has been shown that the C-terminal 192 amino acids of invasin are essential for binding of beta1 integrin receptors and subsequent uptake. By analyzing the internalization of latex beads coated with invasin derivatives, an additional domain of invasin was shown to be required for efficient bacterial internalization. A monomeric derivative encompassing the C-terminal 197 amino acids was inefficient at promoting entry of latex beads, whereas dimerization of this derivative by antibody significantly increased uptake. By using the DNA-binding domain of lambda repressor as a reporter for invasin self-interaction, we have demonstrated that a region of the invasin protein located N-terminal to the cell adhesion domain of invasin is able to self-associate. Chemical cross-linking studies of purified and surface-exposed invasin proteins, and the dominant-interfering effect of a non-functional invasin derivative are consistent with the presence of a self-association domain that is located within the region of invasin that enhances bacterial uptake. We conclude that interaction of homomultimeric invasin with multiple integrins establishes tight adherence and receptor clustering, thus providing a signal for internalization.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/genetics , Integrin beta1/metabolism , Yersinia pseudotuberculosis/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Line , Cell Membrane Permeability , Cross-Linking Reagents , Genes, Reporter , Microspheres , Molecular Sequence Data , Mutation , Peptide Fragments/metabolism , Phagocytosis/genetics , Protein Binding , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics
17.
Curr Opin Microbiol ; 2(1): 30-4, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10047559

ABSTRACT

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.


Subject(s)
Caenorhabditis elegans Proteins , Escherichia coli Proteins , Legionella pneumophila/physiology , Periplasmic Proteins , Virulence Factors , Amoeba/microbiology , Animals , Bacterial Proteins/genetics , Carrier Proteins/genetics , Culture Media , Cytotoxins/biosynthesis , Genes, Bacterial/genetics , Humans , Legionella pneumophila/genetics , Legionella pneumophila/growth & development , Legionella pneumophila/pathogenicity , Macrophages/microbiology , Microtubule-Associated Proteins/genetics , Repressor Proteins/genetics , Sodium Chloride , U937 Cells , Virulence
18.
J Biol Chem ; 273(48): 31837-43, 1998 Nov 27.
Article in English | MEDLINE | ID: mdl-9822651

ABSTRACT

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.


Subject(s)
Adhesins, Bacterial , Antigens, CD/physiology , Bacterial Adhesion/physiology , Bacterial Proteins/metabolism , Integrins/physiology , Point Mutation , Yersinia pseudotuberculosis/physiology , Amino Acid Sequence , Antigens, CD/chemistry , Antigens, CD/genetics , Cell Adhesion Molecules/metabolism , Humans , Integrin alpha3 , Integrin alpha3beta1 , Integrins/chemistry , Integrins/genetics , K562 Cells , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transfection , Kalinin
19.
Proc Natl Acad Sci U S A ; 95(23): 13658-63, 1998 Nov 10.
Article in English | MEDLINE | ID: mdl-9811856

ABSTRACT

High-efficiency entry of the enteropathogenic bacterium Yersinia pseudotuberculosis into nonphagocytic cells is mediated by the bacterial outer membrane protein invasin. Invasin-mediated uptake requires high affinity binding of invasin to multiple beta1 chain integrin receptors on the host eukaryotic cell. Previous studies using inhibitors have indicated that high-efficiency uptake requires tyrosine kinase activity. In this paper we demonstrate a requirement for focal adhesion kinase (FAK) for invasin-mediated uptake. Overexpression of a dominant interfering form of FAK reduced the amount of bacterial entry. Specifically, the autophosphorylation site of FAK, which is a reported site of c-Src kinase binding, is required for bacterial internalization, as overexpression of a derivative lacking the autophosphorylation site had a dominant interfering effect as well. Cultured cells expressing interfering variants of Src kinase also showed reduced bacterial uptake, demonstrating the involvement of a Src-family kinase in invasin-promoted uptake.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/physiology , Cell Adhesion Molecules/physiology , Protein-Tyrosine Kinases/physiology , Yersinia pseudotuberculosis Infections/microbiology , Yersinia pseudotuberculosis/physiology , Animals , Cells, Cultured , Chick Embryo , Fibroblasts/enzymology , Fibroblasts/microbiology , Focal Adhesion Protein-Tyrosine Kinases , Phosphorylation , Signal Transduction
20.
Biochemistry ; 37(31): 10945-55, 1998 Aug 04.
Article in English | MEDLINE | ID: mdl-9692987

ABSTRACT

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.


Subject(s)
Adhesins, Bacterial , Bacterial Proteins/metabolism , Cell Adhesion Molecules/metabolism , Integrins/genetics , Integrins/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Animals , Cell Line , Drosophila , Genetic Vectors/chemical synthesis , Genetic Vectors/metabolism , Humans , Integrin alpha3beta1 , Integrins/metabolism , Leukemia, Erythroblastic, Acute , Protein Binding , Rats , Recombinant Fusion Proteins/biosynthesis , Solubility , Titrimetry , Tumor Cells, Cultured , Yersinia/physiology , Kalinin
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