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R R Isberg

Publications and source records attributed to R R Isberg.

At least 37 records · Page 2Linked to original sources
PubMed1998

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

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.

Adhesins, Bacterial↗
PubMed1998

SWIM analysis allows rapid identification of residues involved in invasin-mediated bacterial uptake.

The Yersinia pseudotuberculosis invasin protein promotes bacterial uptake into normally non-phagocytic cells. Combinations of six alanine substitutions in a region of invasin previously shown to be important for bacterial internalization were analyzed using binomial and codon mutagenesis strategies. A single pool of mutants, potentially containing 64 derivatives with various combinations of alanine substitutions, was enriched by one passage through HEp2 cells. DNA was isolated from the resulting pool of internalization-competent bacteria and sequenced in a single set of reactions to determine which alanine substitutions maintained activity. Results of the single sequencing run performed on the pool indicated that strains harboring the D911A substitution were absent after enrichment, confirming the importance of an aspartate residue at this site. When single clones were subsequently isolated from the pool, those containing multiple alanine substitutions in invasin showed uptake defects that were additive, with the exception of S904A/M912A and S910A/M912A double mutants. Binomial mutagenesis combined with a pooled enrichment and sequencing strategy, called 'SWIM' mutagenesis (selection without isolation of mutants), could be applied to any system for which there exists an enrichment scheme, using a single oligonucleotide pool to analyze multiple residues.

Adhesins, Bacterial↗
PubMed1998

Conjugative transfer by the virulence system of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' pneumonia, replicates within alveolar macrophages by preventing phagosome-lysosome fusion. Here, a large number of mutants called dot (defective for organelle trafficking) that were unable to replicate intracellularly because of an inability of the bacteria to alter the endocytic pathway of macrophages were isolated. The dot virulence genes encoded a large putative membrane complex that functioned as a secretion system that was able to transfer plasmid DNA from one cell to another.

Antigens, CD↗
PubMed1998

Evidence for pore-forming ability by Legionella pneumophila.

Legionella pneumophila is the cause of Legionnaires' pneumonia. After Internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L. pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L. pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.

Animals↗
PubMed1998

Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake.

Numerous intracellular bacterial pathogens modulate the nature of the membrane-bound compartment in which they reside, although little is known about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophages and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate trafficking of the L. pneumophila phagosome. Phagosomes containing L. pneumophila dotA+ bacteria exhibited differential trafficking profiles when compared with isogenic dotA mutants. Phagosomes containing dotA mutants showed rapid accumulation of the lysosomal glycoprotein LAMP-1 as early as 5 min after uptake, whereas the majority of wild-type L. pneumophila phagosomes did not acquire LAMP-1. The association of LAMP-1 with phagosomes containing dotA mutant bacteria was concomitant with the appearance of the small GTP-binding protein Rab7 on the vacuolar membrane. These data demonstrate that phagosomes containing replication-competent L. pneumophila evade early endocytic fusion events. In contrast, the kinetics of LAMP-1 and Rab7 association indicate that the dotA mutants are routed along a well-characterized endocytic pathway leading to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establish an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to maintain a replicative phagosome. These data indicate that DotA is one factor that plays a fundamental role in regulating initial phagosome trafficking decisions either upon or immediately after macrophage uptake.

Animals↗
PubMed1998

Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway.

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The delta dotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.

Animals↗
PubMed1997

Transcriptional regulation of the Yersinia pseudotuberculosis pH6 antigen adhesin by two envelope-associated components.

The Yersinia pseudotuberculosis pH6 antigen mediates haemagglutination and adhesion to cultured mammalian cells. The synthesis of pH6 antigen requires the products of the psaEFABC genes in both Yersinia pseudotuberculosis and Escherichia coli. In-frame deletion mutations of psaE and psaF caused defective haemagglutination. In contrast, we showed that the psaABC genes were sufficient for haemagglutination if they were expressed by a heterologous promoter. Environmental regulation of pH6 antigen by temperature and pH occurs via regulation of the major pilus protein PsaA at the transcriptional level. Northern blot analyses indicate that the psaA transcript was absent in either psaE or psaF mutant strains. Primer extension analyses indicate that, in Y. pseudotuberculosis, the transcription of the psaE and psaF genes is constitutive. Alkaline phosphatase fusion studies confirm the topology prediction that PsaE and PsaF are both inner-membrane-associated proteins. PsaE consists of an N-terminal cytoplasmic domain, containing sequence similarity to transcriptional regulators found in two-component systems as well as to the Salmonella typhimurium HIIA protein, with a C-terminal domain that is periplasmically localized. PsaF is predicted to be oriented with most of the protein in the periplasm, the hydrophobic N-terminus being either integrated in the inner membrane or cleaved as a signal peptide.

Adhesins, Bacterial↗
PubMed1997

Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 and 73 amino acids and a carboxyl-terminal cytoplasmic domain of 122 amino acids. Protein fractionation studies were consistent with DotA residing in the inner membrane. An alkaline phosphatase fusion located 9 amino acids upstream from the C terminus of DotA still retained function and was able to restore intracellular growth when harbored by two L. pneumophila dotA mutants. A hybrid protein from which the carboxyl-terminal 48 amino acids of DotA were deleted was unable to complement the intracellular growth defect in the dotA mutants, indicating that this cytoplasmic region is required for function.

Amino Acid Sequence↗
PubMed1997

Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis.

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.

Amino Acid Sequence↗
PubMed1997

Invasin-dependent and invasin-independent pathways for translocation of Yersinia pseudotuberculosis across the Peyer's patch intestinal epithelium.

Yersinia pseudotuberculosis initiates systemic disease after translocation across the intestinal epithelium. Three Y. pseudotuberculosis factors, previously identified by their ability to promote association with cultured cells, were evaluated for their relative roles in translocation. To this end, mutants defective for invasin, YadA, or pH 6 antigen were tested for movement from the intestinal lumen into the subepithelium. Within 45 min after introduction of bacteria into the lumen, wild-type bacteria were found in the Peyer's patch. Mutants expressing defective invasin derivatives were unable to promote efficient translocation into the Peyer's patch and instead colonized on the luminal surface of the intestinal epithelium. In particular, a translocation defect was observed in a Y. pseudotuberculosis strain that expressed an uptake-defective invasin protein retaining considerable receptor binding activity. To attempt to reduce binding to luminal mucus, Y. pseudotuberculosis yadA and inv yadA strains were analyzed. Both strains had reduced mucus binding, with the inv yadA mutant revealing an alternate uptake pathway that was invasin independent. A mutant defective in the production of the pH 6 antigen adhesin also showed reduced binding to luminal mucus, with specific localization of bacteria in M cells. These results indicate that Y. pseudotuberculosis adhesive factors control the site of bacterial interaction within the intestinal environment and that loss of one factor causes drastic changes in the preferred site of localization of the bacterium in this locale.

Adhesins, Bacterial↗
PubMed1996

Analysis of the intracellular fate of Legionella pneumophila mutants.

L. pneumophila is a model organism for investigating the mechanisms by which intracellular pathogens acquire the metabolites needed for replication while evading the microbicidal mechanisms of the macrophage. We determined that intracellular L. pneumophila replicate in close association with the endoplasmic reticulum and suggest that L. pneumophila exploits the macrophage autophagy pathway to establish this specialized vacuole. To identify the bacterial factors required at this step as well as the factors important for other stages of the intracellular pathway, we isolated a collection of bacterial mutants that are defective for growth in macrophages. The ability of the mutant strains to evade fusion with the lysosomes and to establish replication vacuoles was examined by fluorescence microscopic localization of markers for the late endosomes (lgp 120), lysosomes (Texas Red-ovalbumin), endoplasmic reticulum (BiP), and L. pneumophila. By this approach, we identified mutants with distinct intracellular fates; one type does not evade the endocytic pathway, another forms replication vacuoles less efficiently than does wild-type, and a third type forms replication vacuoles but replicates poorly. These mutants are likely to facilitate identification and characterization of the bacterial factors required by L. pneumophila to establish a protected niche for intracellular replication.

Animals↗
PubMed1996

A region of the Yersinia pseudotuberculosis invasin protein that contributes to high affinity binding to integrin receptors.

The entry of Yersinia pseudotuberculosis into cultured mammalian cells is mediated by the bacterial protein invasin. The mammalian receptors for invasin are five beta1 chain integrins. Site-directed mutagenesis of the aspartate and lysine residues in the 192-amino acid integrin binding domain of invasin was performed to identify regions, in addition to the previously characterized 903-913 region, that are important for integrin binding. One mutation, D811A, resulted in depressed ability of invasin to bind purified alpha5beta1 and to promote bacterial entry. Further mutational analysis of Asp-811 indicated that an oxygen-containing side chain is required at this position. A second nearby residue, Phe-808, was also shown to be important for integrin binding, as an alanine substitution at this site had properties similar to the Asp-811 mutation. This mutational analysis has therefore identified a second region that, in conjunction with residues 903-913, is required for wild type levels of integrin binding. The contribution to binding by two noncontiguous sites in the primary sequence parallels results that indicate two domains of fibronectin are involved in integrin binding.

Adhesins, Bacterial↗
PubMed1996

Common entry mechanisms. Bacterial pathogenesis.

The recent identification of E-cadherin as the cell-surface receptor for the Listeria monocytogenes adhesin, internalin, indicates that pathogenic bacteria exploit a variety of host surface molecules for entry and that they may share some common strategies for uptake into host cells.

Adhesins, Bacterial↗
PubMed1996

Mutations in the cytoplasmic domain of the integrin beta1 chain indicate a role for endocytosis factors in bacterial internalization.

Mutations that result in defective beta1-integrin focal adhesion formation were analyzed for effects on bacterial internalization. Mutations in the cytoplasmic domain of the beta1 chain that disrupt the sequence NPIY resulted in integrins deficient in bacterial uptake. Other mutations in the beta1 chain that reduced cytoskeletal association showed enhanced bacterial uptake. Replacement of the NPIY sequence of the beta1 subunit by the endocytosis internalization sequence PPGY resulted in integrin receptors highly proficient in bacterial internalization, yet severely defective in focal contact localization. Electron microscopy indicated that coated structures associated specifically with bacteria-binding beta1-integrins, with an apparent recruitment of coated pits from ventral cell surfaces to apical surfaces corresponding to nascent bacterial phagosomes. Clathrin inhibition studies indicated a role for the adaptor molecule AP2 as well as clathrin in integrin-mediated bacterial internalization. These results indicate that association of beta1-integrins with the cytoskeleton at focal contacts interferes with integrin-mediated bacterial internalization. Also, although actin polymerization is required for bacterial uptake, clathrin is probably involved in bacterial uptake promoted by beta-1-integrins.

Amino Acid Sequence↗