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Biomedical subjects

R R Isberg

Publications and source records attributed to R R Isberg.

At least 55 records · Page 3Linked to original sources
PubMed1996

The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells.

Yersinia pseudotuberculosis inv mutant strains cured of the virulence plasmid exhibit thermoinducible adhesion to cultured mammalian cells. To identify the genes responsible for this phenotype, Y. pseudotuberculosis homologs of the Y. enterocolitica ail and the Y. pestis psa loci were identified. Mutations in the Y. pseudotuberculosis ail and psa loci were constructed and tested for thermoinducible binding. Results of cellular binding assays indicated that only mutations in psa, not in ail, resulted in defects for thermoinducible binding, with inv yadA psa strains showing no detectable cell adhesion. In addition, an inv psa strain was defective for hemagglutination of sheep erythrocytes, in contrast to an inv psa+ strain which was fully competent for hemagglutination. The introduction of a plasmid containing a 6.7-kb KpnI-ClaI fragment of Y. pseudotuberculosis encompassing the psa locus was sufficient to complement both the cell adhesion and hemagglutination defects of the psa mutant. Results from subcloning and transposon mutagenesis indicated that the complete 6.7-kb region was required for thermoinducible binding and hemagglutination.

Amino Acid Sequence↗
PubMed1996

Identification of Legionella pneumophila mutants that have aberrant intracellular fates.

After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.

Animals↗
PubMed1995

Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13.

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene.

Animals↗
PubMed1995

An aspartate residue of the Yersinia pseudotuberculosis invasin protein that is critical for integrin binding.

The Yersinia pseudotuberculosis invasin protein mediates bacterial entry into mammalian cells by binding multiple beta 1-chain integrins. Invasin binding to purified alpha 5 beta 1 integrin is inhibited by Arg-Gly-Asp (RGD)-containing peptides, although invasin contains no RGD sequence. Fifteen mutations that diminished binding and bacterial entry were isolated after mutagenesis of the entire inv gene. All of the mutations altered residues within the C-terminal 192 amino acids of invasin, previously delineated as the integrin binding domain, and 10 of the mutations fell within an 11 residue region. This small region was subjected to site-directed mutagenesis and almost half of the 35 mutations generated decreased invasin-mediated entry. D911 within this region was the most critical residue, as even a conservative glutamate substitution abolished bacterial penetration. Purified invasin derivatives altered at this residue were defective in promoting cell attachment and this defect was reflected in a 10-fold or greater increase in IC50 for integrin binding. D911 may have a function similar to that of the aspartate residue in RGD-containing sequences.

Adhesins, Bacterial↗
PubMed1995

The mechanism of phagocytic uptake promoted by invasin-integrin interaction.

Many pathogenic bacterial species produce factors that promote their internalization by host cells. The crucial components for uptake of one such pathogen, Yersinia pseudotuberculosis, have been identified. Efficient uptake of this microorganism requires tight binding of the bacterial invasin protein to integrins on the cell surface. Internalization also involves coordination of signals responsible for cytoskeletal rearrangements and those involved in receptor-mediated endocytosis. A start is being made to define the proteins that are required for efficient completion of the internalization process.

Journal Article↗
PubMed1995

Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication.

Animals↗
PubMed1994

Binding and internalization of microorganisms by integrin receptors.

Many microbial pathogens bind host-cell integrin receptors. These interactions are promoted either by a host protein binding the microorganism or by a surface-localized ligand encoded by the pathogen. Attachment facilitates extracellular adhesion of the microorganism or internalization by the host cell.

Amino Acid Sequence↗
PubMed1994

Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene.

Legionella pneumophila dot mutations cause defects in intracellular targeting of the microorganism within cultured macrophages. Each of the previously characterized dot mutations was shown to be complemented by a single open reading frame designated dotA. The defects caused by the mutations appear to be due to disrupted function of the predicted 1048-amino-acid residue DotA protein, and not by polarity effects on a downstream gene. Complementation studies indicated that the product of the dotA53 mutation results in a partially functional DotA protein, consistent with a stable N-terminal fragment having biological activity.

Amino Acid Sequence↗
PubMed1993

A 76-amino acid disulfide loop in the Yersinia pseudotuberculosis invasin protein is required for integrin receptor recognition.

The Yersinia pseudotuberculosis invasin protein is a 986-amino acid protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.

Adhesins, Bacterial↗
PubMed1993

Residues added to the carboxyl terminus of the Yersinia pseudotuberculosis invasin protein interfere with recognition by integrin receptors.

Escherichia coli strains encoding the Yersinia pseudotuberculosis invasin protein are efficiently internalized by mammalian cells. Bacterial uptake into cultured cell lines became defective, however, if invasin was altered by fusion of its carboxyl terminus to E. coli alkaline phosphatase or by the addition of two hydrophobic amino acids to its carboxyl-terminal end. Probing with anti-invasin monoclonal antibodies revealed that the amino-terminal end of invasin was properly localized on the bacterial cell surface in strains encoding invasin with 2 additional amino acids, whereas the carboxyl terminus was not accessible to the monoclonal antibody. Therefore, the 2 additional amino acids interfered with the folding or orientation of the carboxyl terminus in the outer membrane. Alkylation experiments in the absence of reduction indicated that this defect was not caused by a gross inability to form a critical disulfide bond. Revertants were selected from a strain encoding this mutant protein by enriching for organisms able to enter cultured mammalian cells. The vast majority of revertants that were isolated following this enrichment contained a stop codon at the usual position found in the wild type inv gene. The most efficient of the remaining revertants resulted in the introduction of a glycine residue at the site of the wild type stop codon, presumably restoring proper conformation of the carboxyl-terminal region.

ATP-Binding Cassette Transporters↗
PubMed1993

Very late antigen 4-dependent adhesion and costimulation of resting human T cells by the bacterial beta 1 integrin ligand invasin.

Bacteria and viruses often use the normal biological properties of host adhesion molecules to infect relevant host cells. The outer membrane bacterial protein invasin mediates the attachment of Yersinia pseudotuberculosis to human cells. In vitro studies have shown that four members of the very late antigen (VLA) integrin family of adhesion molecules, VLA-3, VLA-4, VLA-5, and VLA-6, can bind to invasin. Since CD4+ T cells express and use these integrins, we have investigated the interaction of CD4+ T cells with purified invasin. Although VLA integrin-mediated adhesion of T cells to other ligands such as fibronectin does not occur at high levels unless the T cells are activated, resting T cells bind strongly to purified invasin. The binding of resting T cells to invasin requires metabolic activity and an intact cytoskeleton. Although CD4+ T cells express VLA-3, VLA-4, VLA-5, and VLA-6, monoclonal antibody (mAb) blocking studies implicate only VLA-4 as a T cell invasin receptor. Like other integrin ligands, invasin can facilitate T cell proliferative responses induced by a CD3-specific mAb. These results suggest that the nature of the integrin ligand is a critical additional factor that regulates T cell integrin activity, and that direct interactions of T cells with bacterial pathogens such as Yersinia may be relevant to host immune responses to bacterial infection.

Adhesins, Bacterial↗
PubMed1993

Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density.

Binding of bacteria to beta 1 chain integrin receptors results in either bacterial adherence or uptake by cultured cells (Isberg, 1991). In this report we show that Staphylococcus aureus coated with high affinity ligands for the beta 1 chain integrin family can be internalized efficiently, whereas bacteria coated with low affinity ligands are poorly internalized. Overproduction of the alpha 5 beta 1 integrin increased the efficiency of bacterial internalization, indicating that the uptake efficiency is directly related to the level of expression of the receptor. By using latex beads or S. aureus coated with mAbs directed against the alpha 5 beta 1 integrin, a roughly semi-logarithmic correlation was observed between the affinity of the receptor-ligand interaction and the rate of bacterial internalization. Evidence is presented that high affinity binding of the bacterium allows the microorganism to compete efficiently with receptor-ligand interactions at the basolateral surface of the cell.

Adhesins, Bacterial↗
PubMed1993

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila.

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome-lysosome fusion and association of host cell organelles with bacteria-containing phagosomes ('recruitment'). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome-lysosome fusion to mutants belonging to both phenotypic classes.

DNA Transposable Elements↗
PubMed1993

Cellular internalization in the absence of invasin expression is promoted by the Yersinia pseudotuberculosis yadA product.

The Yersinia pseudotuberculosis invasin protein is able to promote bacterial penetration into mammalian cells. Insertion mutations that eliminate production of this protein show residual internalization that is dependent on the presence of the Yersinia virulence plasmid. An enrichment procedure was used to isolate molecular clones containing regions of the virulence plasmid that confer this low-level uptake on Y. pseudotuberculosis inv mutants. All of the Y. pseudotuberculosis strains isolated from this procedure harbored plasmids containing a region encompassing the yadA gene, which encodes a previously identified adhesin associated with attachment to extracellular matrix proteins. All of the mutations isolated that affected internalization of one of the strains that survived the enrichment disrupted the yadA open reading frame. Furthermore, a strain that contained yadA sequences and no other region of the virulence plasmid was able to promote internalization of a Y. pseudotuberculosis inv mutant. Consistent with these results, an intact virulence plasmid containing an insertion mutation in yadA was as defective as a plasmid-cured strain at promoting uptake of Y. pseudotuberculosis inv mutants. These results indicate that the product of the yadA gene is responsible for the plasmid-dependent entry observed in Y. pseudotuberculosis inv mutants.

Adhesins, Bacterial↗