Biomedical subjects

J M Patti

Publications and source records attributed to J M Patti.

27 records · Page 2Linked to original sources

PubMed1994

Fibronectin receptors from gram-positive bacteria: comparison of active sites.

Many parasitic bacteria express fibronectin binding proteins that are located on the cell surface. These proteins may act as adhesins and mediate the adherence of the microorganisms to fibronectin-containing host tissues. The ligand binding sites in the fibronectin receptor proteins from Gram-positive bacteria are composed of unique 37-48 amino acid long motifs that are repeated 3-4 times. We have now expressed the ligand binding sites of fibronectin receptors from Staphylococcus aureus, Streptococcus dysgalactiae (two receptors), and Streptococcus pyogenes as recombinant proteins. The purified recombinant proteins have the expected molecular weights as indicated by electrospray mass spectroscopy although they migrate abnormally on SDS-PAGE. Each recombinant protein effectively inhibited the binding of 125I-labeled intact fibronectin or the N-terminal fibronectin domain to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus pyogenes. The relative inhibitory potency of the different recombinant proteins was similar for all target bacteria and is reflected in their relative affinities for fibronectin. Synthetic peptides corresponding to the repeat units of the ligand binding site of the fibronectin receptor proteins were shown to inhibit the binding of the N-terminal fibronectin fragment to Streptococcus pyogenes cells. Together with amino acid sequence comparison, these data demonstrate that the repeat motif of the fibronectin receptor of Streptococcus pyogenes conforms to the consensus sequence previously reported for the Staphylococcus aureus receptor and to one of the Streptococcus dysgalactiae receptors (McGavin et al., 1993).

Amino Acid Sequence↗

PubMed1994

Microbial adhesins recognizing extracellular matrix macromolecules.

Microorganisms express a family of cell-surface adhesins that specifically recognize and bind components of the extracellular matrix. Adhesion of microorganisms to host tissues represents a critical phase in the development of many types of infections. Recent studies have focused on the mechanisms of microbial attachment at a molecular level, including the identification of ligand-binding domains in several cell-surface adhesins from Gram-positive bacteria and the construction of adhesin-deficient isogenic mutants.

Adhesins, Bacterial↗

PubMed1994

The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus.

Adhesins, Bacterial↗

PubMed1994

MSCRAMM-mediated adherence of microorganisms to host tissues.

Microbial adhesion to host tissue is the initial critical event in the pathogenesis of most infections and, as such, is an attractive target for the development of new antimicrobial therapeutics. Specific microbial components (adhesins) mediate adherence to host tissues by participating in amazingly sophisticated interactions with host molecules. This review focuses on a class of cell surface adhesins that specifically interact with extracellular matrix components and which we have designated MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). MSCRAMMs recognizing fibronectin-, fibrinogen-, collagen-, and heparin-related polysaccharides are discussed in terms of structural organization, ligand-binding structures, importance in host tissue colonization and invasion, and role as virulence factors.

Adhesins, Bacterial↗

PubMed1993

Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus.

We have recently shown that the expression of a collagen adhesin is both necessary and sufficient to mediate the attachment of Staphylococcus aureus to cartilage, a complex collagen-containing substrate [Switalski, L. M., Patti, J. M., Butcher, W., Gristina, A. G., Speziale, P., & Höök, M. (1993) Mol. Microbiol. 7, 99-107]. We now report on the localization of the ligand binding site within the 135-kDa S. aureus collagen adhesin. Using deletion mutagenesis in combination with Western ligand blot and direct binding assays, the collagen binding domain (CBD) was localized to a 168 amino acid long segment [CBD(151-318)] within the N-terminal portion of the adhesin. Using biospecific interaction analysis, pepsin-digested bovine type II collagen was found to contain eight binding sites for CBD(151-318); two binding sites were of "high" affinity (Kd = 3 microM) and six sites were of low affinity (Kd = 30 microM). Short truncations in the terminal flanking regions of CBD(151-318) resulted in two CBDs (180-318 and 151-297) that lacked collagen binding activity. Analysis by circular dichroism of the recombinant CBDs in the far UV revealed similar secondary structures, predominantly beta-sheet, whereas the near-UV spectra indicated dramatic changes in the degree of intermolecular packing (tertiary structure). The deduced amino acid sequence of the ligand binding domain of the collagen adhesin is presented.

Adhesins, Bacterial↗

PubMed1993

A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin.

Streptococcus pyogenes secretes an extracellular cysteine protease that cleaves human interleukin 1 beta precursor to form biologically active IL-1 beta, a major cytokine mediating inflammation and shock. To further investigate the potential role of the cysteine protease in host-parasite interactions, the enzyme was purified to apparent homogeneity and tested for ability to degrade several human extracellular matrix proteins. Purified protease cleaved fibronectin, apparently at specific sites, and rapidly degraded vitronectin. In contrast, the protease did not have substantial activity against laminin. The cysteine protease also cleaved fibronectin from human umbilical vein endothelial cells grown in vitro. Allelic variation in the cysteine protease structural gene was studied in 67 strains expressing 39 M protein serotypes and five provisional M serologic types, and representing 50 phylogenetically distinct clones identified by multilocus enzyme electrophoresis. The gene is well conserved and allelic variation is due solely to accumulation of point mutations. Based on predicted amino acid sequences, one mature cysteine protease variant would be made by clones expressing serotypes M2, M3, M4, M5, M6, M9, M10, M11, M12, M14, M18, M22, M23, M25, M27, M41, M49, M56, M59, two provisional M types, and two clones non-typeable for M protein. Moreover, 33 of the 39 speB alleles identified encode one of three mature protease variants that differ from one another at only one or two amino acids clustered in a ten-amino acid region. All 39 alleles, and virtually all strains, encode a product that reacts with polyclonal antisera specific for purified cysteine protease. No compelling evidence was found for a primitive differentiation of the speB gene into two distinct classes, as has been proposed for M protein, opacity factor phenotype, and vir regulon architecture. The results demonstrate that the cysteine protease is well conserved in natural populations of S. pyogenes, provide additional evidence that this enzyme is involved in host-parasite interactions, and suggest that the protease plays a role in bacterial dissemination, colonization, and invasion, and inhibition of wound healing.

Alleles↗

PubMed1993

A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage.

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187-amino-acid repeat motif. Collagen receptor-positive strains adhered to both collagen substrata and cartilage in a time-dependent process. Collagen receptor-specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.

Arthritis, Infectious↗

PubMed1992

Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesin.

Some strains of Staphylococcus aureus bind collagen with a high degree of specificity and affinity. This interaction can represent a mechanism of substrate adhesion and may be an important step in the pathogenesis of osteomyelitis and infectious arthritis. We now report on the cloning, sequencing, and expression of a gene name cna, encoding a S. aureus collagen adhesin. The cna gene was isolated from a lambda GT11 S. aureus genomic library and encodes an 1185 amino acid polypeptide. The deduced amino acid sequence reveals several structural characteristics similar to previously described Gram-positive bacterial cell surface proteins. Antibodies raised against the native collagen adhesin from S. aureus recognize the recombinant collagen adhesin. Collagen binding activity can be detected in a lysate obtained from Escherichia coli cells, which harbor the cloned cna gene on an expression plasmid. Collagen-binding proteins can be detected in the lysate when analyzed by a Western blot type assay in which the membrane-transferred proteins are probed with radioactively labeled collagen. Finally, the bacterial lysate containing the recombinant adhesin can effectively inhibit the binding of soluble collagen to cells of S. aureus.

Adhesins, Bacterial↗

PubMed1987

Model for studying bacterial adherence to skin wounds.

The adherence of bacterial pathogens to wounded skin is probably the first step in wound infection. This report describes the development of a bioassay to simulate the adherence of bacteria to wounds. The adherence of bacteria was examined by exposing wounds to known quantities of pathogens, washing the wounds with distilled water, and quantitating the number of adherent bacteria per cm2 of tissue. Our studies focused on the effects of naturally occurring mediators of bacterial adherence, such as wound fluid, serum, and fibronectin. Bacterial adherence was shown to be challenge dependent. Addition of wound fluid, serum, and heat-inactivated serum was shown to reduce the adherence of Pseudomonas aeruginosa to the wound surface compared with that of a saline control. Additional fibronectin treatment of the wound had no effect on the adherence of Staphylococcus aureus. The ability to identify what affects the binding of bacteria to wounded skin can lead to a better understanding of wound infection.

Animals↗