scholarly journals Analysis of Functional Domains of theEnterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

2001 ◽  
Vol 183 (19) ◽  
pp. 5659-5667 ◽  
Author(s):  
C. M. Waters ◽  
G. M. Dunny
Keyword(s):  
Cell Surface ◽  
High Efficiency ◽  
Cell Surface Hydrophobicity ◽  
Surface Protein ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Host Cells ◽  
Functional Domains ◽  
Aggregation Substance ◽  
C Terminus

ABSTRACT Pheromone-inducible aggregation substance (AS) proteins ofEnterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

scholarly journals Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in Streptococcus gordonii Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties

1999 ◽  
Vol 181 (10) ◽  
pp. 3087-3095 ◽  
Author(s):  
Roderick McNab ◽  
Helen Forbes ◽  
Pauline S. Handley ◽  
Diane M. Loach ◽  
Gerald W. Tannock ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insertional Mutagenesis ◽  
Cell Surface Hydrophobicity ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Streptococcus Gordonii ◽  
Adhesive Properties ◽  
Specific Antibodies ◽  
Structural And Functional Properties ◽  
Human Fibronectin

ABSTRACT It has been shown previously that inactivation of thecshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 ± 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces ofcshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entirecshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologousEnterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity overE. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression inE. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stainedE. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 ± 9.1 nm long that were absent from controlE. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordoniiadhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral “sanguis-like” streptococci.

scholarly journals Inhibition of Hydrophobic Protein-MediatedCandida albicans Attachment to Endothelial Cells during Physiologic Shear Flow

2001 ◽  
Vol 69 (5) ◽  
pp. 2815-2820 ◽  
Author(s):  
Pati M. Glee ◽  
Jim E. Cutler ◽  
Evelyn E. Benson ◽  
Robert F. Bargatze ◽  
Kevin C. Hazen
Keyword(s):  
Endothelial Cells ◽  
Shear Flow ◽  
Cell Surface ◽  
Protective Efficacy ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Host Cells ◽  
Yeast Cells ◽  
Cytokine Activation ◽  
Endothelial Monolayers

ABSTRACT Adhesion interactions during hematogenous dissemination ofCandida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1β activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.

scholarly journals Global Cell Surface Conformational Shift Mediated by a Candida albicans Adhesin

2004 ◽  
Vol 72 (9) ◽  
pp. 4948-4955 ◽  
Author(s):  
Jason M. Rauceo ◽  
Nand K. Gaur ◽  
Kyeng-Gea Lee ◽  
John E. Edwards ◽  
Stephen A. Klotz ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Cell Surface Hydrophobicity ◽  
Cell Aggregation ◽  
Surface Hydrophobicity ◽  
Cellular Adhesion ◽  
Metabolic Inhibitors ◽  
Signal Transduction Inhibitors ◽  
Ordered Aggregation ◽  
Mediate Cell

ABSTRACT Candida albicans maintains both commensal and pathogenic states in humans. Both states are dependent on cell surface-expressed adhesins, including those of the Als family. Heterologous expression of Als5p at the surface of Saccharomyces cerevisiae results in Als5p-mediated adhesion to various ligands, followed by formation of multicellular aggregates. Following adhesion of one region of the cell to fibronectin-coated beads, the entire surface of the cells became competent to mediate cell-cell aggregation. Aggregates formed in the presence of metabolic inhibitors or signal transduction inhibitors but were reduced in the presence of 8-anilino-1-naphthalene-sulfonic acid (ANS) or Congo Red (CR), perturbants that inhibit protein structural transitions. These perturbants also inhibited aggregation of C. albicans. An increase in ANS fluorescence, which accompanied Als-dependent cellular adhesion, indicated an increase in cell surface hydrophobicity. In addition, C. albicans and Als5p-expressing S. cerevisiae showed an aggregation-induced birefringence indicative of order on the cell surface. The increase in birefringence did not occur in the presence of the aggregation disruptants ANS and CR. These results suggest a model for Als5p-mediated aggregation in which an adhesion-triggered change in the conformation of Als5p propagates around the cell surface, forming ordered aggregation-competent regions.

scholarly journals Heterologous Inducible Expression ofEnterococcus faecalis pCF10 Aggregation Substance Asc10 inLactococcus lactis and Streptococcus gordonii Contributes to Cell Hydrophobicity and Adhesion to Fibrin

2000 ◽  
Vol 182 (8) ◽  
pp. 2299-2306 ◽  
Author(s):  
Helmut Hirt ◽  
Stanley L. Erlandsen ◽  
Gary M. Dunny
Keyword(s):  
Cell Surface ◽  
Cell Surface Hydrophobicity ◽  
Regulatory System ◽  
Surface Hydrophobicity ◽  
Bacterial Cells ◽  
Streptococcus Gordonii ◽  
Cell Surfaces ◽  
Gene Encoding ◽  
Aggregation Substance

ABSTRACT Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis andStreptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces ofE. faecalis and L. lactis but not on that ofS. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactisrevealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

scholarly journals Enterococcus faecalis strains show culture heterogeneity in cell surface charge

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 807-814 ◽  
Author(s):  
Annet E. J. van Merode ◽  
Henny C. van der Mei ◽  
Henk J. Busscher ◽  
Karola Waar ◽  
Bastiaan P. Krom
Keyword(s):  
Zeta Potential ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Ph Dependence ◽  
Cell Surface Hydrophobicity ◽  
Surface Protein ◽  
Opportunistic Pathogen ◽  
Surface Hydrophobicity ◽  
Biliary Stents ◽  
Abiotic Surfaces

Adhesion of micro-organisms to biotic and abiotic surfaces is an important virulence factor and involves different types of interactions. Enterococcus faecalis, a human commensal and an important opportunistic pathogen, has the ability to adhere to surfaces. Biliary stents frequently become clogged with bacterial biofilms, with E. faecalis as one of the predominant species. Six E. faecalis strains isolated from clogged biliary stents were investigated for the presence of specific biochemical factors involved in their adhesion: aggregation substances (Aggs) and the enterococcal surface protein (encoded by the esp gene). In addition, physico-chemical factors involved in adhesion (zeta potential and cell surface hydrophobicity) were determined, as well as the influence of ox bile on these properties. Two-thirds of the biliary stent isolates displayed culture heterogeneity in the pH dependence of their zeta potentials. Moreover, 24 out of 46 clinical isolates of E. faecalis, including 11 laboratory strains, also displayed such heterogeneity. The culture heterogeneity was demonstrated to be a stable trait, not caused by quorum sensing, not plasmid mediated, and independent of the presence of esp and Agg. Data presented show that culture heterogeneity in zeta potential enhances adhesion to an abiotic surface. A higher prevalence of culture heterogeneity in zeta potential in pathogenic as compared to non-pathogenic isolates could indicate that this phenomenon might play a role in virulence and putatively in pathogenesis.

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