Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

ABSTRACT pAD1 is a 60-kb hemolysin-bacteriocin plasmid in Enterococcus faecalis that encodes a conjugative mating response to a peptide sex pheromone, cAD1, secreted by plasmid-free bacteria. The pheromone response is regulated by two proteins: TraE1, which positively regulates all or most conjugative structural genes, and TraA, which negatively regulates traE1. TraA binds to pAD1 DNA at theiad (encoding the inhibitor peptide iAD1) promoter but is released upon binding to imported pheromone. This leads to enhanced transcription through two closely spaced downstream terminators (t1 and t2) into traE1. TraE1 is believed to then upregulate itself from a site located within t2; thus, a small amount of transcription through t1-t2 could lead to overall induction. It is important therefore that the t1-t2 terminators be tightly controlled to keep the response shut down in the absence of pheromone. A small (200-nucleotide) RNA molecule designated mD is encoded just upstream of t1 by a determinant (traD) oriented in the direction opposite to that of transcripts utilizing t1. mD is expressed at high levels in the uninduced state, but it decreases significantly upon induction. Here we present results of genetic studies relating to the activity of t1-t2 and show that mD strongly enhances transcriptional termination at t1. The mD activity is shown to influence transcription well downstream and can affect the determinant for aggregation substance asa1. The phenomenon is specific in that there is no effect of mD on the unrelated pheromone-responding plasmids pPD1 and pCF10.

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Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of Enterococcus faecalis Infective Endocarditis

Infection and Immunity ◽

10.1128/iai.69.5.3305-3314.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3305-3314 ◽

Cited By ~ 27

Author(s):

John K. McCormick ◽

Helmut Hirt ◽

Christopher M. Waters ◽

Timothy J. Tripp ◽

Gary M. Dunny ◽

...

Keyword(s):

Infective Endocarditis ◽

Enterococcus Faecalis ◽

Rabbit Model ◽

Surface Protein ◽

Conjugative Plasmid ◽

Aggregation Substance ◽

Exposed Region ◽

Correct Sequence

ABSTRACT The aggregation substance (AS) surface protein fromEnterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS44–331) was cloned with a C-terminal histidine tag translational fusion and expressed fromEscherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS44–331 reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS44–331 bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS44–331were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS44–331 were challenged intravenously withE. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due toE. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

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Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.01837-08 ◽

2009 ◽

Vol 191 (10) ◽

pp. 3237-3247 ◽

Cited By ~ 61

Author(s):

Kimberly A. Kline ◽

Andrew L. Kau ◽

Swaine L. Chen ◽

Adeline Lim ◽

Jerome S. Pinkner ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Virulence Factors ◽

Transmembrane Helix ◽

Spatial Relationship ◽

Sortase A ◽

Aggregation Substance ◽

Retention Signal ◽

Positively Charged ◽

Pilus Assembly

ABSTRACT Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

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Enterococcal Aggregation Substance and Binding Substance Are Not Major Contributors to Urinary Tract Colonization by Enterococcus faecalis in a Mouse Model of Ascending Unobstructed Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.72.4.2445-2448.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2445-2448 ◽

Cited By ~ 13

Author(s):

James R. Johnson ◽

Connie Clabots ◽

Helmut Hirt ◽

Christopher Waters ◽

Gary Dunny

Keyword(s):

Urinary Tract ◽

Enterococcus Faecalis ◽

Recombinant Strain ◽

Plasmid Segregation ◽

Cognate Receptor ◽

Aggregation Substance ◽

Binding Substance ◽

Lower Urinary

ABSTRACT Isogenic Enterococcus faecalis strains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS+, constitutive EBS+) failed to outcompete isogenic derivative INY3000 (AS− EBS−) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS+, EBS+) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by E. faecalis and that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.

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Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy)

Journal of Medical Microbiology ◽

10.1099/jmm.0.05038-0 ◽

2003 ◽

Vol 52 (6) ◽

pp. 491-498 ◽

Cited By ~ 109

Author(s):

I. Duprè ◽

S. Zanetti ◽

A. M. Schito ◽

G. Fadda ◽

L. A. Sechi

Keyword(s):

Enterococcus Faecalis ◽

Enterococcus Faecium ◽

Virulence Genes ◽

Protein Gene ◽

Surface Protein ◽

Virulence Determinants ◽

Colonization Factor ◽

Aggregation Substance ◽

Epithelial Cell Lines ◽

Surface Protein Gene

Enterococci are widely distributed in the environment; within the human body, they are normal commensals of the oral cavity, gastrointestinal tract and vagina. In recent years, enterococci have become one of the most frequent causes of acquired nosocomial infections worldwide. The molecular mechanism of virulence of these bacteria is still not completely understood. The aims of this work were to characterize phenotypically 47 isolates of Enterococcus faecalis and Enterococcus faecium collected in Sardinia (Italy) by their abilities to adhere to different epithelial cell lines (Vero and Caco-2 cells) and to associate their phenotypes with the presence of known virulence genes detected within their genomes by PCR. The following genes were amplified: AS (aggregation substance), esp (surface protein gene), ace (accessory colonization factor), efaA (E. faecalis endocarditis antigen) and gelE (gelatinase). The virulence genes were detected in E. faecalis isolates only, with the exception of esp, which was found in both species. The phenotypic and genotypic results were also compared with the susceptibility of isolates to various antibiotics.

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Adhesion to Bile Drain Materials and Physicochemical Surface Properties of Enterococcus faecalis Strains Grown in the Presence of Bile

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.3855-3858.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 3855-3858 ◽

Cited By ~ 20

Author(s):

Karola Waar ◽

Henny C. van der Mei ◽

Hermie J. M. Harmsen ◽

John E. Degener ◽

Henk J. Busscher

Keyword(s):

Surface Properties ◽

Enterococcus Faecalis ◽

Photoelectron Spectroscopy ◽

Surface Protein ◽

Lower Surface ◽

Contact Angles ◽

Surface Proteins ◽

Water Contact ◽

Zeta Potentials ◽

Aggregation Substance

ABSTRACT The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.

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