Analysis of Functional Domains of the Enterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

Abstract

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for highefficiency 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.Enterococcus faecalis has become a growing health concern as a mediator of the spread of antibiotic resistance and a leading agent of nosocomial infections (for review, see reference 10). The surface protein aggregation substance (AS) appears to play a role in both antibiotic resistance spread and in the pathogenesis of enterococcal infections. Expression of AS, which is encoded on the sex pheromone plasmids of E. faecalis, is induced by small 7-to 8-amino-acid peptide pheromones (26). AS on the surface of the donor cell then binds its receptor, enterococcal binding substance, on the recipient cell, mediating close cell contact that leads to conjugative transfer of the plasmid. It is thought that AS has no role in forming the DNA channel machinery, as efficient conjugation can occur if AS is expressed on either the donor or recipient cells (26).Over 20 different pheromone plasmids have been identified. Often, these pheromone plasmids express antibiotic resistance genes and other virulence factors, and many clinical isolates have multiple pheromone plasmids (36). The AS genes from the three most-studied plasmids, Asa1 from pAD1, Asp1 from pPD1, and Asc10 from pCF10 (encoded by the prgB gene), have been sequenced and show high identity (see below). The gene encoding Asa373, the AS protein of the pheromone plasmid pAM373, has also been sequenced but shows little homology with the other known AS proteins and appears to aggregate through a different mechanism (21). Expression of AS, which is normally tightly controlled in laboratory cultures, is induced in serum (13).A number of functions of AS that may contribute to virulence have been identified. A major function of AS is host cell adhesion. Kreft et al. fou...

Conjugative Plasmid Transfer in Gram-Positive Bacteria

“…However, parallels to the Ti plasmids appear to exist, insofar as many of the components of the pheromone-mediated conjugation system also seem to be involved in host-parasite interactions of enterococci (95,217,226). Hirt et al (95) demonstrated that E. faecalis cells harboring pCF10 showed significantly increased virulence in a rabbit endocarditis model.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…PrgB is a large ϳ137-kDa protein with homologs identified primarily in related pheromone-inducible conjugative plasmids in en- terococci. In addition to an N-terminal signal sequence and C-terminal LPXTG cell wall anchor motif, PrgB contains internal RGD motifs and aggregation domains shown to be important for the binding of lipoteichoic acid, adherence to eukaryotic cells, and bacterial internalization by epithelial cells (see below) (65,67,221,266,284,285,286). prgB mutants show reductions in pCF10 transfer efficiencies by several orders of magnitude, although transfer still occurs on solid surfaces.…”

Functional Analysis of TraA, the Sex Pheromone Receptor Encoded by pPD1, in a Promoter Region Essential for the Mating Response in Enterococcus faecalis

“…Once recipient cells acquire pX, they start to produce a pheromone inhibitor (iX) to block the activity of cX and to prevent selfclumping of donor cells (7,20,23). If plasmid-free cells secreting cX get close to donor cells, the cX outcompetes iX and triggers the mating processes including the synthesis of a surface adhesin, so-called "aggregation substance" (AS) (13,14,15,16,39). AS expressed on the surface of donor cells leads to binding to recipient cells and induces mating aggregates in which the plasmid is transferred at high frequency.…”