[PubMed] [CrossRef] [Google Scholar] 14. immunization is effective in preventing Hyperforin (solution in Ethanol) such contamination, likely by interfering with bacterial adherence. INTRODUCTION Enterococci are among the first microbial colonizers of the infant gastrointestinal tract and, in healthy adults, they are part of the commensal intestinal microbiota (1). However, since the middle to late 1970s, enterococci have emerged as opportunistic pathogens and major causes of health care-associated infections, most notably urinary tract infections (UTIs) and bacteremia, second only to spp. in the hospital setting (2,C5). Among cases of enterococcal endocarditis, has been recognized as the most abundant species, accounting for more than 90% of isolates from this contamination (2, 3, 6). Treatment of enterococcal endocarditis has been clinically challenging due to the emergence of strains resistant to commonly used antibiotics, i.e., aminoglycosides, Hyperforin (solution in Ethanol) although more recent use of ampicillin in combination with ceftriaxone seems to have alleviated this concern (7). The associated mortality remains high, with rates between 9 and 15% in Europe and the United States, respectively (8, 9). Adherence to and colonization of host tissue components, such as platelets and the extracellular matrix (ECM), are abilities that facilitate the early steps toward the development of infective endocarditis. To date, a variety of factors involved in the pathogenesis of experimental aortic valve contamination have been identified; these include MSCRAMM (microbial surface components recognizing adhesive matrix molecules) proteins such as the adhesin to collagen, Ace, and the endocarditis and biofilm-associated pili, Ebp (10,C12). Fibronectin is usually a glycoprotein consisting of two protein chains of approximately 250 kDa each, covalently linked by disulfide bonds, that is present in soluble forms in blood plasma and other body fluids. An insoluble, fibrillar form Hyperforin (solution in Ethanol) of fibronectin is present in Hyperforin (solution in Ethanol) the ECM and is known to support bacterial adherence (13). A recent study from Torelli et al. reported the presence of a fibronectin-binding protein in JH2-2, designated enterococcal fibronectin-binding protein A (EfbA) (14). Similar to the pneumococcal adherence and virulence factor A (PavA) of (17), FbpA of (18), the SmFnB (19), and the group B streptococcus SfbA (20), collectively referred to as PavA-like proteins, several of which were shown to be involved in bacterial adherence to fibronectin and/or to mediate virulence in experimental models (15,C21). JH2-2 EfbA consists of an N-terminal predicted domain name involved in fibronectin binding (FbpA; amino acids Hyperforin (solution in Ethanol) 4 to 429), followed by a domain name of unknown function (Duf814; amino acids 448 to 533) often observed in association with FbpA. EfbA was shown to be a serum-inducible protein displayed around the outer surface of JH2-2 and the derived recombinant protein (rEfbA) bound Vegfa to immobilized human fibronectin. An infective endocarditis has not previously been reported. In the present study, we generated a nonpolar deletion mutant of OG1RF, restored the gene in its initial chromosomal location, and evaluated the ability of these derivatives to infect damaged heart valves in a rat model, adhere to immobilized fibronectin, and form biofilm. Finally, we provide evidence that EfbA is usually a protective antigen that could be used as a vaccine to combat experimental endocarditis. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used in the present study are described in Table 1. Unless otherwise specified, were cultured at 37C in brain heart infusion (BHI; Difco Laboratories) broth and agar or in BHI supplemented with 40% horse serum (BHIS) (Sigma, St. Louis, MO). strains were produced at 37C in Luria-Bertani broth and agar (Difco Laboratories). The concentrations of the antibiotics used for selection were as follows: for in-frame deletion mutant of OG1RFThis study????????TX5708OG1RFin the TX5707 genetic background; carries a silent nucleotide mutation in the Leu79 codon for differentiation from OG1RFThis study????????CK111Conjugative donor; carries in for pHOU1 replication23Plasmids????pHOU1Plasmid for markerless allelic exchange in deletion; contains a 1,029-bp BamHI/PstII fragment encompassing the flanking region of the gene cloned into pHOU1This study????pTX5708Construct for reconstitution of geneThis study Open in a separate windows aAbbreviations: Ampr, ampicillin resistant; Fusr, fusidic acid resistant; Genr, gentamicin resistant; Rifr, rifampin resistant. Construction of isogenic mutants. OG1RF isogenic mutants were constructed using pHOU1, a vector that contains a mutated deletion mutant, two fragments.