The reaction combination was kept at 4C and stirred for 4 h or overnight. of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications. == INTRODUCTION == Enterococci, once regarded as commensal organisms of intestinal origin with minimal clinical importance, have emerged as significant nosocomial pathogens causing endocarditis as well as bloodstream, wound, and urinary tract infections (1,2). Recent data cite enterococci as accounting for 12% of all hospital infections, becoming the second most commonly isolated nosocomial Flurbiprofen Axetil pathogen behind staphylococci (3). Theendocarditis andbiofilm-associatedpili (Ebp) ofEnterococcus faecalishave been extensively studied for their role as virulence factors important for establishing contamination. As with all orthologous pilus structures in other Gram-positive pathogens, includingCorynebacterium diphtheriae,Actinomyces naeslundii,Streptococcus pyogenes(group A streptococcus [GAS]),Streptococcus agalactiae(group B streptococcus [GBS]), Flurbiprofen Axetil andStreptococcus pneumoniae(4), the Ebp pili are terminally anchored to the peptidoglycan surface of the bacterium, allowing pili to readily interact with the external environment (5). EbpA, Flurbiprofen Axetil EbpB, and EbpC are the three structural pilin components which make up the pilus unit, with EbpC being the major component of the shaft (6,7). As its name suggests, the presence of Ebp has been associated with enterococcal contamination, specifically, with the ability of enterococci to form a biofilm or establish endocarditis contamination. Biofilms, surface-adherent cells covered in a matrix of polysaccharide, DNA, and/or protein, provide protection for the microbe, making Rabbit polyclonal to ADD1.ADD2 a cytoskeletal protein that promotes the assembly of the spectrin-actin network.Adducin is a heterodimeric protein that consists of related subunits. them more resistant to the immune response or antimicrobial attack. However, high-titer antibody responses to all three proteins of the Ebp have been found in patients withE. faecalisendocarditis, demonstrating that pili are readily expressed at some point during contamination and can be recognized by the host immune system (8). Biofilms are associated with native valve endocarditis, in addition to assisting with the establishment of contamination on artificial surfaces of a number of medical devices, such as catheters and artificial heart valves (9). In endocarditis, enterococci either adhere to the heart’s valve or inner endothelial lining directly or adhere to a sterile platelet/thrombin vegetation to establish contamination. The resulting contamination is usually a cumulative attachment of microbes, fibrin, platelets, and other host cells to the contamination site, which results in significant individual mortality. Adherence assays have exhibited that disruption of theebpA,ebpB, orebpCgene (6) or deletion of genes which regulate the expression of Ebp (10) inhibits the ability ofE. faecalisto establish a biofilm on a polystyrene surface. Furthermore, disruption ofebpgenes has been shown to significantly reduce the ability ofE. faecalisto form vegetations in a rat endocarditis model, which provides direct evidence of the importance of the pili in endocarditis (6). As with many gene disruption experiments in microbes evaluating their effect on pathogenicity, a logical next and often more challenging step is usually to identify an agent which, upon administration, can similarly cause attenuation. Given the huge success of recombinant IgG monoclonal antibody (MAb) therapy in malignancy and inflammatory/autoimmune disease (11,12) and the increasing battle against the emergence of multiantibiotic-resistant bacteria, the use of MAb drugs for passive protection against Gram-positive nosocomial pathogens has gained considerable interest in recent years (1320). We have previously explained a panel of MAbs against the major pilin protein EbpC, from which MAb 69 was selected on the basis of its affinity to recombinant EbpC antigen and its ability to identify surface-displayed EbpC (10). Here we demonstrate the ability Flurbiprofen Axetil of this MAb to bind to the polymer structure of the pili, functionally inhibit the establishment ofE. faecalisadhesionin vitro, prevent endocarditis infectionin vivo, and, using positron emission tomography (PET) molecular imaging studies of infected animals, validate that MAb 69 directly targets and recognizes pili at the site of endocarditis contamination. These results identify Ebp to be a viable MAb target for preventing infection byE. faecalis. In addition, we demonstrate that our MAb localizes to the established infection. Such localization may provide a means to readily monitor drug treatment in animal models of established infection and could ultimately allow identification (via imaging) and, possibly, treatment of patient populations through MAb therapy or MAb-targeted payload delivery. == MATERIALS AND METHODS == == Materials and strains. == The bacterial strains used in this study includedE. faecalisstrains OG1RF (wild type) and OG1RF ebpABC, which have been previously described (6,10). Recombinant EbpC (rEbpC) protein was produced as previously described (9). == TEM and immunogold labeling. == Transmission electron microscopy (TEM) experiments were carried out as.