Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalis is associated with host cell adherence and endocarditis. The Fsr quorum-sensing system of E. faecalis, a two-component signal transduction system, has also been repeatedly linked to virulence in E. faecalis, due in part to the transcriptional induction of an extracellular metalloprotease, gelatinase (GelE). In this study, we discovered that disruption of the Fsr pathway significantly increased the levels of Ace on the cell surface in the latter phases of growth. Furthermore, we observed that, in addition to fsrB mutants, other strains identified as deficient in GelE activity also demonstrated a similar phenotype. Additional experiments demonstrated the GelE-dependent cleavage of Ace from the surface of E. faecalis, confirming that GelE specifically reduces Ace cell surface display. In addition, disruption of the Fsr system or GelE expression significantly improved the ability of E. faecalis to adhere to collagen, which is consistent with higher levels of Ace on the E. faecalis surface. These results demonstrate that the display of Ace is mediated by quorum sensing through the action of GelE, providing insight into the complicated world of Gram-positive pathogen adhesion and colonization.

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.Enterococcus faecalis, a normal commensal of the human gastrointestinal tract, is also an opportunistic pathogen and a major cause of nosocomial infections. E. faecalis is one of many Gram-positive pathogens recently discovered to possess surface pili. These Gram-positive pili are distinct from Gramnegative pili in their structure and mechanism of assembly (35,42). Pilus expression has been closely associated with virulence in multiple human pathogens, including group A Streptococcus (33), group B Streptococcus (24), and Corynebacterium diphtheriae (43). In E. faecalis, the biofilm-associated pili (Ebp) are considered to be among its major virulence factors and play an important role in biofilm formation and the development of endocarditis (35). Mutations in ebp structural genes have been shown to significantly reduce E. faecalis biofilm formation in vitro (35) and to decrease the ability of E. faecalis to form vegetations in a rat endocarditis model (20). The Ebp pilus also plays a role in murine urinary tract infection (UTI), as was demonstrated using an ascending UTI model (40), which provides further evidence for the importance of pili in bacterial infection.Three genes encoding the E. faecalis pilus proteins (ebpA, -B, and -C) are located in the same operon (35). Ebp pili are formed by the cross-linking of all three pilus proteins (35). EbpR, encoded by the gene upstream of ebpABC,...

A summary of the properties of CGP 51901 is shown in Table 3. On the basis of its binding to IgE and IgE-secreting cells and its activity in vitro and in vivo, CGP 51901 is expected to be able to decrease serum IgE by direct clearance of IgE and by reduction of the numbers and productivity of IgE-secreting cells. The end result of reduction of IgE in the circulation and on mast cells is expected to be the attenuation of IgE-mediated reactions and the improvement in allergy symptoms. The effective serum concentration of CGP 51901 is expected to be in the range 1-10 micrograms/ml. Because CGP 51901 is an antibody specific for IgE, it is expected to be highly selective in its activity. Because IgE does not appear to be essential and because CGP 51901 has been rigorously tested to confirm its non-anaphylactic nature, this treatment is not expected to have any adverse effects. Therefore, CGP 51901 is expected to be safe and to have a good probability of being effective when it is tested in human clinical trials.

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cPassive protection, the administration of antibodies to prevent infection, has garnered significant interest in recent years as a potential prophylactic countermeasure to decrease the prevalence of hospital-acquired infections. Pili, polymerized protein structures covalently anchored to the peptidoglycan wall of many Gram-positive pathogens, are ideal targets for antibody intervention, given their importance in establishing infection and their accessibility to antibody interactions. In this work, we demonstrated that a monoclonal antibody to the major component of Enterococcus faecalis pili, EbpC, labels polymerized pilus structures, diminishes biofilm formation, and significantly prevents the establishment of a rat endocarditis infection. The effectiveness of this anti-EbpC monoclonal provides strong evidence in support of its potential as a preventative. In addition, after radiolabeling, this monoclonal identified the site of enterococcal infection, providing a rare example of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications.

The proliferation of most carcinomas is associated with an overexpression of epithelial cell adhesion molecule (EpCAM), a 40-kDa type I transmembrane protein found on epithelial cells yet absent from other cell types. The absence of EpCAM in normal lymphatics makes it an attractive marker for studying lymph node (LN) metastases of carcinomas to improve LN staging accuracy. Herein, we developed and quantitatively compared dual-labeled monoclonal antibodies (mAbs) of varying affinities against EpCAM for both noninvasive and intraoperative detection of metastatic LNs in prostate cancer. Methods: A panel of hybridoma-derived anti-EpCAM mAbs was generated and screened. Two high-affinity candidate mAbs with specificity for nonoverlapping epitopes on the EpCAM extracellular domain were chosen for further evaluation. After conjugation with DOTA for 64 Cu radiolabeling and IRDye 800CW as a fluorophore, dual-labeled specific or isotype control mAb was administered intravenously to male nu/nu mice at 10-12 wk after orthotopic implantation of DsRedexpressing PC3 cells. Within 18-24 h, noninvasive small-animal PET/CT and in vivo, in situ, and ex vivo DsRed reporter gene and near-infrared fluorescence (NIRF) imaging were performed to detect primary tumors and metastatic LNs. Using DsRed fluorescence as the true indicator of cancer-positive tissue, we performed receiver operating characteristic curve analyses of percentage injected dose per gram measured from quantitative small-animal PET/CT and fluorescence intensity measured from semiquantitative NIRF imaging for each LN examined to compare mAb sensitivity and specificity. Results: mAbs 7 and 153 generated in-house were found to have higher affinity than commercial mAb 9601. Accuracy, as a function of sensitivity and specificity, for the detection of cancer-positive LNs during in vivo small-animal PET/CT was highest for mAbs 7 (87.0%) and 153 (78.0%) and significantly greater (P , 0.001) than random chance (50.0%). Rates for mAb 9601 (60.7%) and control mAb 69 (27.0%) were not significantly different from chance. Similarly, mAb 7 had significant detection accuracy by NIRF imaging (96.0%, P , 0.001). Conclusion: mAbs 7 and 153 are attractive, high-affinity candidates for further multimodal imaging agent optimization aimed at enhancing sensitivity and specificity for detection of metastatic LNs in prostate cancer. Fully quantitative NIRF imaging is needed for comprehensive analyses of NIRF-labeled agent accuracy for intraoperative guidance. Dual -labeled contrast agents promise the advantage of noninvasive imaging via nuclear techniques and molecularly guided surgical resection via near-infrared fluorescence (NIRF) imaging (1). Currently, nodal staging of most cancers is performed after lymph node (LN) biopsy and dissection for subsequent pathologic examination. As in most cancers, imaging of LN involvement in prostate cancer (PCa) lacks sensitivity and specificity (2) and as a result, extended pelvic LN dissection, which provides higher staging accuracy (3), is rapidly becomi...

NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. Enterococcus faecalis, a commensal organism of the gastrointestinal tract, is also known to be an opportunistic pathogen, a major cause of hospital-acquired infections, and a growing public health concern due to its increasing resistance to multiple antibiotics. A group of surface proteins, the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), play an important role in the virulence of E. faecalis by encouraging adherence and colonization to host tissue. Among them, Ace (adhesin of collagen from E. faecalis) plays an important role in adherence, presumably by mediating binding to extracellular matrix proteins, including both collagen and laminin (1). Ace has significant sequence homology with and a structural organization similar to that of Cna, a known collagen binding virulence factor of Staphylococcus aureus (2), and homology to Acm, a collagen adhesion protein in Enterococcus faecium (3). The association of Ace expression with virulence has been reported in many publications, including recent reports that an ace deletion mutant was significantly attenuated in both a rat endocarditis model and a murine urinary tract infection model, suggesting that Ace is involved in the pathogenesis of E. faecalis in both infectious endocarditis and urinary tract infections (4-6).The surface expression of Ace has been shown to be regulated by many factors, including growth phase, temperature, and medium components, such as serum and collagen. As previously reported, Ace is maximally displayed...