RESUMO
Passive 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.
Assuntos
Anticorpos Monoclonais/imunologia , Enterococcus faecalis/imunologia , Fímbrias Bacterianas/imunologia , Infecções por Bactérias Gram-Positivas/prevenção & controle , Imunização Passiva/métodos , Animais , Anticorpos Monoclonais/administração & dosagem , Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Endocardite Bacteriana/imunologia , Endocardite Bacteriana/microbiologia , Endocardite Bacteriana/prevenção & controle , Proteínas de Fímbrias/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , RatosRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Enterococcus faecalis/genética , Biblioteca Gênica , Teste de Complementação Genética , Mutação , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , VirulênciaRESUMO
Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalisis 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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Enterococcus faecalis/genética , Gelatinases/metabolismo , Percepção de Quorum/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Adesão Celular , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Citometria de Fluxo , Gelatinases/genética , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Fenótipo , Deleção de Sequência , Transdução de SinaisRESUMO
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.
Assuntos
Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/genética , Enterococcus faecalis/fisiologia , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Anticorpos Monoclonais , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidade , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutagênese Insercional , Fenótipo , Filogenia , VirulênciaRESUMO
Ace (Adhesin to collagen from Enterococcus faecalis) is a cell-wall anchored protein that is expressed conditionally and is important for virulence in a rat infective endocarditis (IE) model. Previously, we showed that rats immunized with the collagen binding domain of Ace (domain A), or administered anti-Ace domain A polyclonal antibody, were less susceptible to E. faecalis endocarditis than sham-immunized controls. In this work, we demonstrated that a sub nanomolar monoclonal antibody (mAb), anti-Ace mAb70, significantly diminished E. faecalis binding to ECM collagen IV in in vitro adherence assays and that, in the endocarditis model, anti-Ace mAb70 pre-treatment significantly reduced E. faecalis infection of aortic valves. The effectiveness of anti-Ace mAb against IE in the rat model suggests it might serve as a beneficial agent for passive protection against E. faecalis infections.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Valva Aórtica/microbiologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Transporte/antagonistas & inibidores , Endocardite/prevenção & controle , Enterococcus faecalis/imunologia , Infecções por Bactérias Gram-Positivas/prevenção & controle , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Aderência Bacteriana/efeitos dos fármacos , Modelos Animais de Doenças , Enterococcus faecalis/isolamento & purificação , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/imunologia , Masculino , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).
Assuntos
Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Robótica/instrumentação , Animais , Escherichia coli/genética , Humanos , Imunoensaio , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Cinética , Biblioteca de Peptídeos , Software , Ressonância de Plasmônio de SuperfícieRESUMO
AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.
Assuntos
Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Metaloproteases/metabolismo , Zinco/metabolismo , Western Blotting , Separação Celular , Citometria de FluxoRESUMO
Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.
Assuntos
Enterococcus faecalis/enzimologia , Ribonucleases/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Genes Bacterianos , Microscopia Imunoeletrônica , RNA Mensageiro/genética , Ressonância de Plasmônio de Superfície , VirulênciaRESUMO
In this protocol, we describe the application of using a high affinity monoclonal antibody generated against the major pilin protein component of the pilin structure of Enterococcus faecalis as a PET imaging agent for enterococcal endocarditis detection. The anti-pilin -mAb 64Cu conjugate was able to specifically label enterococcal endocarditis vegetation in vivo in a rodent endocarditis model. By targeting pili, a covalently linked surface antigen extending from the bacterial surface, we provided evidence that gram-positive pilin represent a logical surface antigen to define or target an infectious agent for molecularly guided imaging. Our goal in providing a detailed protocol of our efforts is to enable others to build upon this methodology to answer pertinent translational and basic research questions in the pursuit of diagnosis and treatment of infective endocarditis.
Assuntos
Anticorpos Monoclonais , Endocardite Bacteriana/diagnóstico por imagem , Endocardite Bacteriana/metabolismo , Imunoconjugados , Imagem Molecular/métodos , Animais , Biomarcadores , Modelos Animais de Doenças , Endocardite Bacteriana/microbiologia , Citometria de Fluxo/métodos , Microscopia Eletrônica , Tomografia por Emissão de Pósitrons , Ratos , Microtomografia por Raio-XRESUMO
BACKGROUND: Evidence suggests lymphatic function mediates local rheumatoid arthritis (RA) flares. Yet biologics that target the immune system are dosed systemically via the subcutaneous (SC) administration route, thereby inefficiently reaching local lymphatic compartments. Nanotopography has previously been shown to disrupt tight cellular junctions, potentially enhancing local lymphatic delivery and potentially improving overall therapeutic efficacy. METHOD: We first characterized nanotopography (SOFUSA™) delivery of an anti-TNF drug, etanercept, by comparing pharmacokinetic profiles to those obtained by conventional SC, intravenous (IV), and intradermal (ID) routes of administration, and assessed uptake of radiolabeled etanercept in draining lymph nodes (LNs) in single dosing studies. We then compared etanercept efficacy in a progressive rat model of collagen-induced arthritis (CIA), administered systemically via SC route of administration; via the regional lymphatics through ID delivery; or through a nanotopography (SOFUSA™) device at 10, 12, and 14 days post CIA induction. Measurements of hind limb swelling and near-infrared fluorescence (NIRF) imaging of afferent lymph pumping function and reflux were conducted on days 11, 13, and 18 post CIA induction and compared to untreated CIA animals. Univariate and multivariate analysis of variance were used to compare the group differences for percentage swelling and lymphatic contractile activity. RESULTS: Even though all three modes of administration delivered an equal amount of etanercept, SOFUSA™ delivery resulted in increased lymphatic pumping and significantly reduced swelling as compared to untreated, ID, and SC groups. Pharmacokinetic profiles in serum and LN uptake studies showed that using the nanotopography device resulted in the greatest uptake and retention in draining LNs. CONCLUSIONS: Locoregional lymphatic delivery of biologics that target the immune system may have more favorable pharmacodynamics than SC or IV administration. Nanotopography may provide a more efficient method for delivery of anti-TNF drugs to reverse impairment of lymphatic function and reduce swelling associated with RA flares.
Assuntos
Antirreumáticos/administração & dosagem , Artrite Experimental/tratamento farmacológico , Sistemas de Liberação de Medicamentos/instrumentação , Etanercepte/administração & dosagem , Nanotecnologia/instrumentação , Animais , Antirreumáticos/farmacocinética , Artrite Reumatoide/tratamento farmacológico , Etanercepte/farmacocinética , Masculino , Ratos , Ratos Endogâmicos LewRESUMO
The detection of methamphetamine and other chemically related illicit drugs relies extensively on immunoassays. Here we report the cloning and affinity maturation of an anti-methamphetamine antibody which is being employed in the current commercial assays. An anti-methamphetamine scFv was cloned from hybridoma cells, expressed in bacteria and its affinity towards methamphetamine and N-ethylamphetamine (ethamphetamine) was determined by Surface Plasmon Resonance (SPR). The anti-methamphetamine scFv gene was subjected to random mutagenesis by error prone PCR and variants with improved affinity were isolated from the resulting library by a novel screening methodology termed Anchored Periplasmic Expression (APEx) [Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L., Rogers, G.K. (2004). Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.]. The isolated clones exhibited improved affinity to these illicit drugs, yet maintained low cross-reactivity to over-the-counter drugs. In addition, all clones displayed improved expression characteristics in Escherichia coli. The affinity improved scFv antibodies are thus likely to be useful in methamphetamine class immunodiagnostics.
Assuntos
Região Variável de Imunoglobulina/genética , Metanfetamina/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/genética , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Hibridomas/imunologia , Região Variável de Imunoglobulina/metabolismo , Técnicas In Vitro , Metanfetamina/análise , Camundongos , Dados de Sequência Molecular , Mutagênese , Biblioteca de Peptídeos , Periplasma/imunologia , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.
Assuntos
Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Periplasma/metabolismo , Sequência de Aminoácidos , Citometria de Fluxo , Proteínas de Membrana/química , Dados de Sequência Molecular , Conformação ProteicaRESUMO
UNLABELLED: The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of ß-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. IMPORTANCE: Enterococcus faecalis is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in E. faecalis pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in E. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of E. faecalis to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.
Assuntos
Aderência Bacteriana , Códon de Iniciação , Enterococcus faecalis/genética , Enterococcus faecalis/fisiologia , Fibrinogênio/metabolismo , Proteínas de Fímbrias/genética , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/patogenicidade , Proteínas de Fímbrias/metabolismo , Mutagênese Sítio-Dirigida , Virulência , beta-Galactosidase/genéticaRESUMO
INTRODUCTION: Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared (64)Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. METHODS: DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with (64)Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant (Kd) and maximum number of binding sites (Bmax) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. RESULTS: (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2-3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. (64)Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while (64)Cu-DOTA-mAb7 had higher Kd and Bmax values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for (64)Cu-NODAGA-mAb7. (64)Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo. This was further confirmed by the higher blood activity of (64)Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for (64)Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with (64)Cu-NODAGA-mAb7 in our prostate cancer model. CONCLUSIONS: To the best of our knowledge, this study represents the first comparison of (64)Cu-labeled DOTA and NODAGA immunoconjugates in vivo. Our results show favorable in vivo performance for (64)Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.
Assuntos
Acetatos/química , Quelantes/química , Radioisótopos de Cobre , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/química , Transporte Biológico , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Masculino , Camundongos , Distribuição TecidualRESUMO
PURPOSE: Monoclonal antibodies (mAbs) have been shown preclinically as reliable targeting moieties for antigen imaging using near-infrared fluorescence (NIRF) molecular imaging. However, crystallizable fragment-gamma receptor (FcγRs) expressed on immune cells also bind mAbs through defined epitopes on the constant fragment (Fc) of IgG. Herein, we evaluate the potential impact Fc interactions have on mAb agent imaging specificity. PROCEDURE: Through the removal of conserved glycans within the Fc domain, shown to have Fc/FcγR interactions, we evaluate their impact on non-specific binding/accumulation of a NIRF-labeled mAb-based imaging agent in lymph nodes (LNs) in inflamed animals and in an orthotopic prostate cancer animal model of LN metastasis. RESULTS: Deglycosylation of a murine mAb against the human epithelial cell adhesion marker using endoglycosidase EndoS significantly reduced non-specific binding in the LNs of inflamed animals and in cancer-negative LNs of tumor-bearing animals. Sensitivity remained unchanged while improvement in imaging specificity increased imaging accuracy. CONCLUSION: The reduction of non-specific binding through deglycosylation of a mAb-based imaging agent shows that reducing Fc/FcγR interactions can improve imaging accuracy.
Assuntos
Anticorpos Monoclonais/metabolismo , Glicosídeo Hidrolases/metabolismo , Imagem Molecular/métodos , Animais , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial , Glicosilação , Humanos , Inflamação/patologia , Linfonodos/patologia , Macrófagos/metabolismo , Masculino , Camundongos Nus , Ligação Proteica , Curva ROC , Receptores de IgG/metabolismo , Espectrometria de FluorescênciaRESUMO
The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Proteínas de Fímbrias/metabolismo , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Parede Celular/genética , Parede Celular/metabolismo , Modelos Animais de Doenças , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Proteínas de Fímbrias/genética , Camundongos , Mutação , Polimerização , Subunidades ProteicasRESUMO
Dual-labeled compounds containing nuclear and near-infrared fluorescence contrast have the potential to molecularly guide surgical resection of cancer by extending whole-body diagnostic imaging findings into the surgical suite. To simplify the dual labeling process for antibody-based agents, we designed a multimodality chelation (MMC) scaffold which combined a radiometal chelating agent and fluorescent dye into a single moiety. Three dye-derivatized MMC compounds were synthesized and radiolabeled. The IRDye 800CW conjugate, 4, had favorable optical properties and showed rapid clearance in vivo. Using 4, an epithelial cell adhesion molecule (EpCAM) targeting MMC-immunoconjugate was prepared and dual-labeled with (64)Cu. In vitro binding activity was confirmed after MMC conjugation. Multimodal imaging studies showed higher tumor accumulation of (64)Cu-7 compared to nontargeted (64)Cu-4 in a prostate cancer model. Further evaluation in different EpCAM-expressing cell lines is warranted as well as application of the MMC dual labeling approach with other monoclonal antibodies.
Assuntos
Quelantes/química , Diagnóstico por Imagem/métodos , Corantes Fluorescentes , Animais , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Molécula de Adesão da Célula Epitelial , Fluorescência , Corantes Fluorescentes/administração & dosagem , Humanos , Masculino , Camundongos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , Radioisótopos/farmacocinéticaRESUMO
PURPOSE: Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. EXPERIMENTAL DESIGN: An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. RESULTS: Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. CONCLUSIONS: NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Imagem Molecular/métodos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Benzenossulfonatos , Linhagem Celular Tumoral , Progressão da Doença , Molécula de Adesão da Célula Epitelial , Humanos , Indóis , Linfonodos/patologia , Masculino , Camundongos , Reprodutibilidade dos Testes , Proteína Vermelha FluorescenteRESUMO
Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.
Assuntos
Enterococcus faecalis/genética , Técnicas Genéticas , Genoma Bacteriano , Mutagênese Insercional , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Infecção Hospitalar/microbiologia , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , CamundongosRESUMO
PURPOSE: The aim of this study was to develop and characterize a novel peptide imaging agent for noninvasive near-infrared fluorescence imaging of protein transport by the lymphatics. An imaging agent consisting of a cyclic albumin-binding domain (cABD) peptide, with sequence, Arg-Leu-Ile-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Lys, was conjugated to a near-infrared fluorophore, IRDye800CW, allowing for enhanced vascular uptake, retention, and fluorescence imaging. PROCEDURE: Characterization of the cABD-IRDye800 peptide conjugate was performed using fluorescence spectroscopy to assess optical properties and SDS-PAGE and Biacore binding assays to determine binding affinity and specificity. Fluorescence imaging of normal C57BL/6 mice was conducted to monitor lymphatic uptake and retention. RESULTS: cABD-IRDye800 exhibited approximately six times greater fluorescent yield and greater stability than indocyanine green, an agent previously used in humans to image lymphatic vasculature. The agent exhibited affinity for albumin with IC(50) and Kd in the nanomolar range and demonstrated superior retention characteristics within mouse lymphatics when compared with IRDye800CW. CONCLUSIONS: cABD-IRDye800 has utility for assessing lymphatic function in mouse models of human lymphatic disease and the potential for use in clinical diagnostic imaging of the lymphatic vasculature.