RESUMO
Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the ß1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Heme/química , Mutação de Sentido Incorreto , Guanilil Ciclase Solúvel/química , Substituição de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Heme/genética , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Guanilil Ciclase Solúvel/genéticaRESUMO
MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.
Assuntos
GMP Cíclico/metabolismo , Eletrólitos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptídeos/farmacologia , Propionatos/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/fisiologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Cinética , Ratos , Ratos Sprague-Dawley , Receptores de EnterotoxinaRESUMO
Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 µM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.
Assuntos
Colo/enzimologia , Guanilato Ciclase/metabolismo , Mecanorreceptores/enzimologia , Reto/enzimologia , Vias Aferentes/enzimologia , Animais , Linhagem Celular Tumoral , Colo/inervação , Ativação Enzimática/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reto/inervaçãoRESUMO
Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.
Assuntos
Constipação Intestinal/tratamento farmacológico , Síndrome do Intestino Irritável/tratamento farmacológico , Peptídeos/farmacologia , Alquilação , Animais , Área Sob a Curva , Disponibilidade Biológica , Biotransformação , Constipação Intestinal/complicações , AMP Cíclico/metabolismo , Fezes/química , Feminino , Trânsito Gastrointestinal/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Síndrome do Intestino Irritável/complicações , Masculino , Peptídeo Hidrolases/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyRESUMO
Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC(15305RM) and SCC(15305cap). The SCC(15305cap) element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD(Ssp), capL(Ssp), and capK(Ssp)) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI(Ssp) show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: -4)-beta-Glc-(1-3)-Sug-(1-4)-beta-GlcA-(1- | beta-GlcNAc-(1-2) Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.
Assuntos
Cápsulas Bacterianas/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Staphylococcus/metabolismo , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Dados de Sequência Molecular , Família Multigênica/genéticaRESUMO
The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Agonistas da Guanilil Ciclase C/farmacologia , Mucosa Intestinal/metabolismo , Peptídeos/farmacologia , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Guanilato Ciclase/metabolismoRESUMO
Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC50:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 µg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.
Assuntos
Trânsito Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Laxantes/farmacologia , Peptídeos/farmacologia , Receptores Acoplados a Guanilato Ciclase/agonistas , Receptores de Peptídeos/agonistas , Animais , Ligação Competitiva , Disponibilidade Biológica , Linhagem Celular , Células Cultivadas , Constipação Intestinal/tratamento farmacológico , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/citologia , Secreções Intestinais/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Laxantes/química , Laxantes/metabolismo , Laxantes/farmacocinética , Masculino , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacocinética , Conformação Proteica , Estabilidade Proteica , Ratos , Receptores de EnterotoxinaRESUMO
AIMS: Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). MAIN METHODS: Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. KEY FINDINGS: Linaclotide inhibited in vitro [(125)I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [(125)I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3',5'-monophosphate and accelerated gastrointestinal transit. SIGNIFICANCE: Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.
Assuntos
Fármacos Gastrointestinais/farmacologia , Peptídeos/farmacologia , Receptores de Peptídeos/agonistas , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Feminino , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/farmacocinética , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trânsito Gastrointestinal/efeitos dos fármacos , Guanilato Ciclase/genética , Mucosa Intestinal/metabolismo , Secreções Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/genéticaRESUMO
The peptide uroguanylin regulates electrolyte transport in the intestine and kidney. Human uroguanylin has 2 conformations that can be stably isolated because of their slow interconversion rate. The A isomer potently activates the guanylate cyclase C receptor found primarily in the intestine. The B isomer, by contrast, is a very weak agonist of this receptor, leading to a widely held assumption that it is physiologically irrelevant. We show here, however, that human uroguanylin B has potent natriuretic activity in the kidney. Interestingly, uroguanylin A and B both induce saluretic responses, but the activity profiles for the 2 peptides differ markedly. The uroguanylin B dose-response curve is sigmoidal with a threshold dose of approximately 10 nmol/kg of body weight, whereas uroguanylin A has a comparable threshold but a bell-shaped dose-response curve. In addition, our study indicates a unique interplay between the A and B isoforms, such that the A form at high concentrations antagonizes the natriuretic action of the B form. These data show that the kidney contains a uroguanylin receptor of which the pharmacological profile does not match that of the well-defined intestinal uroguanylin receptor (guanylate cyclase C), an observation consistent with previous studies showing that the kidney of the guanylate cyclase C knockout mouse remains responsive to uroguanylin. The results presented here also support the unconventional notion that distinct conformations of a single endocrine peptide can elicit different responses in different tissues.
Assuntos
Peptídeos Natriuréticos/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular/efeitos dos fármacos , Guanilato Ciclase/fisiologia , Humanos , Masculino , Isoformas de Proteínas , Ratos , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacos , Sódio/urinaRESUMO
Most Staphylococcus aureus express a serotype 5 or 8 capsular polysaccharide (CP). However, 20-25% of human isolates and up to 86% of bovine strains of S. aureus are non-typeable (NT), i.e. non-reactive with antibodies to CP types 1, 2, 5 or 8. A vaccine that targets the S. aureus CP would not protect against NT strains. The aim of this study was to characterize NT S. aureus isolates at the molecular level to explain their lack of type 5 or 8 capsule production. The cap5(8) locus was present in all 22 NT clinical isolates from humans, eight of 21 bovine isolates, and in all eight sequenced strains. NT strains positive for the cap5(8) transcript had mutations within essential capsule genes and could be complemented in trans. S. aureus strains with reduced cap5(8) transcript had mutations within the cap5A promoter, decreased RNAIII levels, or a truncated arlR gene product. More than one mutation was identified in several isolates. The cap5(8) locus was replaced by IS257 in 13 of 21 NT bovine isolates of S. aureus. Lack of capsule expression in NT S. aureus can be explained by multiple mechanisms, and the data argue against the existence of capsule serotypes other than 1, 2, 5 and 8.
Assuntos
Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Polissacarídeos Bacterianos/genética , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Bovinos , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Mutação Puntual , Regiões Promotoras Genéticas/genética , Transcrição GênicaRESUMO
T cells are critical for the formation of intraabdominal abscesses by Staphylococcus aureus. We hypothesized that T cells modulate the development of experimental staphylococcal infections by controlling polymorphonuclear leukocyte (PMN) trafficking. In models of staphylococcal s.c. abscess formation, hindpaw infection, and surgical wound infection, S. aureus multiplied in the tissues of WT C57BL/6J mice and elicited a marked inflammatory response. CD4(+) alphabeta T cells homed to the surgical wound infection site of WT animals. In contrast, significantly fewer S. aureus were recovered from the tissues of mice deficient in alphabeta T cells, and the inflammatory response was considerably diminished compared with that of WT animals. Alphabeta T cell receptor (-/-) mice had significantly lower concentrations of PMN-specific CXC chemokines in wound tissue than did WT mice. The severity of the wound infection was enhanced by administration of a CXC chemokine and abrogated by antibodies that blocked the CXC receptor. An acapsular mutant was less virulent than the parental S. aureus strain in both the s.c. abscess and the surgical wound infection models in WT mice. These data reveal an important and underappreciated role for CD4(+) alphabeta T cells in S. aureus infections in controlling local CXC chemokine production, neutrophil recruitment to the site of infection, and subsequent bacterial replication.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiocinas CXC/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/fisiologia , Infecção da Ferida Cirúrgica/imunologia , Infecção da Ferida Cirúrgica/microbiologia , Animais , Cápsulas Bacterianas/metabolismo , Linfócitos T CD4-Positivos/citologia , Movimento Celular , Modelos Animais de Doenças , Membro Posterior/imunologia , Membro Posterior/microbiologia , Membro Posterior/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Infecção da Ferida Cirúrgica/patologiaRESUMO
Staphylococcus aureus is responsible for a wide range of infections, including soft tissue infections and potentially fatal bacteremias. The primary niche for S. aureus in humans is the nares, and nasal carriage is a documented risk factor for staphylococcal infection. Previous studies with rodent models of nasal colonization have implicated capsule and teichoic acid as staphylococcal surface factors that promote colonization. In this study, a mouse model of nasal colonization was utilized to demonstrate that S. aureus mutants that lack clumping factor A, collagen binding protein, fibronectin binding proteins A and B, polysaccharide intercellular adhesin, or the accessory gene regulator colonized as well as wild-type strains colonized. In contrast, mutants deficient in sortase A or clumping factor B (ClfB) showed reduced nasal colonization. Mice immunized intranasally with killed S. aureus cells showed reduced nasal colonization compared with control animals. Likewise, mice that were immunized systemically or intranasally with a recombinant vaccine composed of domain A of ClfB exhibited lower levels of colonization than control animals exhibited. A ClfB monoclonal antibody (MAb) inhibited S. aureus binding to mouse cytokeratin 10. Passive immunization of mice with this MAb resulted in reduced nasal colonization compared with the colonization observed after immunization with an isotype-matched control antibody. The mouse immunization studies demonstrate that ClfB is an attractive component for inclusion in a vaccine to reduce S. aureus nasal colonization in humans, which in turn may diminish the risk of staphylococcal infection. As targets for vaccine development and antimicrobial intervention are assessed, rodent nasal colonization models may be invaluable.