Nasal colonisation by Staphylococcus aureus depends upon clumping factor B binding to the squamous epithelial cell envelope protein loricrin.
ABSTRACT: Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the "dock, lock and latch" mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfB?S. aureus to colonise the nares of wild-type and loricrin-deficient (Lor?/?) mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfB? mutant colonised wild-type mice less efficiently than the parental ClfB? strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB? mutant in the Lor?/? mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lor?/? mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.
Project description:Staphylococcus aureus expresses a number of cell wall-anchored proteins that mediate adhesion and invasion of host cells and tissues and promote immune evasion, consequently contributing to the virulence of this organism. The cell wall-anchored protein clumping factor B (ClfB) has previously been shown to facilitate S. aureus nasal colonization through high affinity interactions with the cornified envelope in the anterior nares. However, the role of ClfB during skin and soft tissue infection (SSTI) has never been investigated. This study reveals a novel role for ClfB during SSTIs. ClfB is crucial in determining the abscess structure and bacterial burden early in infection and this is dependent upon a specific interaction with the ligand loricrin which is expressed within the abscess tissue. Targeting ClfB using a model vaccine that induced both protective humoral and cellular responses, leads to protection during S. aureus skin infection. This study therefore identifies ClfB as an important antigen for future SSTI vaccines.
Project description:BACKGROUND: Nasal carriers not only pose serious threat to themselves but also to the community by playing an active role in the dissemination of serious and life threatening S. aureus especially MRSA strains. The present study focuses on the use of broad spectrum lytic phage as decolonising agent. In addition, the combined use of lytic phage with mupirocin has also been investigated as an effective decolonising regimen. The effect of phage on the adherence, invasion and cytotoxic effect of MRSA strains on nasal epithelial cells was studied in an ex-vivo model of cultured murine nasal epithelial cells. This was followed by demonstration of therapeutic potential of phage along with mupirocin in decolonising the nares of BALB/c mice using a nasal model of MRSA colonisation. RESULTS: Phage was able to significantly reduce the in vitro adherence, invasion and cytotoxicity of MRSA 43300 as well as other clinical MRSA strains on murine nasal epithelial cells as compared to untreated control. Also, the frequency of emergence of spontaneous mutants decreased to negligible levels when both the agents (phage and mupirocin) were used together. CONCLUSION: Phage MR-10, given along with mupirocin showed an additive effect and the combination was able to effectively eradicate the colonising MRSA population from the nares of mice by day 5.
Project description:One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.
Project description:The study objective was to determine the prevalence of Staphylococcus aureus colonisation in the nares and oropharynx of healthy persons and identify any risk factors associated with such S. aureus colonisation. In total 263 participants (177 adults and 86 minors) comprising 95 families were enrolled in a year-long prospective cohort study from one urban and one rural county in eastern Iowa, USA, through local newspaper advertisements and email lists and through the Keokuk Rural Health Study. Potential risk factors including demographic factors, medical history, farming and healthcare exposure were assessed. Among the participants, 25.4% of adults and 36.1% minors carried S. aureus in their nares and 37.9% of adults carried it in their oropharynx. The overall prevalence was 44.1% among adults and 36.1% for minors. Having at least one positive environmental site for S. aureus in the family home was associated with colonisation (prevalence ratio: 1.34, 95% CI: 1.07-1.66). The sensitivity of the oropharyngeal cultures was greater than that of the nares cultures (86.1% compared with 58.2%, respectively). In conclusion, the nares and oropharynx are both important colonisation sites for healthy community members and the presence of S. aureus in the home environment is associated with an increased probability of colonisation.
Project description:Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is common among men who have sex with men (MSM) in the USA. It is unknown whether this is also the case in Amsterdam, the Netherlands.Cross-sectional study.Sexually transmitted infection outpatient low-threshold clinic, Amsterdam, the Netherlands.Between October 2008 and April 2010, a total of 211 men were included, in two groups: (1) 74 MSM with clinical signs of a skin or soft tissue infection (symptomatic group) and (2) 137 MSM without clinical signs of such infections (asymptomatic group).S aureus and MRSA infection and/or colonisation. Swabs were collected from the anterior nasal cavity, throat, perineum, penile glans and, if present, from infected skin lesions. Culture for S aureus was carried out on blood agar plates and for MRSA on selective chromagar plates after enrichment in broth. If MRSA was found, the spa-gene was sequenced.Associated demographic characteristics, medical history, risk factors for colonisation with S aureus and high-risk sexual behaviour were collected through a self-completed questionnaire.The prevalence of S aureus colonisation in the nares was 37%, the pharynx 11%, the perianal region 12%, the glans penis 10% and in skin lesions 40%. In multivariable analysis adjusting for age, anogenital S aureus colonisation was significantly associated with the symptomatic group (p=0.01) and marginally with HIV (p=0.06). MRSA was diagnosed in two cases: prevalence 0.9% (95% CI 0.1% to 3.4%)). Neither had CA-MRSA strains.CA-MRSA among MSM in Amsterdam is rare. Genital colonisation of S aureus is not associated with high-risk sexual behaviour.
Project description:OBJECTIVE:To investigate the conjunctival and nasal flora and the antibiotic susceptibility profiles of isolates from patients undergoing cataract surgery. DESIGN:Observational and cross-sectional study. SETTING:A single-centre study in Taiwan. PARTICIPANTS:128 consecutive patients precataract surgery. PRIMARY AND SECONDARY OUTCOME MEASURES METHODS:Conjunctival and nasal cultures were prospectively obtained from 128 patients on the day of cataract surgery before instillation of ophthalmic solutions in our hospital. Isolates and antibiotic susceptibility profiles were identified through standard microbiological techniques. Participants were asked to complete a questionnaire on healthcare-associated factors. RESULTS:The positive culture rate from conjunctiva was 26.6%, yielding 84 isolates. Coagulase-negative Staphylococci were the most commonly isolated organisms (45.2%), and 35% of staphylococcal isolates were methicillin-resistant. Among staphylococcal isolates, all were susceptible to vancomycin, and 75%-82.5% were susceptible to fluoroquinolones. Methicillin-resistant isolates were significantly less susceptible than their methicillin-sensitive counterparts to tobramycin, the most commonly used prophylactic antibiotic in our hospital (28.6% vs 69.2%; p=0.005). The positive culture rate from nares for Staphylococcus aureus was 21.9%, and six isolates were methicillin-resistant. No subjects had S. aureus colonisation on conjunctiva and nares simultaneously. There were no associated risk factors for colonisation of methicillin-resistant Staphylococci. CONCLUSION: The most common conjunctival bacterial isolate of patients undergoing cataract surgery was coagulase-negative Staphylococci in Taiwan. Because of predominant antibiotic preferences and selective antibiotic pressures, Staphylococci were more susceptible to fluoroquinolones but less to tobramycin than in other reports. Additionally, methicillin-resistant Staphylococci exhibited co-resistance to tobramycin but not to fluoroquinolones.
Project description:Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.
Project description:Staphylococcus aureus prefers the human anterior nares as its habitat, but nothing is known about the nutritional situation in this ecological niche. Analysis of nasal secretions showed a complex mixture of nutrients at low concentration. Based on these findings a synthetic nasal medium (SNM) was composed, mimicking nasal secretions. We used microarrays in order to investigate pathways and expression patterns important in a synthetic medium mimicking nasal secretions compared to standard laboratory complex medium Overall design: Staphylococcus aureus USA300 was inoculated in complex medium (BM) or synthetic nasal medium (SNM) to an OD578nm of 0.005 and grown under aerobic conditions until OD578nm of 0.02. RNA was stabilized and extracted at this early growth phase and hybridization was made on Affymetrix microarrays. The aim was to identify genes which are important during growth under the limited conditions present during colonization of human nares
Project description:Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen ? (Fg ?) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg ? and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.
Project description:Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel β-sheet complementation as observed for the clumping factor A·γ-chain peptide complex. The β-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.