SummaryStaphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surfaceexpressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro . By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis , we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.

The chemical structure of free lipid A isolated from rough-and smooth-form lipopolysaccharides (R-LPS and S-LPS, respectively) of the human gastroduodenal pathogen Helicobacter pylori was elucidated by compositional and degradative analysis, nuclear magnetic resonance spectroscopy, and mass spectrometry. The predominant molecular species in both lipid A components are identical and tetraacylated, but a second molecular species which is hexaacylated is also present in lipid A from S-LPS. Despite differences in substitution by acyl chains, the hydrophilic backbone of the molecules consisted of ␤(1,6)-linked D-glucosamine (GlcN) disaccharide 1-phosphate. Because of microheterogeneity, nonstoichiometric amounts of ethanolaminephosphate were also linked to the glycosidic hydroxyl group. In S-LPS, but not in R-LPS, the hydroxyl group at position 4 was partially substituted by another phosphate group. Considerable variation in the distribution of fatty acids on the lipid A backbone was revealed by laser desorption mass spectrometry. In tetraacyl lipid A, the amino group of the reducing GlcN carried (R)-3-hydroxyoctadecanoic acid ( Helicobacter pylori is a prevalent pathogen of humans, and chronic infection of the gastric mucosa by the bacterium causes recurrent gastroduodenal inflammatory disease (5, 59). H. pylori is the primary cause of active chronic gastritis in humans and plays a pivotal role in the development of peptic ulcer disease (13, 31). Moreover, persistent infection with H. pylori is considered a risk factor for the development of adenocarcinoma and lymphoma of the stomach (11,41).Like the cell envelopes of other gram-negative bacteria, that of H. pylori contains lipopolysaccharides (LPSs). Chemically, LPSs are composed of a poly-or oligosaccharide covalently linked to a lipid component, lipid A (33, 51). Fresh clinical isolates of H. pylori produce high-molecular-weight smoothform LPSs (S-LPSs), which consist of an O side chain, a core oligosaccharide, and lipid A (34, 36). However, strains of H. pylori that have been subcultured many times on conventional solid media may produce low-molecular-weight rough-form LPSs (R-LPSs) that lack the O side chain (36). Nevertheless, the phase shift from S-to R-LPS can be reversed when the strains are grown in liquid media, and thus expression of S-LPS can be stabilized in vitro (38 (33,45,51,64), it has been hypothesized that H. pylori LPS and, in particular, its lipid A component have evolved their present structure as a consequence of adaptation to chronic infection of the gastric mucosa (28). Preliminary investigations have indicated that the lipid A component of H. pylori LPS contains fatty acids longer than those encountered in enterobacterial lipid A and that the lipid A is underphosphorylated (28,34,37). To clarify the molecular basis for the lower immunological activities of H. pylori LPS, we have investigated in detail the primary structure of its lipid A moiety. Investigations were initially undertaken with free lipid A liberated by acid hydrolysis from R-...

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.

Smooth- and rough-form lipopolysaccharides from phenol-water extraction of cells from Helicobacter pylori type strain NCTC 11637 were isolated as the water-soluble component of high-M(r) and water-insoluble low-M(r) gel. Structural investigations were performed on the intact water-soluble smooth-form lipopolysaccharide, various oligosaccharides formed as chemical and enzymic degradation products, and three oligosaccharide fractions liberated by acetic acid hydrolysis from the water-insoluble rough-form lipopolysaccharide. A structure is proposed for the complete polysaccharide component of the smooth-form lipopolysaccharide comprising the O antigen chain, an intervening region, and the inner core oligosaccharide on the basis of 1H and 13C NMR experiments, fast atom bombardment/mass spectrometry, and methylation linkage analysis of permethylated oligo- and polysaccharide derivatives. The most striking feature of the O antigen region in the lipopolysaccharide is the presence of extended chains with fucosylated and nonfucosylated N-acetyllactosamine (LacNAc) units that mimic human cell surface glycoconjugates in normal human granulocytes. The chains are terminated by di- or trimeric Lewis(x) (Le(x)) determinants, which are also found in tumor-associated carbohydrate antigens in many adenocarcinomas.