Characterization of theN-Acetylglucosaminyltransferase Activity Involved in the Biosynthesis of the Staphylococcus epidermidisPolysaccharide Intercellular Adhesin

Abstract

The polysaccharide intercellular adhesin (PIA) is an important factor in the colonization of medical devices by Staphylococcus epidermidis. The genes encoding PIA production are organized in the icaADBC (intercellular adhesion) operon. To study the function of the individual genes, we have established an in vitro assay with UDP-N-acetylglucosamine, the substrate for PIA biosynthesis, and analyzed the products by thin-layer chromatography and mass spectrometry. IcaA alone exhibited a low N-acetylglucosaminyltransferase activity and represents the catalytic enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA and icaB and overlaps both genes. N-Acetylglucosamine oligomers produced by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that react with PIA-specific antiserum synthesized. IcaA and IcaD are located in the cytoplasmic membrane, and IcaC also has all the structural features of an integral membrane protein. These results indicate a close interaction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complete PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate carrier is not involved. IcaAD represents a novel protein combination among ␤-glycosyltransferases.In recent years, Staphylococcus epidermidis has emerged as a frequent cause of nosocomial infections in association with indwelling medical devices such as intravascular catheters, cerebrospinal fluid shunts, prosthetic heart valves, prosthetic joints, artificial pacemakers, and chronic ambulatory peritoneal dialysis catheters (reviewed in Refs. 1 and 2). The virulence of S. epidermidis in these infections is thought to be based on its ability to colonize medical devices by forming a biofilm composed of multilayered cell clusters embedded in a slime matrix (3). As shown by electron microscopy studies, surface colonization takes place in two steps (4). The first step is primary adhesion of some bacteria, which is followed by proliferation of the cells to multilayered clusters. Factors reported to contribute to primary attachment of S. epidermidis cells to a polymer surface include unspecific hydrophobic interactions (5, 6), a capsular polysaccharide/adhesin (PS/A) (7, 8), proteinaceous cell-surface antigens (SSP-1 and SSP-2) (9, 10), and the autolysin AtlE identified by our group (11).A characteristic feature of the second phase of biofilm formation is intercellular adhesion, which results in the formation of large cell clusters by clinical S. epidermidis strains. This reaction is associated with the production of the polysaccharide intercellular adhesin (PIA) 1 located at the cell surface (12). PIA consists of two structurally related homoglycans, polysaccharides I and II, composed of at least 130 2-deoxy-2-amino-Dglucopyranosyl residues that are mostly (Ͼ80%) N-acetylated. The residues are ␤-1,6-linked (13), ...

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface

“…In Y. pseudotuberculosis, mutation of the hmsT gene similarly prevents biofilm formation on C. elegans (Darby et al, 2002). It is known that HmsT is a positive regulator of the hmsHFRS operon in Y. pestis, and homologues of HmsF and HmsR are required for the synthesis of polysaccharide components of biofilms of Staphylococcus epidermidis (Gerke et al, 1998). We have also identified a Y. pseudotuberculosis YPIII pIB1 hms gene (hmsF; The murine lethal toxin (phospholipase D) encoded on the 100 kb plasmid pMT1 in Y. pestis also plays a role in the survival of Y. pestis in the midgut of fleas (Hinnebusch et al, 2002).…”

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

“…A model for PIA biosynthesis by the ica (intercellular adhesin) gene products has been developed, based on several genetic and biochemical studies (reviewed in Götz 2002). The glycosyl transferase activity of IcaA, in conjunction with IcaD, first forms intracellular oligomers of 10-20 β-1,6-linked N-acetylglucosamine residues (Gerke et al 1998). The oligomers are then further polymerized and transported through the cell membrane via IcaC, where the polymer is partially deacylated by the polysaccharide deacetylase activity of the cell-surface protein IcaB (Vuong et al 2004a).…”

The role of σ B in persistence of Staphylococcus epidermidis foreign body infection

“…The attachment step is affected by multiple factors, including hydrophobic interactions, presence of host proteins, and specific surface proteins such as the autolysin AtlE and the capsular polysaccharide adhesin PS/A (Muller et al, 1993). In the accumulation phase, polysaccharide intercellular adhesin (PIA), synthesized by products of the icaADBC operon, mediates cell-to-cell adhesion and is essential for bacterial cell accumulation (Dunne, 2002;Gerke et al, 1998). Expression of the icaADBC operon depends on a variety of environmental conditions, and at least four unlinked genes (icaR, s B , the purR locus and SarA) are known to control PIA synthesis at the level of transcription (Conlon et al, 2002;Mack et al, 2000Mack et al, , 2007.…”