EVIDENCE FOR AUTOLYSIN-MEDIATED PRIMARY ATTACHMENT OF STAPHYLOCOCCUS-EPIDERMIDIS TO A POLYSTYRENE SURFACE

Biofilm formation on a polymer surface which involves initial attachme nt and accumulation in multilayered cell clusters (intercellular adhes ion) is proposed to be the major pathogenicity factor in Staphylococcu s epidermidis foreign-body-associated infections. We have characterize d two distinct classes of biofilm-negative Tn917 mutants in S. epiderm idis affected in initial attachment (class A) or intercellular adhesio n (class B). mut1 (class A mutant) lacks five surface-associated prote ins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be c omplemented by transformation with a 16.4 kb wild-type DNA fragment. T he complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. S ubcloning experiments revealed that the 60 kDa protein is sufficient f or initial attachment, Immunofluorescence microscopy using an antiseru m raised against the 60 kDa protein showed that this protein is locate d at the cell surface, DNA-sequence analysis of the complementing regi on revealed a single open reading frame which consists of 4005 nucleot ides and encodes a deduced protein of 1335 amino acids with a predicte d molecular mass of 148 kDa. The amino acid sequence exhibits a high s imilarity (61% identical amino acids) to the atl gene product of Staph ylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by prot eolytic processing. The 120 kDa protein missing from mut1 presumably r epresents the unprocessed amidase and glucosaminidase domain after pro teolytic cleavage of the signal- and propeptide. The 45 and 38 kDa pro teins are probably the degradation products of the 60 and 52 kDa prote ins, respectively. Additionally, AtlE was found to exhibit vitronectin -binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during l ater stages of adherence. Our findings provide evidence for a new func tion of an autolysin (AtlE) in mediating the attachment of bacterial c ells to a polymer surface, representing the prerequisite for biofilm f ormation.