Abstract
Some strains of Staphylococcus aureus bind collagen with a high degree of specificity and affinity. This interaction can represent a mechanism of substrate adhesion and may be an important step in the pathogenesis of osteomyelitis and infectious arthritis. We now report on the cloning, sequencing, and expression of a gene name cna, encoding a S. aureus collagen adhesin. The cna gene was isolated from a lambda GT11 S. aureus genomic library and encodes an 1185 amino acid polypeptide. The deduced amino acid sequence reveals several structural characteristics similar to previously described Gram-positive bacterial cell surface proteins. Antibodies raised against the native collagen adhesin from S. aureus recognize the recombinant collagen adhesin. Collagen binding activity can be detected in a lysate obtained from Escherichia coli cells, which harbor the cloned cna gene on an expression plasmid. Collagen-binding proteins can be detected in the lysate when analyzed by a Western blot type assay in which the membrane-transferred proteins are probed with radioactively labeled collagen. Finally, the bacterial lysate containing the recombinant adhesin can effectively inhibit the binding of soluble collagen to cells of S. aureus.
Highlights
Some strains of Staphylococcus aureusbind collagen with a high degree of specificity and affinity
Taken together thesedatademonstrated thatthe collagen-binding component acts as an adhesin and mediates the attachment The pathogenesis of bacterial infections typically involves of staphylococcal cells to collagen containing substrata. a multitude of virulence factors which participate in an often In this paper we report on the molecular cloning, sequencsurprisingly sophisticated cascade of interplays between the ing, and expression of the cna gene fromS. aureus strain FDA host and prokaryotic organism
Collagen-rich tissues like bone and cartilage are common sites of staphylococcal infection, with S. aureus implicated in approximately 80% of the acute and chronic cases of osteomyelitis (Waldvogel and Vasey, 1980)
Summary
S. aureus Strain FDA 574 Expresses a Collagen AdhesinInitial experiments showed that S. aureus strain FDA574 bound '261-labeled collagentype I1 in a time andtemperaturedependent manner. PstI which resulted in two smaller fragments of2.9 and 1.7 contain restriction sites for EcoRI and PstI, respectively, as kb in length Each of these fragmentswere subcloned into the described under "Experimental Procedures." The amplified plasmid vector pUC 18 and introduced into E. coli TG-1 to cna gene was purified, digested with EcoRI and PstI, and generate clones pCOL 10 and pCOL 11, respectively. A Coomassie-stained protein corresponding to the exprotein harbored a recombinant plasmid with an insert of 4 pected molecular weight of the expressed collagen adhesin kb One such clone called pCOL 20 was chosen for further could be detected in the lysate of the induced bacteria The same immunoreactive band can be seen in thepost-induction lysate (Fig. 4B, lane g) along with a number of smaller molecular weight proteins These smaller bands probably represent proteolytic degradation fragments of theintact collagen adhesin. No collagen-binding proteins could be detected inthe lysate prepared frompKK2233(JM105) (Fig. 7, lane e )
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