Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation

Abstract

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> or = 68% similarity) and is preceded by a gene similar to icaR (> or =70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.

FIG. 1

Organization of the S. caprae autolysin AtlC and percent amino acid identity to the corresponding domains of S. aureus Atl (38), S. epidermidis AtlE (18), and S. saprophyticus Aas (20). , Glycine-tryptophan (GW) dipeptide; , active enzymatic center. The enzymatic domains (AA and GL) were deduced from the similarities to those of the staphylococcal Atl-related autolysins. Abbreviations: AA, N-acetylmuramoyl-l-alanine amidase; GL, N-acetylglucosaminidase; R, repeats.

FIG. 2

Bacteriolytic enzyme profile of S. caprae strain 96007 on an SDS-polyacrylamide gel containing dried S. caprae 96007 cells (1 μg · ml⁻¹) as a substrate. Bands with lytic activity were observed as clear zones in the opaque gel and as dark bands after photography against a dark background. The sizes of marker proteins (in kilodaltons) are indicated on the left, and those of the bacteriolytically active proteins are shown on the right.

FIG. 3

Screening for fibronectin-binding proteins according to the Western affinity blotting technique described previously (1). Lanes: 1, surface proteins released from an LiCl extract of S. aureus strain DU5883 (pFNBA4) (17) used as positive control; 2, proteins released from uninduced culture of E. coli M15 harboring pREP4 and pIP1818; 3, 66-kDa protein resulting from purification on Ni-nitrilotriacetic acid resin (Qiagen) of the proteins released from an IPTG-induced culture of E. coli M15 harboring pREP4 and pIP1818; 4: proteins released from an LiCl extract of S. caprae isolate 96007.

FIG. 4

Map of the ica locus and surrounding chromosomal region in S. caprae strain 96007 (accession number AF246926). The IcaC product is truncated. The fragments reported below the map were cloned into pUC18 for sequencing; their sizes and the designation of the recombinant plasmids are given.