Bap, a Staphylococcus aureus surface protein involved in biofilm formation

Abstract

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.

FIG. 1

Biofilm formation phenotype. Differences between the wild-type strain V329 and mutants m556 and m3591 in the capacity to form a 24-h biofilm on polystyrene microtiter plates after staining with safranin. The ELISA plate colors correspond to OD₄₉₀s of 1.3, 0.17, and 0.18, respectively.

FIG. 2

(A) SDS-PAGE of protein extracts of wild-type strain V329, m556, RN4220, SA113, and the corresponding complemented strains: m556 pBT2:Bap (m556, lane +), RN4220 pBT2:Bap (RN4220, lane +), and SA113 pBT2:Bap (SA113, lane +). Note that a double protein band of 230 and 240 kDa is present in the wild-type strain as well as in all the strains harboring plasmid pBT2:Bap. (B) Study of the presence of Bap by Western blotting. An approximately 240-kDa band is recognized by polyclonal antibodies against the first 640 aa of Bap only in the wild-type strain V329 and complemented strains: m556 pBT2:Bap (m556, lane +), SA113Δica pBT2:Bap (SA113Δica, lane +), and SA113 pBT2:Bap (SA113, lane +).

FIG. 3

pBT2:Bap complementation studies involving biofilm formation in biofilm-defective mutant m556, non-biofilm-forming strain RN4220, and biofilm-forming strain SA113 and its ica-defective mutant SA113Δica. Biofilm formation capacity differences correspond to 24-h biofilm formed on polystyrene microtiter plates after staining with 0.1% safranin. The ELISA plates and mean OD₄₉₀s obtained are shown. (A) ELISA wells corresponding to wild type V329, m556 (−) and m556 pBT2:Bap (+), RN4220 (−) and RN4220 pBT2:Bap (+), SA113Δica (−) and SA113Δica pBT2:Bap (+), and SA113 (−) and SA113 pBT2:Bap (+). (B) Mean optical density. Bars represent the mean values, and error bars represent the standard errors of the means (n = 5). Significant differences in adherence (P < 0.01) were noted between complemented and noncomplemented strains, as well as between the wild type and isogenic mutants.

FIG. 4

Primary attachment assay. Significant (P < 0.01) differences were detected between wild-type strain V329 and isogenic mutant m556. No differences were found between S. aureus SA113 and isogenic mutant SA113Δica. Bars represent the mean values, and error bars represent the standard errors of the means (n = 3).

FIG. 5

Dot blot analysis of S. aureus PIA-PNSG acummulation induced by Bap expression. S. aureus V329 (blot A1) and m556 (blot B1) produced low levels of PIA-PNSG. Complementation of S. aureus SA113 with pBT2-Bap plasmid strongly increased the levels of PIA-PNSG (blot B2) with respect to the wild-type strain (blot A2). PIA-PNSG was not detected in S. aureus SA113Δica (blot A3).

FIG. 6

Phenotypic differences between the wild-type strain V329 and the defective mutant m556. (A) CRA colony morphology; (B) capacity to form a 24-h biofilm on PVC plastic, as observed by phase-contrast microscopy (magnification, ×1,000); (C) capacity to form a 48-h biofilm on the surface of a glass container (visual observation).

FIG. 7

(A) Structural analysis of Bap. S represents the signal sequence. The positions of the LPXTG motif and the A, B, C, and D domains are shown. Region A (aa 45 to 360) contains two short repeats of 32 aa (A₁ and A₂) separated by 26 aa. Region B (aa 361 to 818) represents the remaining part of the N-terminal domain. A spacer region (aa 819 to 947) followed by region C (aa 948 to 2139) consisting of 258-nt tandem repeats constitutes the central domain of Bap. The carboxy-terminal region of Bap consists of region D (aa 2148 to 2208), which contains three short repeats of 18 aa followed by an incomplete D repeat, and the LPXTG motif. (B) Alignment of the amino acid residues within repeat blocks A, C, and D of the Bap protein. Substitutions are shown in boldface type.

FIG. 8

Recovery of S. aureus V329 and its isogenic Bap mutant m3591 from implanted subcutaneous catheter in a mouse foreign body infection model. Bars represent the mean of CFU collected from catheters, and the error bars represent the standard errors of the means (n = 9). At day 10, differences between wild type and mutant were detected (P < 0.05). Bacteria were not detectable in control animals at the end of the experimental period.