Accumulation-associated protein enhances Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in a rat catheter model

Abstract

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces.

FIG 1

Domain architecture of accumulation-associated protein (Aap) varies among S. epidermidis strains. Published Aap sequences were used to create diagrams for strains NCTC 11047 (GenBank accession no. HM587132.1), RP62A (AJ249487.1), 5179 (AY359815.1), and ATCC 12228 (NP_763730.1). Draft genomic sequencing results from S. epidermidis 1457 were used to construct the 1457 Aap diagram (GenBank accession no. KJ920749). Chevrons (><) indicate regions amplified for the construction of 1457 Δaap.

FIG 2

S. epidermidis strain 1457 produces and processes Aap in vitro. Antiserum raised against the recombinant B domain of Aap specifically binds to an approximately 150-kDa band present on the cell wall of bacteria grown in standard medium (left lane). The ∼250-kDa band corresponding to full-length Aap is apparent when bacteria are cultured in medium containing the protease inhibitor α₂-macroglobulin (α₂m; right lane). While Aap runs at a higher apparent molecular mass (∼250 kDa) than that predicted from its sequence (174 kDa), this is common of cell wall-associated proteins and may be a consequence of cell wall fragments remaining covalently bound to the protein.

FIG 3

Deletion of aap decreases static biofilm formation in a PIA-negative background. Isogenic ica and aap allelic replacement mutants were constructed in the S. epidermidis 1457 background. Bacteria were grown statically in TSB for 24 h in a 96-well plate. Adherent cells were stained with crystal violet, and the absorbance was measured at 595 nm on a multiplate reader. The deletion of icaADBC (Δica) significantly impaired biofilm formation compared to that for 1457 (P < 0.0001), while no statistical difference was noted between 1457 and 1457 Δaap with regard to biofilm formation. However, a significant difference was detected between 1457 Δica and 1457 Δica Δaap (P < 0.01), suggesting that the presence of PIA masks the accumulation function of Aap in this assay. The biofilm phenotype was complementable in 1457 Δica Δaap with an icaADBC plasmid (pNF110). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (n ≥ 4 biological replicates).

FIG 4

Flow cell biomass is decreased in 1457 Δica Δaap compared to that in 1457 Δica. S. epidermidis 1457 Δica (A) and 1457 Δica Δaap (B) were grown for 4 days in 2% TSB at a flow rate of 3.75 rpm. Biofilms were poststained for live (Syto9; green) and dead (ToPro3; red) cells and then visualized by confocal light microscopy. Magnification, ×60. The close proximity of live and dead cells causes regions containing dead cells to appear yellow. (C) COMSTAT analysis of live cell biomass from three-dimensional biofilm reconstructions demonstrating decreased biomass in 1457 Δica Δaap compared to that in 1457 Δica (P < 0.001) (n ≥ 3).

FIG 5

Flow cell biomass is decreased in 1457 Δaap compared to that in 1457. S. epidermidis 1457 (A) and 1457 Δaap (B) were grown for 2 days in 2% TSB at a flow rate of 3.75 rpm. Biofilms were poststained for live (Syto9; green) and dead (ToPro3; red) cells and then visualized by confocal light microscopy. Magnification, ×20. (C) COMSTAT analysis of three-dimensional biofilm reconstructions demonstrating decreased biomass in 1457 Δaap compared to that in 1457 (P < 0.05) (n = 5).

FIG 6

A foreign body and immunosuppression are required for S. epidermidis infection. Prior to bacterial inoculation, rats were divided into four groups: catheter plus cyclophosphamide (CP), catheter only, CP only, and no catheter or CP (no treatment). Three days after infection, rats were sacrificed and the catheter (A), blood (B), and spleen (C) were harvested and plated for bacterial load. Bacterial burdens from the catheter plus CP rats were significantly higher than those in the other three groups (P < 0.05). Bacterial counts were normalized (log₁₀ + 1) due to complete clearance of bacteria in some animals. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Cath, catheter; tx, treatment.

FIG 7

Aap is required for bacterial adherence in a rat model of intravenous catheter infection. Rats with implanted jugular catheters were immunosuppressed with cyclophosphamide and then inoculated via the tail vein with 10⁹ CFU of S. epidermidis 1457, 1457 Δica, 1457 Δaap, or 1457 Δica Δaap. Three days postinfection, the catheter (A), blood (B), and spleen (C) were harvested and plated to assess bacterial burden. Both Aap-deficient strains (1457 Δaap and 1457 Δica Δaap) had significantly lower bacterial burdens on the catheter and in the blood than strains with Aap (1457 and 1457 Δica). Similar titers were obtained from the spleen for all four strains, suggesting the differences in catheter and blood counts were not due to decreased fitness of the mutants. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 8

The A domain of Aap mediates initial attachment to plastic surfaces. Complementation of 1457 Δaap (dark gray circles) with full-length aap (pRBaap; black squares) (A) but not the B domain alone (pRBaapDomB; black squares) (B) enhances initial attachment. (C) Expression of the A domain (pCNaapDomA; black diamonds) in S. carnosus TM300 (dark gray circles) mediates attachment to a plastic surface, while in trans expression of the B domain (pRBaapDomB; light gray squares) does not.

FIG 9

Model of Aap dual functionality. S. epidermidis expressing full-length Aap attaches to a foreign body through unknown interactions within the A domain. Following binding, Aap is cleaved by staphylococcal or host-derived proteases, enabling intercellular accumulation via the B domain.