Localized tufts of fibrils on Staphylococcus epidermidis NCTC 11047 are comprised of the accumulation-associated protein

Abstract

Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 +/- 10.8 nm long) are usually in a lateral position on the cells. Fibrillar (Fib(+)) and nonfibrillar (Fib(-)) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% Fib(+) cells and the less hydrophobic cells from the buffer phase entirely comprised Fib(-) cells. The Fib(+) and Fib(-) subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, Fib(+), and Fib(-) cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and Fib(+) cells that were absent from the Fib(-) cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogold staining with anti-Aap antibodies labeled tuft fibrils of the WT and Fib(+) cells but not the cell surface of Fib(-) cells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and Fib(+) S. epidermidis NCTC 11047 populations. Reverse transcription-PCR showed that aap was transcribed in both Fib(+) and Fib(-) cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

FIG. 1.

Micrographs of S. epidermidis NCTC 11047 cells (a to e) and S. epidermidis RP62A (f) negatively stained with 2% (wt/vol) methylamine tungstate. (a) One lateral tuft of fibrils on one side of the septum. No fibrils were detected on the remaining cell surface (not shown in the image). (b) Two lateral tufts of fibrils, one on either side of the septum. (c) Four lateral tufts symmetrically positioned on either side of the septum. (d) Tuft of fibrils showing a few longer individual fibrils (arrows) projecting through the mass of shorter fibrils. (e) Dividing cell without any tufts of fibrils. (f) Cell of strain RP62A carrying a tuft of fibrils. Bars, 100 nm.

FIG. 2.

Schematic diagram of the different positions occupied by fibrillar tufts on 25% of S. epidermidis NCTC 11047 cells in relation to the septum of the dividing cell. (a to d) The majority of tufts were in the lateral position. (e and f) A minority of tufts were in a polar position.

FIG. 3.

Change in percentages of Fib⁺ cells (triangles) and Fib⁻ cells (squares) of S. epidermidis NCTC 11047 subpopulations after repeated hydrophobic and hydrophilic enrichments in the hexadecane partition assay.

FIG. 4.

Comparison of cell surface hydrophobicities of S. epidermidis NCTC 11047 WT, Fib⁺, and Fib⁻ subpopulations after partitioning between hexadecane and SPB, dH₂O, or PBS. Error bars are standard errors of the means.

FIG. 5.

SDS-PAGE analysis of cell surface polypeptides of S. epidermidis strains, extracted using lysostaphin (100 μg ml⁻¹, 4 h, 37°C) and run on a 10% SDS-polyacrylamide gel stained with Coomassie brilliant blue. (A) Standard molecular mass markers (kDa). (B) S. epidermidis NCTC 11047 WT cells. (C) S. epidermidis NCTC 11047 Fib⁻ subpopulation. (D) S. epidermidis NCTC 11047 Fib⁺ subpopulation. Both NCTC 11047 WT and Fib⁺ subpopulations express two high-molecular-mass (280- and 230-kDa) polypeptides (indicated by arrows), which are absent from the Fib⁻ subpopulation.

FIG. 6.

Electron micrographs of S. epidermidis NCTC 11047 cells labeled with anti-Aap antibodies and 10-nm colloidal gold-conjugated secondary antibody negatively stained with 2% (wt/vol) methylamine tungstate. (a) Fib⁺ cell labeled on the tuft fibrils with anti-Aap A-region antibody (top cell) attached to a Fib⁻ cell with no tuft and no gold labeling. (b) Fib⁺ cell labeled with anti-Aap B-region antibody revealing a very extensive fibrillar tuft. (c) WT cells labeled with anti-A antibody, showing a labeled cell with a tuft (Fib⁺) attached to a cell with no labeling and no tuft (Fib⁻). (d) Fib⁻ cell showing no specific gold attached after labeling with anti-A antibody. Bars, 100 nm.