Group B streptococci and other gram-positive cocci bind to cytokeratin 8

Abstract

Group B streptococci (GBS) adhere to surface receptors present on epithelial cells; these receptors include fibronectin and laminin. To identify other possible receptors, plasma membranes from A549 cells, a respiratory tract epithelial cell line, were prepared. These plasma membranes were tested in a protein blot analysis using radiolabeled GBS as a probe. GBS adhered to two species, with molecular masses of 50 kDa (p50) and 57 kDa (p57). We concluded that p50 and p57 correspond to two forms of cytokeratin 8 (CK8) on the basis of the following results: (i) protein blot results demonstrated that p50 and p57 exactly comigrated with two forms of CK8 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE); (ii) p50 and p57 exactly comigrated with CK8 after separation by two-dimensional PAGE; (iii) CK8 in solution bound to GBS, as demonstrated by immunoblot analysis of proteins from A549 lysates that bound to GBS in a liquid-phase assay; and (iv) radiolabeled GBS bound to A549 lysate-derived CK8 that had been captured in anti-CK8-coated microtiter wells. CK8 bound to COH1-13, an acapsular mutant of COH1, demonstrating that adherence is not mediated by capsular polysaccharide. Trypsin-treated GBS did not bind to CK8, indicating that adherence is mediated via a protein on the surface of GBS. Soluble CK8 bound to six of six GBS strains tested. Soluble CK8 also bound to Staphylococcus aureus, Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. We hypothesize that adherence of GBS to cytokeratin may be important for maintenance of colonization at sites of keratinized epithelium, such as the vagina, or for adherence of these bacteria to damaged epithelial cells at other sites.

FIG. 1

Protein blot analysis of A549 membranes. A549 membrane proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed as follows: lane 1, ³⁵S-labeled COH1; lane 2, M20 (anti-CK8); lane 3, uchm-1 (anti-CD14); and lane 4, no primary antibody. (Lanes 2 to 4 are immunoblots.)

FIG. 2

Two-dimensional protein blot analysis of A549 membranes. A549 membranes were separated by the method of O'Farrell (11), transferred to a PVDF membrane, and probed. (A) Immunoblot probed with M20 (anti-CK8). (B) Protein blot probed with ³⁵S-labeled COH1.

FIG. 3

Protein blot analysis of A549 proteins that bind to COH1 in solution. Lanes: 1 to 3, protein blots probed with ³⁵S-labeled COH1; 4 to 6, immunoblots probed with M20 (anti-CK8). Lanes 1 and 4 contain proteins eluted from COH1 alone; lanes 2 and 5 contain proteins eluted from COH1 incubated with A549 lysates, and lanes 3 and 6 contain whole A549 membranes.

FIG. 4

COH1 adherence to CK8 captured by anti-CK8. ³H-labeled COH1 was allowed to bind to wells coated with anti-immunoglobulin; then no specific antibody (none), MOPC21 (an isotype control antibody), or M20 (anti-CK8) was added, followed by A549 lysate (+). Negative-control wells (−) did not have lysate added. Percent adherence was calculated as described in Materials and Methods.

FIG. 5

CK8 binding to trypsin-treated COH1. COH1 was treated as indicated below and then incubated with A549 lysates. Bound proteins were eluted and analyzed by immunoblotting with M20 (anti-CK8). Lane 1, PBS-treated COH1; lane 2, trypsin-treated COH1; lane 3, trypsin- and soybean trypsin inhibitor-treated COH1; lane 4, A549 whole-cell lysate.

FIG. 6

CK8 binding to different GBS strains. Various GBS strains were incubated with A549 lysate, and bound proteins were eluted and analyzed by immunoblotting with M20 (anti-CK8). Strains used were as follows: lane 1, A909 (type Ia); lane 2, DK14 (type Ib); lane 3, DK23 (type II); lane 4, NT6 (type VI); lane 5, NCTC 10/84 (type V); lane 6, COH1 (type III); and lane 7, COH1-13 (acapsular mutant of COH1). Lane 8 contained A549 whole-cell lysate.

FIG. 7

CK8 binding to five species of gram-positive cocci. Various strains were incubated with A549 lysates, and bound proteins were eluted and analyzed by immunoblotting with M20 (anti-CK8). Lane 1, COH1 (Streptococcus agalactiae); lane 2, RH110 (E. faecalis); lane 3, LM0230 (L. lactis); lane 4, SA25923 (Staphylococcus aureus); lane 5, CS101 (Streptococcus pyogenes); lane 6, A549 whole-cell lysate.