Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis

Abstract

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.

FIG. 1.

Alignment of Ply from VGS with two pneumococcal Ply sequences. The epitope recognized by MAb PLY-7 is shaded.

FIG. 2.

Western Blot analysis of toxins from VGS strains TIGR4 (T), R75II (75), R76 (76), and the negative control NCTC 10712 (NC). The samples were cell lysates from 15 ml of mid-log-phase cultures. Pneumolysin was detected with an anti-rabbit pneumolysin PAb (Ply) and an anti-mouse pneumolysin MAb (PLY-7).

FIG. 3.

Expression of Ply in culture media by strains COL15 and R75I. Bacteria were grown in BHI. Samples were withdrawn every hour, and the OD₆₀₀ was determined (a). The cells were centrifuged down, and the pneumolysin concentration in supernatants was measured by ELISA (b).

FIG. 4.

Western blot analysis of toxin expression during in vitro growth of strains TIGR4 (T), R75I (R), COL15 (C), and the negative controls lysate buffer (NCI) and BHI (NCII). The cells were collected from 15 ml of mid-log-phase cultures, resuspended in 1 ml of lysis buffer, and lysed by sonication (intracellular). The extracellular sample is unconcentrated culture medium (BHI). Toxin was detected with an anti-rabbit pneumolysin PAb.