Proteins PblA and PblB of Streptococcus mitis, which promote binding to human platelets, are encoded within a lysogenic bacteriophage

Abstract

The binding of platelets by bacteria is a proposed central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 (an endocarditis isolate) was recently shown to be mediated in part by the surface proteins PblA and PblB. The genes encoding PblA and PblB are clustered with genes nearly identical to those of streptococcal phages r1t, 01205, and Dp-1, suggesting that pblA and pblB might reside within a prophage. To address this possibility, cultures of SF100 were exposed to either mitomycin C or UV light, both of which are known to induce the lytic cycle of many temperate phages. Both treatments caused a significant increase in the transcription of pblA. Treatment with mitomycin C or UV light also caused a substantial increase in the expression of PblA and PblB, as detected by Western blot analysis of proteins in the SF100 cell wall. By electron microscopy, phage particles were readily visible in the supernatants from induced cultures of SF100. The phage, designated SM1, had a double-stranded DNA genome of approximately 35 kb. Southern blot analysis of phage DNA indicated that pblA and pblB were contained within the SM1 genome. Furthermore, Western blot analysis of phage proteins revealed that both PblA and PblB were present in the phage particles. These findings indicate that PblA and PblB are encoded by a lysogenic bacteriophage, which could facilitate the dissemination of these potential virulence determinants to other bacterial pathogens.

FIG. 1

Map of the pblAB locus in S. mitis strains SF100 and PS344. The pblAB locus is an apparent mosaic of streptococcal phage sequences. ORF1, ORF2, ORF3, and pblA are similar to sequences of the L. lactis phage r1t. pblB is similar to an S. thermophilus phage 01205 gene encoding a tail fiber protein. ORF4, ORF5, ORF6, and ORF8 are not similar to any sequences reported in GenBank. ORF7 and lys are similar to sequences of the S. pneumoniae phage Dp-1. In PS344, the pblAB locus was replaced with the integrative vector pVA891. EcoRI sites (E), as well as select BglII (B) and HindIII (H) sites, are indicated.

FIG. 2

Induction of pblA transcription by mitomycin C and UV light. S. mitis strains carrying a lacZ transcriptional fusion in pblA in the forward orientation (strain PS291) or in the reverse orientation (strain PS294) were treated with mitomycin C (+MC) or UV light (+UV). Transcription was monitored by measuring the production of β-galactosidase. An asterisk denotes a value that was significantly different (P < 0.0001) from that of untreated cultures (control).

FIG. 3

Western blot analysis of PblA and PblB production in uninduced and induced cultures. Proteins were extracted from untreated cultures of PS344 (lane 1), SF100 (lane 2), and cultures of SF100 treated with mitomycin C (lane 3) or UV light (lane 4). Blots were probed with either anti-PblA serum (A) or anti-PblB serum (B).

FIG. 4

Electron micrographs of purified phage SM1 (A) and SM1ΔAB (B). Note the absence of tails in particles of phage SM1ΔAB. Bar, 100 nm.

FIG. 5

Ethidium bromide stain of phage genomic DNA. DNA from phage SM1 (lanes 1 and 2) or from phage SM1ΔAB (lanes 3 and 4) was digested with EcoRI and then separated by electrophoresis though a 0.7% agarose gel. The samples in lanes 2 and 4 were heated for 10 min at 70°C prior to loading, which led to the shift of a large submolar fragment (large arrow) to two smaller fragments (small arrows).

FIG. 6

Southern blot of phage SM1 genomic DNA and SF100 chromosomal DNA. Lanes 1, 3, and 5 contain phage DNA. Lanes 2, 4, and 6 contain chromosomal DNA. DNA was digested with EcoRI, and membranes were hybridized with probes corresponding to pblA (lanes 1 and 2), pblB (lanes 3 and 4), or pblT (lanes 5 and 6). One of two pblB fragments is a different size in the phage DNA (lane 3) than in the SF100 chromosomal DNA (lane 4), which indicates the presence of an attachment site within these fragments.

FIG. 7

Western blot analysis of PblA and PblB in purified phage. Lanes 1 and 3 contain phage SM1, and lanes 2 and 4 contain phage SM1ΔAB. Lanes 1 and 2 were probed with anti-PblA serum. Lanes 3 and 4 were probed with anti-PblB serum.