The blp bacteriocins of Streptococcus pneumoniae mediate intraspecies competition both in vitro and in vivo

Abstract

The introduction of the conjugate seven-valent pneumococcal vaccine has led to the replacement of vaccine serotypes with nonvaccine serotypes of Streptococcus pneumoniae. This observation implies that intraspecies competition between pneumococci occurs during nasopharyngeal colonization, allowing one strain or set of strains to predominate over others. We investigated the contribution of the blp locus, encoding putative bacteriocins and cognate immunity peptides, to intraspecies competition. We sequenced the relevant regions of the blp locus of a type 6A strain able to inhibit the growth of the type 4 strain, TIGR4, in vitro. Using deletional analysis, we confirmed that inhibitory activity is regulated by the function of the response regulator, BlpR, and requires the two putative bacteriocin genes blpM and blpN. Comparison of the TIGR4 BlpM and -N amino acid sequences demonstrated that only five amino acid differences were sufficient to target the heterologous strain. Analysis of a number of clinical isolates suggested that the BlpMN bacteriocins divide into two families. A mutant in the blpMN operon created in the clinically relevant type 19A background was deficient in both bacteriocin activity and immunity. This strain was unable to compete with both its parent strain and a serotype 4 isolate during cocolonization in the mouse nasopharynx, suggesting that the locus is functional in vivo and confirming its role in promoting intraspecies competition.

FIG. 1.

(A) Table summarizing results of agar overlay assays with deletion mutants of the blp locus. Plus signs designate definite zones of inhibition, and empty cells designate combinations that were not tested. (B) Alignment of BlpM and BlpN amino acid sequences from type 6A and TIGR4 strains and the chimeric proteins from 6AblpMNO^TIGR. Shaded amino acids are nonconserved, and arrows designate putative cleavage sites of preproteins. (C) Photographs of results of selected overlay assays. Pictures a to g demonstrate test strains with zones of inhibition, while pictures h to m demonstrate test strains lacking inhibition. Pictures a and b are shown with a TIGR4 overlay, and pictures c to m are shown with an overlay of 6AΔblpMNO. Test strains: a and c, type 6A; b and f, type 19A; d, 6AblpMNO^WT; e, 6AΔblpO; g, 19AblpMNO^WT; h, 6AΔblpMNO; i, 6AΔblpM; j, 6AΔblpN; k, TIGR4; l, 6AblpMNO^TIGR4; m, 19AΔblpMNO.

FIG. 2.

Graphical demonstration of the blp locus in a type 6A strain and comparison with the corresponding portion of the TIGR4 genome. Solid arrows represent coding sequences for double glycine-containing proteins, vertically striped arrows represent genes of unknown function, checked arrows represent transport genes, the white box represents an insertion sequence element, and gray boxes represent the conserved putative BlpR binding sites designating the start sites of operons. The gap in 6A designates an unsequenced region.

FIG. 3.

Amino acid alignment of sequences of BlpM and BlpN from a selection of clinical isolates of the serotypes indicated. Shaded amino acids are areas of nonconservation. Arrows designate putative cleavage sites of preproteins.

FIG. 4.

19AΔblpMNO is outcompeted by TIGR4 (A) or its parent type 19A strain (B) during mouse nasopharyngeal colonization. Six-week-old BALB/c mice were challenged intranasally with single or dual inoculations of the type 19A parental strain (19A; open circles), the 19AblpMNO^WT corrected mutant (19A; closed circles), 19AΔblpMNO (closed diamonds), and TIGR4 (19Ablp-; closed triangles) (A) or with single or dual inoculations of the type 19A strain and19AΔblpMNO (B). The colonizing strain is depicted on the x axis and was detected in lavage fluid at 4 days postinoculation at the density indicated (y axis). Coinoculated strains are shown in parentheses. Statistical analysis was done by the Mann-Whitney test, and horizontal lines indicate median values. Dashed lines denote the limit of detection.