Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements

Abstract

Natural competence for genetic transformation is the best-characterized feature of the major human pathogen Streptococcus pneumoniae. Recent studies have shown the virulence of competence-deficient mutants to be attenuated, but the nature of the connection between competence and virulence remained unknown. Here we document the release, triggered by competent cells, of virulence factors (e.g., the cytolytic toxin pneumolysin) from noncompetent cells. This phenomenon, which we name allolysis, involves a previously undescribed bacteriocin system consisting of a two-peptide bacteriocin, CibAB, and its immunity factor, CibC; the major autolysin, LytA, and lysozyme, LytC; and a proposed new amidase, CbpD. We show that CibAB are absolutely required for allolysis, whereas LytA and LytC can be supplied either by the competent cells or by the targeted cells. We propose that allolysis constitutes a competence-programmed mechanism of predation of noncompetent cells, which benefits to the competent cells and contributes to virulence by coordinating the release of virulence factors.

Fig. 1.

Competence-dependent induction of β-hemolysis involves Ply release. (A) R841 mutant (RT) but not R800 wild-type (wt) cells display spontaneous β-hemolysis on 5.5% horse blood CAT-agar (1×= 1.5 × 10⁴ cells per plate). (B) CSP-induced β-hemolysis on RT mutant cells. One-microliter spots (indicated by white tips) containing 1-100 ng of CSP were deposited immediately after plating (1×= 2.5 × 10⁴ cells per plate), and plates were incubated overnight at 37°C. (C) Inactivation of the ply gene or antibodies to Ply abolish the formation of HH. R1065 (RT ply⁺) but not R1130 (RT ply^-) cells display spontaneous HH (1× = 2.5 × 10⁴ cells per plate). CSP (1 μl, 100 ng, white tips) and PLY-4 monoclonal mouse antibody (5 μl, 4 μg, black tips) (53) were spotted immediately after plating. Note the zone of inhibition of β-hemolysis around the PLY-4 antibody spot.

Fig. 2.

Genetic requirements for the release of Ply. (A) The absence of both LytA and LytC in strain R1332 (lytAC^-) reduced HH formation (Left), whereas inactivation of cibC in its derivative R1414 (lytAC^- cibC^-) exacerbated it (Right). The 1× concentration corresponds to 2.5 × 10⁵ cells per plate. CSP (1 μl, 100 ng) was deposited immediately after plating (indicated by white tips). (B) Organization of the bacteriocin system operon, cibABC. Open bars indicate the limits of cibAB and cibC precise deletions. mariner minitransposon insertions are also indicated (only cotranscribed orientations of inserted minitransposons with respect to the targeted ORF were retained to minimize polar effects). Insertions occurred, respectively, after positions 108, 142, 142, 262, 280, 296, 347, 424, 427, and 430 with respect to the ATG of cibA. Arrows indicate primers used for mariner mutagenesis and for transcriptional studies. (C) Strain D39K, the parent of the laboratory strain R800, also forms HH. The 1× concentration corresponds to 8 × 10² cells per plate.

Fig. 3.

Competent cells cause noncompetent cells to release Ply. Mixtures (2 × 10⁷ total cells) of R1130 (indicated as C ply^-), a ply^- derivative of the RT strain R1065, and 1, 5, or 10% (indicated as 1 + 99, 5 + 95, and 10 + 90, respectively) of either the competence-deficient and ply⁺ strain R1240 (NC) (A), the RT and ply⁺ strain R1065 (C) (B), or the RT and ply⁺, but cibC mutant strain R1135 (C cibC^-) cells (C) were plated on 5.5% horse blood CAT-agar. CSP (1 μl, 100 ng) was deposited immediately after plating (white tips).

Fig. 4.

Proposed model for predation of noncompetent cells through allolysis in S. pneumoniae. (a) Precompetent (pC) cells differ from competent (C) cells by the presence of the CbpD amidase, the two-peptide bacteriocin, CibAB, and its immunity factor, CibC. (b) We propose that cell-to-cell contacts between C and NC cells allow CibAB to interact with the latter, whereas competent cells are protected by CibC. (c) CibAB then triggers the action of CbpD, as well as LytA and LytC (two hydrolases present at the surface of both NC and C cells) on the cell wall of NC cells. (d) Cell wall disruption results in release of cell components, including Ply, TA, LTA, and chromosomal DNA.