SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model

Abstract

The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

FIG. 1.

Representative growth curves of strains 603 and Pn-20 and their mutants under different conditions. (A and B) Optical density (A) and cell viability (B) over time of strains 603 (squares) and 603ΔlytA (triangles), under microaerophilic conditions, in BHI (solid lines) or in BHI supplemented with catalase (dotted lines). (C) Cell viability over time of Pn-20 (diamonds), Pn-20ΔlytA (triangles), and Pn-20ΔspxB (circles), under microaerophilic conditions, in BHI (solid lines) and in BHI supplemented with catalase (dotted lines) and of Pn-20ΔspxB in BHI supplemented with exogenous H₂O₂ (×). (D) Cell viability over time of these strains under anaerobic conditions. Each experiment was repeated independently at least three times.

FIG. 2.

Quantification of cells at stationary phase with features of apoptosis. The percentage of cells with features of apoptosis was determined by annexin V-FITC staining 6 h into stationary phase (A) or by PI staining of permeabilized cells 12 h into stationary phase (B). Data for Pn-20 in BHI and in BHI supplemented with catalase, Pn-20ΔspxB in BHI, Pn-20ΔspxB in BHI supplemented with 1 mM exogenous H₂O₂, and heat-treated Pn-20 (60°C for 1 h) are shown. **, P < 0.01; ***, P < 0.001.

FIG. 3.

Transmission electron micrographs showing the morphological appearance of a typical Pn-20 cell at different growth phases (magnification, ×18,500). (A) Exponential phase. Cells have a normal appearance. (B) Six hours into stationary phase. Cells have an apoptotic appearance, with condensation of DNA. (C) Twelve hours into stationary phase. Cells have a ghost cell appearance, with ruptured membranes and clearing of cytoplasm. (D) Heat-killed cells. No condensation of DNA at the cell periphery is seen; rather, DNA is at the center of the dead cells. Arrows point to areas of condensed DNA.

FIG. 4.

Transmission electron micrographs showing the morphological appearance of Pn-20 cell cultures (magnification, ×4,800). (A) Exponential phase (<5% appear apoptotic); (B) 6 h into stationary phase (>90% appear apoptotic); (C) 12 h into stationary phase (>99% appear apoptotic or have a ghost cell appearance). (D to F) TEM of bacteria 6 h into stationary phase; (D) Pn-20 in culture supplemented with catalase; (E) Pn-20ΔspxB; (F) Pn-20ΔlytA.

FIG. 5.

Density of S. pneumoniae 603 and 603ΔspxB colonization determined 7 days after intranasal inoculation of infant Sprague-Dawley rats. Group 1 was inoculated with 1 × 10⁷ CFU of strain 603. Group 2 was inoculated with a mixture of 5 × 10⁶ CFU of 603 and 603ΔspxB; 2a shows colonization by strain 603 as determined by plating on streptomycin blood agar plates, and 2b shows colonization by 603ΔspxB as determined by plating on kanamycin blood agar plates. Group 3 was inoculated with 1 × 10⁷ CFU of strain 603ΔspxB. Solid lines indicate group medians (CFU/nasal wash). The dashed line indicates the limit of detection. P values were determined by Mann-Whitney tests for differences in the distributions of CFU/nasal wash between groups.