Person:

Malley, Richard

CD4(+) T-cell-dependent acquired immunity confers antibody-independent protection against pneumococcal colonization. Since this mechanism is poorly understood for extracellular bacteria, we assessed the antigen specificity of the induction and recall of this immune response by using BALB/c DO11.10Rag(-/-) mice, which lack mature B and T cells except for CD4(+) T cells specific for the OVA(323-339) peptide derived from ovalbumin. Serotype 6B Streptococcus pneumoniae strain 603S and unencapsulated strain Rx1Delta lytA were modified to express OVA(323-339) as a fusion protein with surface protein A (PspA) (strains 603OVA(1) and Rx1Delta lytAOVA(1)) or with PspA, neuraminidase A, and pneumolysin (Rx1Delta lytAOVA(3)). Whole-cell vaccines (WCV) were made of ethanol-killed cells of Rx1Delta lytA plus cholera toxin (CT) adjuvant, of Rx1Delta lytAOVA(1) + CT (WCV-OVA(1)), and of Rx1Delta lytAOVA(3) + CT (WCV-OVA(3)). Mice intranasally immunized with WCV-OVA(1), but not with WCV or CT alone, were protected against intranasal challenge with 603OVA(1). There was no protection against strain 603S in mice immunized with WCV-OVA(1). These results indicate antigen specificity of both immune induction and the recall response. Effector action was not restricted to antigen-bearing bacteria since colonization by 603S was reduced in animals immunized with vaccines made of OVA-expressing strains when ovalbumin or killed Rx1Delta lytAOVA(3) antigen was administered around the time of challenge. CD4(+) T-cell-mediated protection against pneumococcal colonization can be induced in an antigen-specific fashion and requires specific antigen for effective bacterial clearance, but this activity may extend beyond antigen-expressing bacteria. These results are consistent with the recruitment and/or activation of phagocytic or other nonspecific effectors by antigen-specific CD4(+) T cells.

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.