SpxB Is a Suicide Gene ofStreptococcus pneumoniaeand 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 ⌬spxB 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 ⌬lytA 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 ⌬spxB 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.Streptococcus pneumoniae is a gram-positive human pathogen that colonizes the nasopharynx of most children during infancy. Despite the availability of effective antibiotics and vaccines, pneumococci remain a major cause of morbidity and mortality, causing pneumonia, bacteremia, and meningitis. It is estimated that over 850,000 children die annually of pneumococcal infections worldwide (3).S. pneumoniae grown in broth dies spontaneously when reaching stationary phase. How this process occurs and why it would persist in pneumococci are controversial and unresolved issues. Almost a century ago, McLeod and Gordon suggested that this phenomenon is due to accumulation of self-produced H 2 O 2 (18); however, their hypothesis was not widely accepted. In 1970, Tomasz et al. (39) reported that several antibiotic groups induce their bactericidal effect through the induction of lytA and that with loss of LytA function, pneumococci fail to lyse and lose viability at a substantially lower rate. Despite a subsequent report demonstrating that a LytA-independent component was responsible for cell death (19), the phenomenon of spontaneous death, or "suicide," has been conventionally attributed to autolysis by the major autolysin of S. pneumoniae, an N-acetylmuramoyl-L-alanine amidase (LytA) (11,14). LytA appears to be constitutively expressed (9, 11), but its activity is likely under the control of a complex and only p...

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

“…Given these examples of pathogens that have evolved mechanisms to induce DNA damage in mammalian cells, it seemed plausible that the host genome might be an intended target of S. pneumoniae. Indeed, in the case of S. pneumoniae, although it was known that the spxB gene worsens infection (51,52), our work points specifically to the ability of the spxB gene to induce DNA damage as the underlying driver of spxB-associated virulence. Furthermore, to date, the link between pathogen-induced DNA damage and disease has been focused primarily on cancer, whereas here we show an example of a pathogen-secreted DNA-damaging factor that has a direct association with pathogen-induced morbidity and mortality during infection.…”

“…of having spxB in a nasopharyngeal colonization model (52). It has been reported that spxB inactivation in acapsular pneumococcal serotype 2 strain reduces its adherence to alveolar epithelial cells (28).…”

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

“…According to the Centers for Disease Control (CDC), ϳ5 million fatal cases of pneumococcal pneumonia in children under the age of five are reported globally each year (37). Pneumococci have an unusual lifestyle because they produce high levels of hydrogen peroxide that provides a competitive advantage for the organism during colonization of the nasopharynx (1,2). In other organisms, it has been reported that exposure to increased levels of hydrogen peroxide can enhance cellular mistranslation rates both in vivo and in vitro (3,4).…”

“…Following this process, the aminoacyl-tRNA is released from the synthetase and bound by elongation factor Tu (EF-Tu) 2 for delivery to the ribosome and use in protein synthesis (6,7). Aminoacyl-tRNA synthetases are usually highly selective for their cognate tRNA due to the availability of a large surface area for recognition, identity elements within the tRNA molecule itself, and also kinetic proofreading during the aminoacylation reaction (8 -10).…”

Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis

“…LytA, an N -acetylmuramyl-l-alanine amidase, is important for peptidoglycan remodeling during growth [36] and appears to be constitutively expressed [37,38]. Death or suicide of S. pneumoniae in the stationary phase has been attributed to autolysis by LytA [38,39], however, it has been proposed that spontaneous pneumococcal death is due to hydrogen peroxide (H 2 O 2 ), a by-product of aerobic metabolism by SpxB, not LytA [40]. Taken together, not only cell wall attached proteins but also exposed Ccs4 may interact with host cells when host-pneumococcal contact induces LytA expression, causing degradation of the cell wall peptidoglycan and capsule.…”