Abstract
Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.
Highlights
Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that resides in the human nasopharynx
Pneumocin expression is controlled by a pheromone-induced two-component system (BlpR/H) but the triggers for the system are poorly understood
We show that the pheromone-induced twocomponent system driving competence for genetic transformation, ComD/E, controls expression of BlpC, the peptide pheromone activating BlpR/H-dependent gene expression
Summary
Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that resides in the human nasopharynx. In most pneumococcal strains the genes mediating export of the blp peptides, encoding the ABC transporter BlpAB, carry frameshift mutation(s) that render these genes non-functional. This paradox raises two questions: 1) are Blp bacteriocins exported in strains lacking a functional transporter? One possibility is that bacteriocins are not exported, but that strains with interrupted blpAB alleles constitute so-called cheater sub-populations, which cannot export pneumocins alone but are able to express immunity in response to co-colonizing strains [7] Such cheaters would only activate blp expression in response to secreted BlpC in their surroundings. These functional data clarify the mechanisms of blp regulation and cast doubt on the validity of the blpA cheater hypothesis
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