The chromosomal relBE2 toxin–antitoxin locus of Streptococcus pneumoniae: characterization and use of a bioluminescence resonance energy transfer assay to detect toxin–antitoxin interaction

Abstract

SummaryProteic toxin-antitoxin (TA) loci were first identified in bacterial plasmids, and they were regarded as involved in stable plasmid maintenance by a socalled 'addiction' mechanism. Later, chromosomally encoded TA loci were identified and their function ascribed to survival mechanisms when bacteria were subjected to stress. In the search for chromosomally encoded TA loci in Gram-positive bacteria, we identified various in the pathogen Streptococcus pneumoniae . Two of these cassettes, sharing homology with the Escherichia coli relBE locus were cloned and tested for their activity. The relBE 2 Spn locus resulted to be a bona fide TA locus. The toxin exhibited high toxicity towards E. coli and S. pneumoniae , although in the latter, the chromosomal copy of the antitoxin relB 2 Spn gene had to be inactivated to detect full toxicity. Cell growth arrest caused by expression of the relE 2 Spn toxin gene could be reverted by expression of the cognate antitoxin, relB 2 Spn , although prolonged exposition to the toxin led to cell death. The pneumococcal relBE 2 Spn locus is the first instance of a chromosomally encoded TA system from Grampositive bacteria characterized in its own host. We have developed a bioluminescence resonance energy transfer (BRET) assay to detect the interactions between the RelB2 Spn antitoxin and the RelE2 Spn toxin in vivo . This technique has shown to be amenable to a high-throughput screening (HTS), opening new avenues in the search of molecules with potential antibacterial activity able to inhibit TA interactions.

Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

“…The number of TAS reported to exist in S. pneumoniae was relatively small, with at most five in strain R6 and six in virulent strain TIGR4, when they were first reported in 2005 to 2006 (117,122). About 4 years later, the number was increased to up to eight TAS in the genome of S. pneumoniae (118).…”

“…Even though TAS of G ϩ bacteria are also widely distributed and have been found in both chromosomes and plasmids, they are comparatively less well characterized. For instance, some of the well-studied TA genes of G Ϫ bacteria have also been discovered in G ϩ bacteria, such as mazEF in S. aureus (40) and relBE and yefMyoeB in S. pneumoniae (117). Nevertheless, there are cases in which specific TAS seem to play different roles in G ϩ and in G Ϫ bacteria: the epsilon-zeta TA cassette stabilizes plasmids in E. coli less efficiently than in Bacillus subtilis.…”

“…In this review, we focus on the TA genes of S. pneumoniae because they constitute a fairly unexplored world: only three pneumococcal TA pairs, namely, RelBE2, PezAT, and YefMYoeB, have been studied so far, and only the PezAT TA pair has been characterized in terms of structure and function (21,82,113,117). Surprisingly enough, an in-depth study of possible TAS present in the genomes of pneumococcal strains that have been sequenced showed that their number is far larger than envisaged (90), and our results presented here show that their number can be even larger.…”

Molecular Control of Bacterial Death and Lysis

“…The number of TA loci varies from none (as in the case of many obligate host-associated bacteria) to as many as 45 found in the Nitrosomonas europaea genome (189). The chromosomal TA modules of E. coli have thus far been the most extensively characterized, but note that chromosomal TA modules in other organisms including Streptococcus mutans (150), Bacillus anthracis (1), S. aureus (66), and S. pneumoniae (123,178) have recently been studied. Several reviews detailing the advances in chromosomal TA module research have been published (33,(57)(58)(59)(60)80); therefore, this review will focus on two well-characterized E. coli TA modules, MazEF and RelBE, as well as on the E. coli TA-like module HipBA, highlighting the evidence for and evidence against their controversial roles in eliciting bacterial cell death.…”

Structure and Function of Bacterial Kid-Kis and Related Toxin-Antitoxin Systems

“…Therefore, at present, the RelE-RelB TA system is considered to be a stress-response element rather than a cell-killing module [102]. However, it was shown that in the case of the homologous pneumococcal RelBE2 system, prolonged exposure of the cells to the toxin resulted in the inability to rescue the cultivability of the cells by subsequent expression of the antitoxin [104].…”