Introduction

Bacteria can protect themselves from antibiotic treatment by becoming resistant or persistent. Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with antibiotic treatment failures and relapsing infections. Among others, type I toxin-antitoxin (TA) systems have been linked to persister cells formation. They are composed of a peptide toxin whose overexpression confers growth stasis or cell death, and of an RNA antitoxin that base-pairs with the toxin mRNA to inhibit its translation. In the Staphylococcus aureus SprG1/SprF1 type I TA system, SprF1 is a multifaceted antitoxin that, in addition to repress SprG1 expression and toxicity, can also bind ribosomes to inhibit global translation and promote persister cells formation [1]. To better understand SprF1 regulatory implication in antibiotic persistence, we aim to identify its RNA direct targets using in silico analysis and MAPS (MS2-affinity purification coupled with RNA sequencing) experiments.

Material and methods

In silico SprF1 targets were identified using three softwares: RNApredator, IntaRNA and CopraRNA. In parallel, a MAPS experiment was carried out to identify in vivo SprF1 targets. To achieve this, an MS2 aptamer was fused at the 5' or 3'-end of SprF1 RNA. These chimeras were cloned in pRMC2 plasmid, allowing anhydrotetracycline (aTc) inducible expression, and transformed into S. aureus HG003 strain. After aTc induction, RNAs were extracted and purified. SprF1 targets were identified by RNA sequencing and differential expression analysis using the HTSeq/DESeq2 pipeline.

Results and conclusion

MAPS enabled to identify, besides SprG1, eleven novel SprF1 targets. These include mRNAs encoding the 50S ribosomal protein L31 (RpmE2) and the membrane protein insertase YidC. Interestingly, yidC is one of the five common mRNA targets found by the three in silico interaction predictions softwares. Based on our in vivo and in vivo/in silico investigations, these two candidates were selected. SprF1 interaction with yidC and rpmE2 was more precisely defined in silico using IntaRNA and confirmed in vitro by gel retardation assays. Next, the functional link between SprF1, these two mRNA targets, and persister cells formation will be studied. Overall, this work could lead to the identification of novel therapeutic strategies to fight against recurrent S. aureus infections.