The 3' UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions

Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3' UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3' UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3' UTR was found to repress dapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3' UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3' UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3' UTR. Our findings reveal an extended regulatory network for vigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate. Overall design: We have used MS2 affinity pull-downs (MAPS, PMID: 25891076) to capture and sequence mRNAs interacting with the 3'UTR of the vigR transcript from Staphylococcus aureus JKD6008. The experiment used the aTc-inducible vector pRAB11 to express the vigR 3'UTR fused to a 5' MS2 RNA sequence. The same vector expressing MS2 RNA but lackign the vigR 3'UTR was used as a control. The MS2 fusion and control plasmods transformed into Staphylococcus aureus JKD6009 ?vigR-3'UTR and grown in BHI media to an OD of 3.0. Biological duplictes were induced for 15 mins with 0.4uM aTc and processed for MAPS as per PMID: 25891076.