The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Profiling the in vivo RNA interactome associated with the endoribonuclease RNase III in Staphylococcus aureus.

Regulatory small RNA (sRNA) have been extensively studied in model Gram-negative bacteria, but the functional characterisation of these post-transcriptional gene regulators in Gram-positives remains a major challenge. Our previous work in enterohaemorrhagic E. coli utilised the proximity-dependant ligation technique termed CLASH (UV-crosslinking, ligation, and sequencing of hybrids) for direct high-throughput sequencing of the regulatory sRNA-RNA interactions within the cell. Recently, we adapted the CLASH technique and demonstrated that UV-crosslinking and RNA proximity-dependant ligation can be applied to Staphylococcus aureus, which uncovered the first RNA-RNA interaction network in a Gram-positive bacterium. In this chapter, we describe modifications to the CLASH technique that were developed to capture the RNA interactome associated with the double-stranded endoribonuclease RNase III in two clinical isolates of S. aureus. To briefly summarise our CLASH methodology, regulatory RNA-RNA interactions were first UV-crosslinked in vivo to the RNase III protein and protein-RNA complexes were affinity-purified using the His6-TEV-FLAG tags. Linkers were ligated to RNase III-bound RNA during library preparation and duplexed RNA-RNA species were ligated together to form a single contiguous RNA 'hybrid'. The RNase III-RNA binding sites and RNA-RNA interactions occurring on RNase III (RNA hybrids) were then identified by paired-end sequencing technology. RNase III-CLASH represents a step towards a systems-level understanding of regulatory RNA in Gram-positive bacteria.

RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3'UTR required for intermediate vancomycin resistance.

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

Networks of Resistance: Small RNA Control of Antibiotic Resistance.

The golden age of antibiotics has passed, and the threat of untreatable antimicrobial resistant infections is now a reality for many individuals. Understanding how bacteria resist antimicrobial treatment and regulate gene expression in response to antibiotics is an important step towards combating resistance. In this review we focus on a ubiquitous class of bacterial gene regulators termed regulatory small RNAs (sRNAs) and how they contribute to antimicrobial resistance and tolerance. Small RNAs have notable roles in modulating the composition of the bacterial envelope, and through these functions control intrinsic antimicrobial resistance in many human pathogens. Recent technical advances that allow profiling of the 'sRNA interactome' have revealed a complex post-transcriptional network of sRNA interactions that can be used to identify network hubs and regulatory bottlenecks. Sequence-specific inhibition of these sRNAs with programmable RNA-targeting therapeutics may present avenues for treating antimicrobial resistant pathogens or resensitizing to our current antibiotics.