RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

Recent insights into the world of dual‐function bacterial sRNAs

“…Thus, the underlying mechanism of how RNAIII stimulates esxA translation may be new for sRNA‐mediated translational activation but remains to be elucidated in detail. Since the agr locus also contributes to the transcription of esxA , a concerted action of agr and RNAIII in regulation of EsxA production at both the mRNA and protein levels as part of a coherent feed‐forward loop is likely (McKellar et al, 2022; Schulthess et al, 2012).…”

“…In addition to the known targets, two new targets have been assigned for RNAIII. Firstly, a segment of helix 9 of RNAIII binds directly to esxA mRNA and stimulates the production of EsxA‐toxin (McKellar et al, 2022). The EsxA toxin is involved in bacterial persistence and dissemination during infection and thus important for intracellular survival of S. aureus in infected epithelial cells by inhibiting apoptosis (Burts et al, 2005; Sundaramoorthy et al, 2008).…”

Advantages and Limitations of UV Cross-linking Analysis of Protein-RNA Interactomes in Microbes

“…Resulting methods, such as CLASH (cross-linking, ligation and sequencing of hybrids), hiCLIP (RNA hybrid and individual-nucleotide resolution CLIP), and RIL-seq (RNA interaction by ligation and sequencing) [46][47][48] , have used RBPs chaperoning RNA-RNA interactions as bait to map and functionally characterise non-coding transcripts. So far, these procedures have been used in systems including mammalian cells, E. coli , Salmonella and S. aureus [47][48][49][50][51][52][53][54][55] , which unravelled large ncRNA-RNA interactomes in these organisms. Eventually, customising present protocols to replace UV irradiation by chemical cross-linking could verify and expand prevailing knowledge on the microbial and metazoan RNA-RNA interactome.…”

Amphiphilic Janus nanoparticles for nitric oxide synergistic photodynamic eradication of MRSA biofilms

“…Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of chronic infections, leading to high morbidity and mortality. 1–5 Biofilms, formed by bacteria that adhere to surfaces and aggregate together within a matrix of extracellular polymeric substances (EPS), create a formidable barrier to effective anti-bacterial treatments and evade the host immune system. 6–10 The formation of biofilms further exacerbates the severity of MRSA infections.…”