The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

1 Abstract Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5’-ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here we report the occurrence of several NAD-RNAs in the opportunistic human pathogen Staphylococcus aureus . Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5’-NAD-capped to a significant extent. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. Reduction of RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects to not seem to be due to changes in RNAIII’s secondary structure upon NAD attachment, as indicated by largely unaltered patterns in in vitro chemical probing experiments. Our study represents a large step towards establishing a biological function of the 5’-NAD cap, which for RNAIII in S. aureus is to modulate the expression of virulence factors. 2 Importance Numerous organisms, including bacteria, are endowed with a 5’-NAD cap in specific RNAs. While the presence of the 5’-NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5’-NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.