## 2 sRNA as Orchestrators of Bacterial Pathogenesis
### Invasion of the Host
#### 2.2.2 Rna Iii  
_Staphylococcus aureus_\(\alpha\)-hemolysin (Hla), a \(\beta\)-barrel pore-forming toxin, is essential to the pathogen's virulence [96]. Secreted as a monomer, Hla oligomerizes into a heptameric pore on host cell membranes, forming a 1-3 nm \(\beta\)-barrel pore that disrupts cellular ionic homeostasis and facilitates the passage of small molecules. At lytic concentrations, Hla causes cell lysis, while sublytic levels alter host signaling, affecting processes such as proliferation, inflammation, cytokine secretion, and cell-cell interactions [97]. Hla targets diverse human cell types, including epithelial, endothelial, and immune cells. Its activity is regulated by the Agr quorum sensing system and involves interactions with host lipids [98], contributing to species- and cell type-specific effects [96]. Structural and mechanistic insights have informed therapeutic strategies aimed at mitigating Hla-mediated diseases.  
The quorum sensing system in \(S\). _aureus_ encodes two divergent RNAs: RNAIII, a central effector, and RNAII, encoding the _agr_-operon [98]. RNAIII, S14-nt sRNA upregulates Hla expression through two distinct mechanisms. First, it binds to the 5\({}^{\prime}\) UTR of Repressor of toxin (Rot) mRNA, a SarA ortholog that represses _hla_ and other virulence factors, such as _hlgB_ and _hlgC_ (encoding gamma-hemolysin components) and _sspB_ and _sspC_ (encoding cysteine protease precursors) [99]. By silencing Rot, RNAIII promotes toxin and virulence factor expression. Second, RNAIII directly binds _hla_ mRNA, exposing the Shine-Dalgarno (SD) sequence and enhancing translation [100].  
Recent studies identified SprY, an sRNA encoded by the prophage \(\varphi\)12, as a novel virulence regulator through its interaction with RNAIII. It acts as an RNA sponge, binding the 13 th stem-loop of RNAIII--the same site targeted by multiple RNAIII-regulated mRNAs, such as _rot_, _spa_, and _eca_[101]. This interaction prevents RNAIII from modulating its targets, enhancing Rot translation and reducing hemolytic activity. SprY is most active during early growth phases, facilitating colonization by suppressing toxin production. Conversely, SprX, another RNAIII-binding sRNA, exerts opposing effects, illustrating a complex network of sRNA interactions in fine-tuning virulence factor expression.  
Additionally, [PERSON] et al. identified 5\({}^{\prime}\)-NAD-capped RNAs in _S. aureus_, including RNAIII, using NAD capture sequencing. RNAIII exhibits variable NAD modification levels [102]. NAD capping reduces \(\alpha\)- and \(\delta\)-hemolysin production and alters the expression of the _splABCD_ operon, which is involved in tissue dissemination. Although in vitro studies suggest that NAD capping does not significantly affect RNAIII's secondary structure or translation, it may modulate regulatory functions in vivo by interacting with other factors. NAD capping is thought to fine-tune bacterial responses to redox state and quorum sensing signals. NAD\({}^{+}\)/NADH 5\({}^{\prime}\) modification of RNA occurs across bacteria, eukaryotes, and viruses, with RNA polymerases incorporating NAD during synthesis initiation. For example, in _E. coli_, RNA NADylation is enhanced under low ATP conditions, weak ATP-RNA polymerase interactions, or DNA supercoiling [103]. Although NADylated RNA remains susceptible to RNase E processing and translation, it undergoes rapid degradation by NudC decapping, a process inhibited by the stringent response molecule ppGpp. This inhibition stabilizes NADylated RNAs during nutrient limitation, extending RNA half-life and facilitating efficient protein expression under stress conditions. In _S. aureus_, this mechanism may provide an energy-efficient strategy for ensuring stable toxin production and stress response under host-induced nutrient limitations.