## 5 Role of sRNA in Quorum Sensing
### PhrS and ReaL  
_P. aeruginosa_ is an opportunistic pathogen that causes severe infections in immunocompromised individuals and cystic fibrosis patients [244]. In _P. aeruginosa_, four interconnected QS systems--Las, Rhl, Pqs, and Iqs--regulate over 300 virulence-related genes [229; 245]. The Las and Rhl systems use N-acyl homoserine lactone (AHL) signals, with _Las1_ producing 3-oxo-C12-HSL to activate _LasR_, leading to the expression of virulence factors like elastases and exotoxins. The Rhl system, with Rhl producing C4-HSL, interacts with Rhl to regulate factors such as rhamnoidyrid and hydrogen cyanide. The Pqs system synthesizes quinolones (PQS signals) via PqsABCD, activating PqsR. PQS acts as a critical link between the Las and Rhl systems, regulating genes involved in biofilm formation, iron acquisition, and the production of the redox-active toxin pyocyanin (PYO) [246; 247]. PYO contributes to pathogenicity by generating ROS, inducing oxidative stress, and damaging host tissues, while also exhibiting antimicrobial activity against competing microorganisms [248]. The Iqs system, with _ambBCDE_, produces IQS signals. These QS systems function hierarchically, with Las at the top, regulating Rhl and Pqs, while Pqs also modulates Las and Rhl.  
The sRNA PhrS plays a central role in modulating QS and virulence in _P. aeruginosa_[249]. Its expression is controlled by the oxygen-responsive transcription factor ANR and is enhanced during hypoxia or the stationary phase. A putative ANR binding site upstream of the _phrS_ promoter has been identified, and _phrS_ expression is abolished in ANR-deficient mutants [249]. Additionally, Hfq indirectly regulates _phrS_ by stabilizing ANR. PhrS promotes the translation of _pqsR_, a key activator of PQS synthesis through structural rearrangements in hypoxic conditions, ultimately enhancing PQS and PYO production. PYO contributes to immune evasion through ROS generation and the modulation of IL-8-driven immune responses. PhrS-deficient strains exhibit impaired PYO production, highlighting its critical role in _P. aeruginosa_ virulence. In addition, PhrS downregulates the expression of cytochrome bo3 oxidase genes (_cyoAB_), facilitating a shift in oxygen utilization pathways under low oxygen [249]. By overcoming the oxygen-dependent suppression of PQS, PhrS supports QS-dependent gene expression under hypoxia, a condition common in biofilms and cystic fibrosis lung infections. Furthermore, PhrS-mediated PQS synthesis promotes extracellular DNA release, a crucial component for biofilm stability. These findings suggest that targeting PhrS may offer a novel strategy to mitigate _P. aeruginosa_ infections.  
In addition to PhrS, the sRNA Real in _Pseudomonas aeruginosa_ plays a role in virulence by linking the Las and Pqs quorum sensing (QS) systems [250]. The deletion of _realL_ reduces virulence in the _Galeria mellonella_ infection model, while its overexpression leads to a hyper-virulent phenotype. ReaL is negatively regulated by the Las QS regulator LasR and enhances the synthesis of the pqs quinolone signal PQS by posttranscriptionally activating PqsC. The region from \(-13\) to \(+32\) of PqsC mRNA is predicted to form a stable stem-loop structure, sequestering the SD sequence and the AUG start codon through base pairing with the RII region, effectively acting as an anti-SD/AUG element and repressing PqsC translation. When ReaL binds to the RI-RII region of PqsC, it induces a conformational change that extrudes a loop containing the SD and AUG sequences. This structural rearrangement facilitates the recruitment of the 30S ribosomal subunit to the mRNA, enabling translation initiation [250]. This affects key virulence traits such as PYO production, biofilm formation, and swarming motility. Additionally, ReaL responds to host-relevant conditions like temperature and oxygen levels and is upregulated in the stationary phase by the alternative \(\sigma\) factor RpoS. These regulatory mechanisms fine-tune PQS synthesis and contribute to _P. aeruginosa_ virulence.