# Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa
## 4 Metabolic Pathways Associated with Pathogenicity
### Virulence Factors
#### 4.1.4 Multiple Signalling Systems in the Regulation of Virulence Genes
aeruginosa_ infectious processes by counter-regulating the expression of virulence factors associated with acute infection, such as T3 SS, type IV pili, and flagellum formation, as well as mechanisms associated with chronic infection, such as T6 SS and biofilm formation [3]. The GaCS/GaCA system consists of a transmembrane hybrid sensor HK, GaCS. The function of GaCS is to phosphorylate the response regulator (RR) and histidine phosphotransfer (Hpt) domains, resulting in their transfer to GaCA in the cytosol. GaCA promotes the transcription of two regulatory sRNAs (RsmZ and RsmY). These two bind to the small RNA-binding protein RsmA, through characteristic GGA motifs, preventing it from attaching to its target mRNAs, avoiding their translation suppression and increasing mRNA turnover, positively regulating the expression of genes related to T3 SS, type IV pili, and flagellum formation, while negatively regulating the expression genes involved in T6 SS and biofilm formation [129; 130]. The GaCS/GaCA regulators are LadS, RetS, and PA1611. LadS, a hybrid histidine kinase, promotes autophosphorylation of GaCS by shuttling additional phosphate groups into its Hpt domain [131]. RetS, a hybrid histidine kinase, captures phosphate groups from the GaCS, preventing the autophosphorylation of GaCS [132]. The PA1611, a hybrid histidine kinase, interacts with RetS, thus preventing RetS from inhibiting GaCS [133]. However, the ligands binding to the sensor domain of GaCS are unknown. Studies in mice have shown that the loss of RsmA results in a reduction in colonization during the early stages of acute infection, but also favors chronic infection, resulting in increased inflammation in the lungs of infected mice [129]. Nucleotide Signals  
Studies related to signal propagation have shown that a group of nucleotide-based molecules also plays an important role in bacterial control [107]. The 3', 5'-cyclic adenosine monophosphate (cAMP) is an essential signaling molecule for the regulation of virulence factors in acute infections [134]. Basically, cAMP binds and activates transcription factors from the CRP family. In the genome of _P. aeruginosa_ PAO1, cAMP synthesis is driven by two adenylate cyclases, CyaA and CyaB [135; 136]. In mice, the enzymatic activity of CyaB is increased in response to specific signals, such as low calcium levels, showing that the most significant contribution to cellular levels of cAMPs is derived from CyaB [137]. The production of various virulence factors such as unregulating exotoxin A, T3 SS, T2 SS, and the _las_ and _rhl_ QS systems, can be directly or indirectly controlled by binding cAMP to the homologous CRP VfR regulator (virulence factor regulator). On the other hand, flagellar gene expression is downregulated by repressing expression of _fleQ_[138; 139]. In _P. aeruginosa_, mutations in _muca_ (virulence factor that regulates alginate) and activation of AlgU inhibit cAMP-VfR signaling, proving that this type of cAMP-VfR signaling works as a complex signalling cascade with many regulatory inputs [107]. The other group is the cellular alarmones ppGpp (5'-diphosphate 3'-diphosphate guanosine) and ppGpp (5'-triphosphate 3'-diphosphate guanosine) [140]. The cellular pool of ppGpp can be controlled by two classes of enzymes: monofunctional synthetases and bifunctional synthetase-hydrolase enzymes. The monofunctional synthetases (RelA proteins) use GTP and ATP to generate ppPGPp, which is then converted in ppGpp. Bifunctional synthetase-hydrolase enzymes (SpoT and Rel proteins) can act on ppGpp and ppGpp, and ppGpp hydrolyses the nucleotides to yield either GDP and pyrophosphate (PPi), whereas ppGpp hydrolyses GTP and PPi [141; 142]. The association of ppGpp with the transcriptional regulator DksA (global regulator of metabolism) allows the interaction between ppGpp and the RNA polymerase, inhibiting the transcription of ribosomal RNA promoters. This inhibition occurs simultaneously with the activation of stress response genes transcription and upregulation of amino acid biosynthesis pathways [140; 143].