Roles Of PNPase In The Virulence And SRNA Stability In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a metabolically versatile Gram-negative bacterium in diverse environment. As an opportunistic pathogen, P. aeruginosa causes acute and chronic infections. In acute infection, the type III secretion system(T3SS) and the motility play important roles. During chronic infection, formation of biofilm protects the bacteria from host immune clearance. Remarkably, high expression of the type VI secretion system(T6SS) is a characteristic of chronic infection. Two small RNAs, RsmY and RsmZ(RsmY/Z), reciprocally regulate acute and chronic infection associated virulence factors in P. aeruginosa.Polynucleotide phosphorylase(PNPase) contains an N-terminal catalytic core and C-terminal RNA binding KH-S1 domains. So far, PNPase has been found to control gene expression mainly through 3 mechanisms: degradation of mRNA, affecting translation and modulating sRNA stability.Previously, we found that a pnp::Tn mutant was defective in the secretion of a T3 SS effector protein ExoS in P. aeruginosa. Here we studied mechanism of the PNPase mediated gene regulation. Firstly, we demonstrated that the KH-S1 domains of PNPase were required for the T3 SS and bacterial virulence. Transcriptome analysis revealed a global role of PNPase in gene regulation. Particularly, the RNA level of exsA was reduced in the ?KH-S1 mutant, which resulted in the defective T3 SS expression. Meanwhile, the pilus biosynthesis genes were down-regulated and T6 SS genes were up-regulated in the ?KH-S1 mutant, which were caused by increased RsmY/Z levels. We found that deletion of the KH-S1 domains did not affect the transcription of RsmY/Z, but increased their stabilities. An in vivo pull-down assay and in vitro electrophoretic mobility shift assay(EMSA) demonstrated a direct interaction between RsmY/Z and the KH-S1 fragment.Overall, this study reveals the roles of PNPase in the regulation of virulence factors and small RNAs' stabilities in P. aeruginosa.