Study On The Role And Mechanism Of Pseudomonas Aeruginosa PpGpp In Regulation Of Its Pathogenicity And Virulence

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen which is widely spread in natural environment,existing in plants,animals and humans.This ubiquitous bacterium can cause a series of bacterial infections in acute and chronic diseases with high morbidity and mortality,especially in the immunocompromised patients.Recently,lots of multi-drug resistant bacteria have developed with the abuse and misuse of antibiotics.The rapid mutagenesis of bacteria,the failure of existing antibiotics and the delay of new antibacterial compounds pose great threats to public health,clinical prevention and treatment of pathogenic organisms.Thus,it is urgent to illuminate the virulence systems and the underlying mechanisms in P.aeruginosa infections,for the sake of developing and exploring potential therapeutic strategies.The pathogenic versatility of P.aeruginosa is attributed to its large arsenal of virulence factors and strong invasiveness,such as pyocynin,pyoverdin,elastase,exotoxin A and phospholipase C.Besides,the alginates and other polysaccharides secreted onto the colonized surface are demonstrated to be involved in biofilm formation,which strengthen the bacteria resistance to antibiotic treatment and human antibacterial defense systems.To quickly sense and adapt to the hostile environment,complex adaptive responses are launched in P.aeruginosa in order to regulate related gene expression and cell-to-cell signaling pathways.Among these regulatory systems,the conservative stringent response is essential for coordinating the nutrient-poor and stressful environments by bacterium,which is mediated by the second messengers guanosine 5’-triphosphate-3’-diphosphate and 5’-diphosphate-3’-diphosphate [(p)pp Gpp].In most Gram-negative bacteria,pp Gpp synthesis is controlled by synthetase Rel A and bifunctional synthetase/hydrolase Spo T.The elevation of pp Gpp levels induced by amino acid/carbon/phosphate starvation and oxidative stress regulates the downstream genes transcription by modulating RNA polymerase activity,which finally facilitates the bacterial adaptation to adverse conditions.Recent studies have shown that the stringent response mediated by pp Gpp is critical for regulating growth,virulence and stress coping in P.aeruginosa.However,the related pathways and specific mechanisms are rarely reported.Therefore,deep investigation of the effects of pp Gpp on bacterial virulence and pathogenesis as well as the potential mechanisms are significant for developing effective drugs and treatment of related infections.To investigate the regulatory role of pp Gpp,the pp Gpp deficient P.aeruginosa Δrel AΔspo T strain(ΔRS)and its complemented strain(ΔRS++)were constructed utilizing the Red homologous recombination technique.Herein,we reported that the ΔRS strain showed pp Gpp sythesis dificiency and decreased cytotoxicity and host pathogenicity compared to wild-type PAO1 and the complemented strain ΔRS++.Moreover,deficient in pp Gpp synthesis led to reduced levels of T3 SS protein,decreased virulence production,impaired swarming motility and biofilm formation compared to PAO1 and ΔRS++ strain.However,the addition of quinolone signal molecules rescued the inhibited synthesis of T3 SS Exo S protein,alginates and pyoverdin and swarming motility in the ΔRS strain.Taken together,these results suggest that pp Gpp is required for virulence regulation in P.aeruginosa,and this may occur in a quinolone quorum-sensing system-dependent manner.Our findings provide new clues for the investigation of complex mechanisms between bacterial adaption and virulence regulation,and also the development of interference strategies against P.aeruginosa infection.