Study On The Role And Mechanism Of PA1864 Gene In Regulating Virulence And Pathogenicity Of Pseudomonas Aeruginosa

Pseudomonas aeruginosa(P.aeruginosa)is one of the major species of Pseudomonas which widely exist in nature environment.P.aeruginosa is a gram-negative opportunistic pathogen that can cause infections in kinds of animals and plants.It is worth mentioning that P.aeruginosa can cause fatal infections in multiple organs of human body,and the P.aeruginosa-associated infection especially occur in patients suffering from immune deficiency,metabolic diseases,and invasive diagnosis and treatment.Due to the strong environmental adaptability,complex virulence system and drug resistance mechanisms of P.aeruginosa,the infections caused by P.aeruginosaare often associated with high mortality.Once the infection is established,the bacteria are difficult to eradicate.Therefore,deeper studies on the mechanisms of P.aeruginosa virulence and pathogenesis are urgently needed,which might provide new theoretical basis for prevention and treatment of P.aeruginosa infections.Bacterial invasiveness,toxins and secretion systems which are the main factors of bacterial virulence are associated with the bacterial host pathogenicity.There are numerous virulence components of P.aeruginosa,including bacterial structure components,such as flagella,pili and lipopolysaccharide,and extracellular secretions of virulence factors/proteins,such as pyocynanin,pyoverdine and proteases.In addition,the type III secretion system(T3SS)of P.aeruginosa also plays a crucial role in establishing acute infection by injecting toxic effector proteins ExoS,ExoT,ExoY and/or ExoU into host cells,by which way to promote host cell apoptosis and inflammatory response.Bacterial virulence can be regulated through a variety of mechanisms.Among these,the quorum sensing(QS)system is one of the most important mechanisms.P.aeruginosa contains the largest bacterial genome,and a lot of gene functions of the bacteria are still unknown at present.PA1864 gene is one of the unknown genes with a length of 651 bp.The gene expression product,PA1864 protein located in the cytoplasm is composed of 216 amino acids with a molecular weight of 23.9kDa.According to the predication of Pseudomonas Genome Database Database,the PA1864 may act as a transcription factor and regulates the virulence and host pathogenicity of P.aeruginosa.In order to evaluate the role of PA1864 gene in the virulence and pathogenicity of P.aeruginosa,the PA1864 gene knockout strain(△ PA1864)was firstly constructed by utilizing the Red homologous recombination technique.The host pathogenicity between wide type PAO1 and △ PA1864 were compared by constructing the acute lung infection mouse models.Results showed that compared to the PAO1 infected group,the mouse suffered from △ PA1864 infection showed increased survival rate,and decreased pulmonary edema and tissue damage.The cytotoxicity of the bacteria on human A549 lung cancer cells analyzed by CCK8 and flow cytometry showed that the knock-out of P.aeruginosa PA1864 gene resulted in impaired cytotoxicity of the bacteria.To further investigate the role of PA1864 gene in regulating the P.aeruginosa virulence-associated traits,the bacterial secretory toxins/proteases and T3 SS were analyzed.Resulted demonstrated that the null-mutation of PA1864 gene led to the increased secretion of pyocyanin and swimming,decreased T3 SS gene and protein expression,whereas it showed marginal effects on the production of pyoverdine and proteases of the bacteria.Moreover,to study the possible mechanisms regarding the role of PA1864 gene in regulating P.aeruginosa virulence,the QS system-related genes and signal molecules were analyzed.Results showed that quinolone signal molecules of thequinolone quorum sensing(pqs)QS system were significantly increased in the △ PA1864 compared with the PAO1 wild-type.This study firstly reveals a role of PA1864 gene in the regulation of bacterial pathogenicity,virulence and the virulence-associated QS regulation system of P.aeruginosa,and found that PA1864 gene might promote P.aeruginosa virulence through the quinolone QS system,providing new clues for the further mechanistic investigation of PA1864 gene on P.aeruginosa virulence and host pathogenicity.