Screening Of LasR Interacting Proteins And Preliminary Study Of Their Functions In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a conditional pathogen with strong environmental adaptability and pathogenicity,and its pathogenicity is regulated by multiple signals.The most important signal regulatory network is the quorum sensing system(QS system).The QS of P.aeruginosa mainly includes the Las system,the Rhl system,and the Quinolone signal(PQS)system.The Las system is composed las R and las I.Deletion of las R usually causes the impairment of multiple virulence factors which it controls,thereby affectes the pathogenicity of P.aeruginosa.In this paper,the yeast two-hybrid technology was used to detect the interaction between Las R protein with other proteins.in P.aeruginosa,and then further research the relationship between these interacting proteins and the virulence of P.aeruginosa,finally construct the regulatory network of Las R.The results of this paper can provide new theoretical basis for controlling the infection of P.aeruginosa.In this paper,the p GBKT7-las R protein bait plasmid was constructed first,and the self-activation and toxicity detection were carried out.The results show that the bait protein Las R has no self-activation phenomenon to yeast and no toxicity to yeast cells.Next,we hybridized the p GBKT7-las R protein bait plasmid with the P.aeruginosa PAO1whole-genome library plasmid,and screened the proteins that interacted with Las R through four-deficiency plate.184 clones were obtained by screened on four-deficiency plates,detected by lac Z reporter gene,and 25 positive clones were obtained after PCR verification to remove false positives.The results of sequencing and BLAST analysis showed that 25 positive clones contained the coding sequences of 19 genes.The recombinant plasmids of these 19 genes(p GADT7-X)and p GBKT7-las R plasmids were co-transformed into yeast for rotation verification,and two proteins that interact with Las R were obtained: Hut G(N-formylglutamic acid amide hydrolase)and PA5509(histidine catabolic enzyme).Finally,the effect of Hut G on the related virulence phenotypes of P.aeruginosa was investigated used knockout strains and the corresponding complemented strains.The results showed that Hut G could inhibit the production of pyocyanin without affecting the bacterial motility and biofilm.In conclusion,two proteins interacted with Las R were obtained by the yeast two-hybrid method: Hut G and PA5509,and Hut G was found to promote the production of pyocyanin from P.aeruginosa.The research results of this paper provide a theoretical basis for the follow-up study of Las R-interacted proteins to analyze the pathogenic mechanism of the bacteria,thus lay a theoretical foundation for the treatment of P.aeruginosa infection.