Study The Function Of PA0667 And PA2734 Involved In The Susceptibility Of Pseudomonas Aeruginosato Carbapenem Antibiotics

Pseudomonas aeruginosa is one of the prime opportunistic bacterium of the leading infection in the world, especially in immunity compromised patients such as burns and organ transplantation. The cure of the infection by Pseudomonas aeruginosa is hard because of its intrinsic and acquired resistance to lots of antibiotics.Carbapenem antibiotics classed to β-lactam antibiotics, are one of the most significant antibiotics against bacterial infection.But in recent yearswith the wide spread use of carbapenems in clinical setting, understanding the knowledge of the mechanisms about carbapenem is of profound meaning and can lay foundation for developing effective therapy alternatives.PA0667 and PA2734 were selected from transposon mutations library we constructed earlier involved in carbapenem resistance. Then we constructed knockout genes of the two genes respectively. Further study was carried on to investigate the function involved in antibiotic resistance.PA0667 is a type IV and function unknown gene. The tolerance of the mutant of PA0667 to biapenem, meropenem and imipenem was increased compared with PAO1.The results show that PA0667 are responsible for the tolerance of PA to carbapenem antibiotics.PA0667 was predicted to encode membrane proteins related to metalloendopeptidases, and the sequence of PA0667 shares 54% similarity to hypothetical protein YebA in E.coli. The growth of mutant 0667 showed no difference with PAO1. The mutant 0667 showed compromised outer membrane integrity and stability, thus implying the PA0667 played vital role in antibiotic resistance. Ampicillin can prevent bacterial synthesis of cell wall of the third and final stage, eventually leading to cell lysis. The growth of the PA0667 mutant and PAO1 were repressed with the presence of ampicillin in LB. The expression of pbpG7,pbpla,pbpB3,pbpA2 in those two mutants were decreased compared with wild type PAO1. According to the results of Chinese Cabbage experiment, Drosophila infection analysis and swarming test, indicate that PA0667 was responsible for the virulence and pathogenicity. In order to further confirm the function of PA0667,we constructed the knockout mutant of operon PA0666-0667,turns out that PA0666-0667 involved in antibiotic resistance and pathogenicity.PA2734 is also a type IV function unknown gene, and predicted to encode the S restrictionendonuclease subunit. According to our results, the expression of plasmid PilD, fliC in PAO1(A2734) were decreased compared with PAO1.In addition, motility analysis of mutant 2734 showed PA2734 may related to virulence. Adhesion assay suggested that the adhesion of the PAO1(A2734) was reduced compared with that of PAO1.And the results indicate the adhesion is important for bacterial’scolonization and diffusion.