Role Of ClpP Gene In The Virulence Properties Of Pseudomonas Aeruginosa

ObjectivePseudomonas aeruginosa(P.aeruginosa) is a gram-negative conditional pathogen. It can cause chronic persistent infection in long-term hospitalization patients especially cystic fibrosis patients. During infection and pathogenic processes, It can expresses varieties of virulence factors —— the cell surface virulence factors( pili, flagella, lipopolysaccharide, alginate) and extracellular toxins( alkaline protease, exotoxin A, elastase, phosphoplipase, pyocyanin and effector proteins secreted by type III secretion system), and is primacy among the pathogens of nosocomial infection.Bacterial pathogenic ability can be affected by external environment change. Maintaining homeostasis is useful to full expression of bacterial virulence factors. Studies showed that Clp P protease played an important role in the homeostasis under stress conditions. Clp P protease is encoded by clp P gene. It was first found in Escherichia coli(E.coli) and combined with AAA+ chaperon like Clp X and Clp A to form a compound. The latter can recognize specific polypeptide tags and then transform these proteins to Clp P. Irreversible damaged proteins produced and accumulated in stress conditions are degraded to small molecule peptides to maintain bacteria homeostasis. Clp P protease of Escherichia coli and Enterobacteriaceae can regulate the type Ⅲ secretion system and other virulence factors to affect bacterial’pathogenicity. But there were few reports about the Clp P of P.aeruginosa. Because P.aeruginosa PAO1 has 70% homology with E.coli, so we speculate that the Clp P of P.aeruginosa has similar biological function with E.coli and has certain regulatory effects in the expression of virulence factors of P.aeruginosa. Therefor, our study plans to construct clp P gene deficient strains and over-expression strains of P.aeruginosa PAO1, to determine the effector genes expression of type III secretion system, pyocyanin, motilities, biofilms and and other virulence factors, thus preliminary discuss the role ofclp P gene in the virulence properties of P.aeruginosa.Methods(1) 3’fanking sequences and 5’flanking sequences of clp P gene and gentamicin resistance cassette(GN) are amplified by PCR and inserted into the suicide plasmid p EX18 Tc, resulting in clp P gene targeting vector. Transformed it into P. aeruginosa PAO1 and obtained clp P gene deficient mutant(Δclp P).(2) The open reading frame of clp P gene was amplified by PCR and inserted into the downstream of lac Z promoter of plasmid p BBR1MCS-2, resulting in clp P gene expression vector. Transformed it into P. aeruginosa Δclp P mutant and obtained clp P gene over-expression strain(S-clp P).(3) Total RNA of P. aeruginosa PAO1 was extracted with Trizol reagent.Detect the expression levels of effector genes(exo S 、 exo T and exo Y) of type Ⅲ secretion system among wild-type, deficient and over-expression strains by real-time PCR.(4) Extract the pyocyanin by chloroform and observe the OD520 to determine the pyocyanin production.(5) Detect the three kinds of motilities(swarming, swimming and twhiching) of wildtype, deficient and over-expression strains by using special culture medium.(6) Cultivate biofilms in vitro for 72 h, then stained by crystal violet and examined by fluorescence microscope to observe the biofilms formation among wild-type, deficient and over-expression strains.(7) The growth ability of wild-type, deficient and over-expression strains were determined by time as X-axis and changes of OD600 during the observation as Yaxis.(8) Comparing the colony counts on LB plates containing certain concentrations of H2O2、Na Cl and acid to observe the tolerance to H2O2、Na Cl and acid among wild-type, deficient and over-expression strains, with the colony counts on LB(p H7.0) plate as reference.Results(1) Enzyme digestion, PCR and sequencing results showed that the clp P gene targeting vector was constructed right. Transformed it into PAO1 and successfully constructed P. aeruginosa Δclp P mutant. Sequencing results showed that the 37bp~453bp of clp P gene had been deleted.(2) Enzyme digestion, PCR and sequencing results showed that the clp P gene expression vector p BB-clp P was constructed right. Transformed it into Δclp P and successfully obtained P. aeruginosa clp P gene over-expression strains S-clp P.(3) Real-time PCR showed that the expression levels of exo S、exo T and exo Y were decreased by 32.2%, 52.4% and 57.7% respectively compared to wild-type, while the value of S-clp P were increased by 4.11, 7.11 and 4.25 times respectively.(4) The pyocyanin production of P. aeruginosa Δclp P mutant was half of that in PAO1. While the pyocyanin production was increased about 2.5-fold in S-clp P compared to that in the wild-type.(5) P. aeruginosa Δclp P mutant has deficiency in the three kinds of motilities, while Sclp P could restore the motilities to the wild-type level.(6) The P. aeruginosa PAO1 Δclp P mutant had decreased ability to form biofilms after 72h’ cultivation compared to wild-type.(7) During 7h’ culture in 37℃, P. aeruginosa Δclp P mutant grew slower than wild- type at logarithmic stage and came up with PAO1 when reached stationary phase. While the growth between Δclp P and S-clp P has no significant difference.(8) The Δclp P mutant strain was more sensitive to H2O2、Na Cl and acid compared to wild-type, while the stress tolerance of S-clp P was nearly the same as wild-type.ConclusionsThe clp P gene plays important roles in the virulence of P. aeruginosa by participating in regulation of effector genes expression of type Ⅲ secretion system, pyocyanin production, biofilms formation, motilities and stress tolerance.