Interaction Mechanism Of Quorum Sensing System-mediated Drug-resistant Pseudomonas Aeruginosa And Escherichia Coli

Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that can thrives in a varity of natural or clinical environments by using the hierarchically arranged quorum sensing(QS)regulation,and has become an important pathogen that harms human health and social development.In bacterial chronic respiratory infections,there are usually complex interactions between pathogenic bacteria that affect the progress of the disease.However,the relationship and potential interaction mechanisms between the dominant pathogen P.aeruginosa and other bacterial pathogens in chronic respiratory infection lesions remain largely unexplored.The current study investigated the interspecific interactions between P.aeruginosa and Escherichia coli in the following aspects:(1)Through the identification of pathogenic bacteria in the sputum samples of patients with clinical chronic obstructive pulmonary disease,the proportion of pathogenic bacteria in the patients was determined.Among 25 sputum samples,Streptococcus species showed the highest detection rate(11 of 25)followed by P.aeruginosa(5 of 25),E.coli(4 of 25),Enterococcus faecalis(4 of 25),Bacillus cereus(2 of 25),Corynebacterium argentoratense(2 of 25),and Staphylococcus epidermidis(1 of 25).The minimal inhibitory concentration(MIC)experiments of commonly used antibiotics on the co-isolated P.aeruginosa COP2 and E.coli COP1512 as well as the model P.aeruginosa strain PAO1 were carried out by using dilution method of microbroth.The results showed that PAO1 showed resistance to ampicillin,COP2 showed resistance to cefotaxime,ampicillin,meropenem,and imipenem,and COP1512 showed resistance to imipenem.(2)By estabolising the on-plate proximity assay,we found that both P.aeruginosa PAO1 and COP2 could inhibited the growth of neighboring E.coli COP1512,and the COP1512 colonies close to PAO1 or COP2 were significantly inhibited with time.On the other side,the presence of E.coli COP1512 had on significant effect on the growth of P.aeruginosa colonies.These results indicated that P.aeruginosa had intrisic competitive advantage over E.coli.(3)The intracellular transcriptional patterns of interacting P.aeruginosa PAO1 and E.coli COP1512 were determined by using RNA-sequencing.The result showed that no significant transcription change was detected in PAO1 in the co-culture with COP1512 after 1 day.However,the transcriptional pattern of P.aeruginosa PAO1 changed significantly after 4 days.When the differentially expressed genes were mapped to the QS regulon of P.aeruginosa,it was found that the expression of the key QS regulatory genes such as lasR and rhlR,the genes involved in PQS and cyanide biosynthesis were all down-regulated.The expression of genes(type IV secretion system)related to the biosynthesis of pyocyanin and H2-T6 SS were increased.By contrast,491 down-regulated and 378 up-regualted genes were detected in E.coli COP1512 that co-cultured with PAO1.The decreased genes of E.coli COP1512 were significantly enriched in the aerobic metabolism of cells,while the increased genes were enriched in the anaerobic metabolism and membrane transport.(4)The gene knockout experiment was performed based on the differentially expressed genes of the P.aeruginosa QS system in the transcriptome results to further study the interaction between QS-deficient strains and E.coli.The results showed that the mutant strains PAO1-ΔlasR,PAO1-ΔrhlR,PAO1-ΔmvfR,and PAO1-ΔmvfRlasR of P.aeruginosa exhibited different degrees of inhibitory effects on E.coli with increasing culture days.Compared with the wild-type PAO1,the inhibitory effect of all mutant strains on E.coli was significantly reduced,especially the PAO1-ΔmvfRlasR double-mutant strain had the weakest inhibitory effect on E.coli.After knocking out the lasR gene of clinically isolated P.aeruginosa COP2,it also showed inhibitory effect on E.coli.Moreover,the study also found that the supernatant of E.coli COP1512 can promote the synthesis of PAO1-ΔlasR pyocyanin.It was found by fluorescence quantitative PCR that the supernatant of E.coli COP1512 could stimulate the expression of rhlR,mvfR and its downstream functional genes phzA and pqsE in P.aeruginosa PAO1,PAO1-ΔlasR.(5)The pathogenicity of P.aeruginosa and E.coli COP1512 to the host was studied by constructing a Caenorhabditis elegans infection model.It was found that the supernatant of COP1512 could enhance the lethality of PAO1,PAO1-ΔlasR to C.elegans.The above research results have revealed the inter-species interaction mechanism of P.aeruginosa and E.coli,the main pathogens of chronic respiratory infections,which is is expected to lay the theoretical and factual basis for the discovery of more complex multi-pathogen interaction mechanisms in the future and the clarification of the formation mechanism of multi-pathogen chronic respiratory infections.