A Preliminary Study On The Inhibitory Effect Of Pseudomonas Aeruginosa Secreted Protein Pec1 On Macrophage Phagocytosis

Objective: This study aims to explore the effect of Pseudomonas aeruginosa secretory protein pec1 on phagocytosis of pulmonary macrophages and its possible mechanism,so as to clarify the molecular basis of Pseudomonas aeruginosa in the process of resisting host immune defense system,and provide theoretical basis for clinical treatment of Pseudomonas aeruginosa infection.Methods:The sequencing primers of PAO1 were designed according to NCBI library,The recombinant protein Pec1 was prepared by PCR amplification,plasmid construction,induced expression and protein purification.The effect of the recombinant protein Pec1 on the proliferation of MH-S cells was detected by CCK-8kit.Neutral red was used to measure the phagocytic function of MH-S cells,and the phagocytosis of heat-inactivated Pseudomonas aeruginosa standard strain PAO1 was observed by Fluorescence microscope.The phagocytic efficiency of MH-S to PAO1and PAO1 knockout strain?pec1 was calculated and compared by colony-counting method;the expression of TLR4 and the phosphorylation of PI3K and Akt in MH-S cells were detected by Western blot.In vivo experiments,rats were infected by intratracheal injection with standard strain PAO1 and?pec1.HLung inflammation was observed by HE staining;the expression of TLR4,phosphorylation of PI3K and Akt in macrophages of lung tissue of rats in each group were observed by immunofluorescence double staining and semi-quantitatively compared by Image J software;the colony culture of lung tissue in each group was counted,and the clearance level of bacteria in the lung of each group was calculated and compared.Results:1.The full-length pec1 gene was about 828 bp amplified by PCR.The recombinant plasmid p MD19-T-pec1 and prokaryotic expression vector p ET-30a-pec1 were successfully constructed,and the recombinant protein Pec1 was expressed and purified,with a molecular weight of 30.5 k Da.2.Pec1 inhibited the proliferation of MH-S cells in a time-dependent and dose-dependent manner within 72 h.3.When Pec1 stimulated MH-S cells at 40,80 and 120 ?g/m L,the results presented that MH-S cells could not only inhibit the phagocytosis of neutral red(P < 0.01),but also inhibit the phagocytosis of heat-inactivated Pseudomonas aeruginosa standard strain PAO1(P < 0.001).Meanwhile,compared to ?pec1 group,the phagocytosis of PAO1 by MH-S cells was significantly reduced(P < 0.001),and the bacterial clearance rate of PAO1 was also significantly decreased(MOI = 10,P < 0.001).4.Compared with the control group,the phosphorylation levels of PI3 K and Akt and the expression levels of TLR4 were significantly decreased after Pec1 stimulated MH-S cells(P < 0.05).PI3 K / Akt agonist could not only reverse the inhibitory effect of Pec1 on phagocytosis of neutral red by MH-S cells(P < 0.001),but also restore the function of decreased phagocytosis of PAO1 in MH-S cells(P < 0.001).TLR4 agonist could promote TLR4 expression,PI3 K and Akt phosphorylation in MH-S cells(P <0.05),and counteract the inhibition of Pec1 pretreatment on TLR4 expression and PI3 K and Akt phosphorylation(P < 0.01);moreover,TLR4 agonist increased the phagocytosis of MH-S cells to neutral red(P < 0.001)and PAO1(P < 0.001).5.The pneumonia model was constructed in rats infected with PA bacteria.The color of lung of PAO1 group was dark red and obviously congested and swollen,while that of ?pec1 group was light red and slightly swollen and hyperemia.The results of HE staining showed that,in PAO1 group,a large number of inflammatory cells were found in alveoli,and alveolar structure was damaged,while in ?pec1 group,there was less inflammatory cell exudation and more complete alveolar structure.Moreover,the semi-quantitative comparison of inflammatory area between the two groups indicated that the inflammation in ?pec1 group was mild(P < 0.05).Besides,double-immunofluorescence staining showed that,by compared with ?pec1 group,the total level of TLR4,PI3 K and Akt phosphorylation in PAO1 group were significantly decreased(P < 0.05);two weeks after infection,the colony count of PAO1 group was noticeably higher than that of ?pec1 group(P < 0.05),and the clearance rate of PAO1 was considerably lower(P < 0.01).Conclusion:1.The prokaryotic expression vector p ET-30a-pec1 was successfully constructed and the Pec1 protein was expressed and purified.2.Pec1 can inhibit the proliferation of MH-S cells.3.Pec1 can inhibit the phagocytosis and clearance of PA by pulmonary macrophages,and its mechanism may be related to the down regulation of TLR4 / PI3 K / Akt signaling pathway.