The Biochemical And Biological Functions Of Legionella Pneumophila Effector Protein MavQ

Legionella pneumophila is a gram-negative pathogen that is widely distributed worldwide.After infecting humans,it induces Legionnaires' disease characterized by fever and respiratory symptoms.Since it was first reported and discovered in 1976,there have been reports of Legionella epidemics around the world.The pathogen widely exists in aquatic environment and artificial water systems under natural conditions.In recent years,with the widespread use of air-conditioning and other artificial water systems,the incidence of Legionnaires' disease has increased year by year,and it has become one of the major threats to human health in modern society.Legionella is an intracellular parasite and its growth in the cell strictly depends on its special Dot/Icm type IV(T4SS)secretion system.When it is swallowed by the host cell,it will form a phagocytosis containing Legionella in the cell,then the Legionella-containing vacuole(LCV)recruits the vesicles transported between the endoplasmic reticulum and the Golgi apparatus to the surface of the LCV to modify the LCV to form a structure similar to the endoplasmic reticulum,in which the bacteria complete the process of proliferation and replication and finally the cells are lysed.The surface of LCV is rich in a large amount of phosphatidylinositol-4-phosphate(PtdIns4P),and many of the effector proteins secreted by Legionella contain PtdIns4 P binding domains,which can be anchored to LCV by binding to the mediator to exert its effect.(1)In this study,it is found through bioinformatics analysis that the Legionella effector MavQ(more regions allowing vacuolar colocalization N)has no homology to proteins with known functions in terms of overall amino acid composition,but there are catalytic motifs Dx Hxx N and IDH at its N-terminus which is similar to eukaryote or prokaryote phosphatidylinositol phosphokinases.And ectopic expression of MavQ in yeast cells can significantly inhibit the growth of yeast,so we chose the yeast system to verify the effect of this potential catalytic domain on MavQ function.After mutating the key amino acids 149 histidine(H),152 aspartyl(N)and 160 aspartic acid(D)to alanine(A),all the mutants can be significantly relieved inhibition of yeast cell growth.It shows that this potential catalytic domain plays an important role in the function of MavQ.Further,we purified the recombinant protein of MavQ through the prokaryotic expression system.Under the conditions of kinase reaction,MavQ is incubated with different types of phosphatidylinositols [PtdIns?PtdIns3P?PtdIns4P?PtdIns5P?PtdIns(3,4)P2?PtdIns(3,5)P2?PtdIns(4,5)P2] and the screening found that in the presence of phosphatidylinositol(PtdIns),MavQ shows significant kinase activity,indicating that phosphatidylinositol is the substrate of MavQ.Furthermore,through thin layer chromatography(TLC)experiments,we find that the monophosphate product produced by MavQ acting on PtdIns can be degraded by the phosphatase MTM,which can hydrolyze phosphatidylinositol-3-phosphate(PtdIns3P).And after adding LepB-N that is a kinase with PtdIns3 P as the substrate to the reaction system of PtdIns and MavQ,the reaction produces an obvious phosphatidylinositol bisphosphate product,which fully proves that MavQ acts on PtdIns to generate PtdIns3 P,and MavQ has phosphatidylinositol 3-phosphate(PI3K)activity.(2)We overexpress of MavQ in HeLa cells and find that it can selectively destroy the intermediate and trans Golgi without affecting the cis Golgi.In addition,MavQ is not sensitive to the broad-spectrum PtdIns3 P inhibitor wortmannin in eukaryotic cells,and can also exert its PI3 K activity in the cell.After wortmannin inhibits the production of endogenous PtdIns3 P,the co-localization of the early endosome marker EEA1 and the PtdIns3 P fluorescent probe 2×FYVE can still be detected in the cells transfected with MavQ.This indicates that MavQ can produce PtdIns3 P on early endosomes.(3)Through the commonly used three-parent hybridization method of Legionella knockout,we successfully construct the mavQ-deficient Legionella strain.In the process of drawing the intracellular growth curve,it is found that compared with the wild-type Legionella,the growth of the mavQ-deficient strain does not change significantly in the cell,indicating that MavQ is not necessary for the growth of Legionella.Furthermore,we prepar mouse-derived anti-MavQ antibodies and find MavQ could be localized to the LCV surface when detected by immunofluorescence.The only proven source of PtdIns4 P on the surface of LCV is that the Legionella effector protein LepB uses PtdIns3 P to generate PtdIns(3,4)P2,further,under the synergistic effect of SidF,the phosphate at position 3 is hydrolyzed to produce PtdIns4 P.The source of LepB's substrate,PtdIns3 P is still unclear.Based on the enzyme activity characteristics of MavQ and the characteristics of localization on the surface of LCV,we use the SidC staining reporter system to identify whether MavQ would provide a substrate for the action of LepB.SidC is localized on the surface of LCV by binding to PtdIns4 P.When mavQ is missing,the amount of SidC on the surface of LCV is significantly reduced.Its effect is the same as that of missing lepB or sidF alone of missing mavQ and lepB.And this reduction can be restored by replenishing mavQ,while the mutants have no effect.Therefore,it is shown that MavQ can act on PtdIns to generate PtdIns3 P to provide a substrate for Legionella effector LepB and then participate in the biosynthesis of PtdIns4 P on the LCV surface.(4)Through GST pull-down and surface plasmon resonance technology,we confirm that MavQ can interact with the Legionella phosphatidylinositol phosphatase effector SidP.However,SidP does not affect the kinase activity of MavQ in vitro and in vivo.And the lack of sidP does not affect the biosynthesis of PtdIns4 P on the LCV surface.This study clarifies for the first time the biochemical and biological functions of the unknown function of Legionella effector MavQ and proves the upstream mechanism of the biosynthesis of PtdIns4 P on the LCV surface.All these provide support for a better understanding of the molecular mechanism of Legionella pneumophila,and further provide a theoretical basis for the treatment and prevention of Legionella pneumophila infection.