Study On The Function Of Pseudomonas Aeruginosa ZmuT In Regulating The Activities Of SodM And FumC1 Mediated By Metal Ions

As a human pathogen of conditional infection,Pseudomonas aeruginosa can cause devastating acute and chronic infections in individuals with impaired immune system.In P.aeruginosa,metal ions have been shown to play an important role in virulence,antibiotic resistance,infection host and colonization,in addition to maintaining normal cell structure and physiological metabolic function.Members of the ZIP（ZRT/IRT-likeprotein）family are zinc ion transporters widely found in organisms,which can transfer Zn²⁺into the cytoplasm from extracellular space or organelles,and later proved that it can also transport other metal ions such as iron,manganese,copper and cobalt.In the previous study,through NCBI-BLAST homology search and comparison,it was found that PA4467（named Zmu T）in P.aeruginosa belongs to the homologous protein Zup T of the ZIP family,and its amino acid sequence is as high as 36.59% and 49.59% with Ec Zup T of E.coli and Bb Zup T of Bordetella bronchiseptica.Moreover,using Phyre2 to analyze the transmembrane helix,it was found that the Zmu T protein contained nine transmembrane helices（TM1 to TM9）and its N-terminal and C-terminal were located in the periplasmic space and cytoplasm,respectively,which was significantly different from the topological structure of Ec Zup T and Bb Zup T.These results suggest that Zmu T may be a unique divalent metal ion transporter.Therefore,this study mainly focused on P.aeruginosa Zmu T transporter.The metal ion transport function of Zmu T protein was analyzed and identified by metal ion regulation of PA4467-PA4471 operon,growth phenotype analysis,test on tolerance to metal ions,calprotectin（CP）resistance test,hydrogen peroxide sensitivity test and larvae of Galleria mellonella infection test.Furthermore,the effect of Zmu T transporter on Sod M and Fum C1 activity was detected by bacterial two-hybrid experiment,protein metal content analysis,growth phenotype analysis and superoxide dismutase activity determination.The results are as follows:（1）The expression response of PA4467-PA4471 operon to divalent metal ions was analyzed by enzyme activity assay of transcriptional fusion of PA4467-PA4471 operon promoter and lac Z.The results showed that the expression of PA4467-PA4471 operon was inhibited by Fe²⁺and Co²⁺.（2）The mutant and genetic complementary strain were constructed by knocking out zmu T based on the mutantΔznu BCΔcnt LΔhmt A（named PAO1Δ3Zn）which lacked three known zinc uptake systems.Through the growth phenotype analysis of mutants and genetic complementary strains under zinc-rich and zinc-limited conditions,the results showed that Zmu T could mediate zinc uptake by P.aeruginosa.（3）Heterologous expression of zum T can significantly enhance the sensitivity of E.coli to divalent metal ions such as Zn²⁺,Mn²⁺,Mg²⁺,Ni²⁺,Cu²⁺,Co²⁺and Ca²⁺.These result suggest that Zum T transporters may be involved in the uptake of these metal ions by P.aeruginosa.（4）Physiological phenotypic experiments such as calprotectin（CP）resistance test,hydrogen peroxide sensitivity test and larvae of Galleria mellonella infection test showed that zmu T mutation could significantly reduce the resistance of P.aeruginosa to calprotectin（CP）and H₂O₂ oxidative stress and its virulence to larvae of Galleria mellonella.（5）The bacterial two-hybrid experiment showed that there was a pairwise interaction among Zmu T,Sod M and Fum C1 proteins in the PA4467-PA4471 operon.The gene truncation assay further showed that the C-terminal domain of Zmu T protein interacted with Sod M and Fum C1 protein,and the C-terminal domain of Sod M interacted with the N-terminal domain of Fum C1.（6）The metal content of Fum C1 protein was analyzed by ICP-MS,and the results showed that calcium was bound to Fum C1 protein.（7）Wild strain PAO1 or PAO1Δsod B was used as the starting strain to further knock out zmu T or sod M,and different mutants were constructed to detect the growth phenotype and superoxide dismutase activity of these mutants.The results showed that the deletion mutation of zmu T caused the functional defect of Sod M,while the addition of Mn²⁺could restore its function.At the same time,the growth level and superoxide dismutase activity of PAO1Δzmu TΔsod B strain were weaker than that of PAO1Δsod B strain.These results suggest that Zmu T affects the function of Sod M at the protein level by affecting Mn²⁺uptake.（8）The functions of Fum A,Fum C1 and Fum C2 fumarase in P.aeruginosa was studied by using the growth phenotype in eutrophic medium TSB.the results showed that the three fumarase were active and complementary in the process of growth,and at least one fumarase could maintain the normal growth of bacteria.The infection experiment of larvae of Galleria mellonella also showed that the virulence of fumarase to P.aeruginosa was necessary,and Fum A and Fum C1 played an important role in this process.（9）The growth phenotype test when different metal ions were added to the metal ion deficient VBMM medium showed that the function of Fum C1 depended on Ca²⁺and the activation of Ca²⁺to Fum C1 was sensitive to H₂O₂,which further confirmed that Fum C1 was a calcitonin.（10）Wild strain PAO1 or PAO1Δfum AΔfum C2 was used as the starting strain to further knock out zmu T or fum C1.The growth phenotypic analysis showed that Zmu T affected the function of Fum C1 at the protein level by affecting the uptake of Ca²⁺,suggesting that the acquisition of Ca cofactors in Fum C1 depends on Zmu T.Based on the above results,we propose a molecular mechanism model of functional correlation between Zmu T and Sod M and Fum C1: When P.aeruginosa infects the host,the host conducts immune defense by iron restriction and production of ROS.Iron restriction and ROS impaired the activity of fumarase Fum A containing 4Fe-4S,and caused Pseudomonas aeruginosa to induce iron starvation and oxidative stress response,thus activating the expression of PA4467-PA4471 operon and producing proteins such as Zmu T,Sod M and Fum C1.Zmu T transports extracellular bivalent metal ions into the cell,and transmits Mn2+ and Ca2+ to Sod M and Fum C1 through protein interaction to activate their functions to replace Sod B and Fum A,which are dysfunctional due to iron deficiency,respectively.