The Legionella Pneumophila Effector Lem8 Regulates Motility Of Host Cells Via Cleavage Of Phldb2

Background and Objective: Legionella pneumophila(L.pneumophila)is a kind of Gram-negative intracellular parasitic bacteria.Its natural host in nature is some aquatic single-celled protists.Opportunistic infection can occur when humans are exposed to infectant water,leading to a severe pneumonia called Legionella pneumonia.After entering the host cell,the bacteria transports over 330 effectors through the Type IV secretion system(T4SS),which targets important life activities in the cell to evade the host immune response and is essential for survival and bacterial replication in the cell.Leg G1,a T4 SS effector of Legionella,was found to promote microtubule polymerization by acting as a guanine nucleotide exchange factor(GEF)towards Ran GTPase,thereby enhancing host cell migration.Surprisingly,L.pneumophila has been shown to inhibit the motility of infected cells through T4 SS,suggesting the existence of other effector proteins that can regulate cell motility.However,the mechanism underlying this process is unknown.The aim of this study is to uncover the regulatory effects of a functionally unknown Legionella effector Lem8(Lpg1290)on the motility of host cells,identify its eukaryotic target(s)and clarify its biochemical activity,so as to make a new supplement to the current knowledge of virulence of Legionella.Materials and Methods: HEK293 T and Hela cells were cultured in DMEM supplemented with 10% fetal bovine serum.Bone marrow cells were isolated from A/J mice aged 6 to 10 weeks and induced to differentiate into macrophages(BMDMs)using L929 cell culture medium.E.coli strain DH5α was used for plasmid construction,and strain BL21(DE3)or XL1-Blue was used for recombinant protein production and purification.All E.coli strains were grown in LB agar plates or LB broth at 37°C.All Legionella strains were derived from Philadelphia strain Lp02 and Dot A-mutant strain Lp03,which were cultured in N-(2-acetyl amino)-2-amino ethanesulfonic acid buffered yeast extract medium(AYE)or charcoal buffered yeast extract plate(CYE).Lem8 encoding gene lpg1290 was knocked out from L.pneumophila genome by homologous recombination,and the gene expression in Legionella was implemented by transformed with p ZL507 plasmid.The protein structure and enzyme activity of Lem8 were analyzed by bioinformatics.The toxicity of Lem8 and its mutants to eukaryotic cells was tested by yeast toxicity assay.The binding of Lem8 and its mutants to 14-3-3-ζ protein was detected by yeast two-hybridization,coimmunoprecipitation and GST pulldown.The expressions of Lem8,14-3-3ζ,Phldb2,ICDH,Tubulin,PGK,GFP,HA,Flag,GST and His were detected by Western blot.The distribution of Lem8 and Phldb2 in cells was detected by immunofluorescence staining.The Lem8 and 14-3-zeζ proteins were purified from E.coli by affinity chromatography.The cysteine protease activity of Lem8 was detected by in vitro biochemical reaction,and the self-cleavage sites of Lem8 were identified by mass spectrometry.The effect of Legionella infection or Lem8 transfection on cell migration was detected by cell scratch test.Results: Bioinformatics analysis showed that t Lem8 harbors a putative Cys280-His391-Asp412 catalytic triad present in a variety of cysteine proteases.Mutation of any of the above three sites could lead to the loss of Lem8 toxicity to yeast.Lem8 binds eukaryotic protein 14-3-3-ζ in a nonphosphorylated form both in vitro and in vivo.Intriguingly,Lem8 undergoes auto-cleavage in the presence of 14-3-3ζ,which cuts off a 52-amino acid length peptide at its carbon terminal.Lem8ΔC52 still has a strong binding capacity to 14-3-3ζ protein.Further study revealed that Lem8 binds to 14-3-3ζ protein through a Coiled coil motif at its amino terminal.In the presence of 14-3-3ζ,Lem8 exhibits protease activity towards Phldb2,which is involved in eukaryotic cell motility,and thus inhibits migration of host cells.Moreover,the self-cleaved form Lem8ΔC52 also exhibits protease activity in binding to the 14-3-3ζ and cleaves Phldb2.Knockout of Lem8 significantly decreased the inhibitory effects of Legionella on the motility of infected cells.Conclusion: Lem8 interacts with the host regulatory protein 14-3-3ζ,which activates its protease activity.Furthermore,Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ.We identified the Pleckstrin homologylike domain-containing protein Phldb2 involved in cytoskeleton organization as a target of Lem8 and demonstrated that Lem8 plays a role in the inhibition of host cell migration by attacking Phldb2.