A Novel FasL-like Lipoprotein And Lipopolysaccharide Of Leptospira Interrogans Synergisticalley Induce The Apoptosis Of Macrophages Through Fas/FasL-caspase-8/-3 Pathway

Background.Leptospira interrogans(L.interrogans)is the major pathogen of leptospirosis,a world-spreading zoonotic infectious disease.L.interrogans serogroup Icterohaemorrhagiae serovar Lai is most popular in China.The pathogen has been confirmed to induce apoptosis of macrophages during infection,but its molecular mechanism remains unknown.Methods.Mouse J774A.1 macrophages-and PMA-induced human THP-1 macrophages-phagocytosis tests and leptospiral phagosomes were detected by laser confocal microscopy and transmission electron microscopy.Apoptosis of phagocytosis-inhibited macrophages induced by L.interrogans strain Lai was confirmed by phagocytosis inhibition test and flow cytometry.Leptospiral outer membrane proteins or lipopolysaccharide(Lep-OMP or Lep-LPS)was extracted from L.interrogans strain Lai.Mouse/human-Fas-captured Lep-OMP was identified by NanoLC-MS/MS.Fas-rLep-OMP047 binding was determined by surface plasmon resonance(SPR)and isothermal titration calorimetry(ITC).Lep-OMP-,Lep-LPS-or rLep-OMP047-induced macrophage apoptosis was detected by flow cytometry.Recombinant Lep-OMP047-induced caspase-8/-3 activation,Lep-LPS-induced Fas/FasL expression and membrane translocation,TLR2/4-dependent JNK/p38MAPK/ERK phosphorylation and c-Jun/ATF2/CHOP nuclear translocation were detected by fluorometric assay,Western Blot assay,qRT-PCR,flow cytometry and confocal microscopy.Results.We observed that L interrogans strain Lai could cause apoptosis of phagocytosis-inhibited macrophages,implying that some leptospiral surface molecules may play a FasL-like role to induce macrophage apoptosis by Fas/FasL-caspase-8/-3 pathway.The mouse or human-Fas-based co-precipitation and NanoLC-MS/MS determined the product of L.interrogans LB047 gene(Lep-OMP047)as the unique mouse or human-Fas-captured leptospiral surface lipoprotein.The recombinant expressed Lep-OMP047(rLep-OMP047)binds strongly to mouse/human-Fas with the equilibrium association constant(KD)values of 5.20×10-6-2.84×10-9 M,measured by SPR and ITC.Flow cytometric examination showed that 5 ?g rLep-OMP047 and 1?g Lep-LPS caused 56.61%and 21.90%early-apoptosis for mouse J774A.1 macrophages,respectively,or 39.55%and 15.80%for PMA-induced human THP-1 macrophages,but the apoptosis was blocked by Fas-IgGs and caspase-8/-3 inhibitors.Moreover,the qRT-PCR and Western Blot assay confirmed that Lep-OMP047 expression was significantly up-regulated during infection of macrophages.Lep-LPS promoted the expression and translocation of Fas/FasL in macrophages.JNK/p38MAPK but not ERK signaling pathways and c-Jun/ATF2 but not CHOP transcription factors mediated Lep-LPS-induced Fas/FasL expression and translocation.However,TLR2 but not TLR4 mediated Lep-LPS-induced JNK/p38MAPK activation.Conclusions.A novel FasL-like surface lipoprotein and LPS of L.interrogans synergistically induce macrophage apoptosis through Fas/FasL-caspase-8/-3 pathway,which contributes to leptospiral survival in hosts and pathogenesis of leptospirosis.