| Background and Objective:Leptospirosis caused by infection of pathogenic Leptospira is a globally-prevailing zoonotic infectious disease.L.interrogans is the most predominant pathogenic Leptospira genospecies.Many animals serve as natural hosts of pathogenic Leptospira,but have no illness or present mild disease.However,the infected hosts can persistently excrete leptospires from urine to contaminate natural body of water to form infection source(infected water).Human individuals have illness after infected with pathogenic Leptospira by contact with the infected water.Pathogenic Leptospira enters human bloodstream to form toxic septicaemia and then diffuse into many internal organs such as lungs,liver and kidneys through small vessels that cause even death.Until now,the mechanism of pathogenic Leptospira through vascular endothelial cells and renal tubular epithelial cells remains unknown.In the present study,we try to determine the molecular mechanism of L.interrogans passing through small vessels for diffusion in vivo and renal tubules for excretion from urine by utilization of cellular endocytic recycling system(ERS)and vesicular transport system(VTS).Methods:Laser confocal microscopy and electron microscopy were used to detect the endocytic vesicles of L.interrogans strain Lai endocytosed by human or mouse vascular endothelial cells,renal tubular epithelial cells and fibroblasts.The mechanism of leptospiral endocytosis was detrmined by using endocytic inhibitors and siRNA-based target gene knockdown.Laser confocal microscopy was also employed to detect that the leptospiral vesicles recruited Rab5/Rab11 proteins in ERS to form recycling endosome(RE),Sec/Exo proteins in ERS to form exocyst complex(EC)and VAMP2/SYN1 proteins in SNARE complex of VTS.Transwell assay and reconstruction of three dimensional images were perfomed to determine the ability of the spirochete through the monolayers of different cells.The transcytosis mechanism of L.interrogans was determined by anthrax toxin(EF/LF+PA)or botulismotoxin(BoNT/D-LC and BoNT/C-LC)inhibition tests and siRNA-based Rabll-,Sec15-,Sec3-,VAMP2-or SYN1-encoding gene knockdown.The intracellular propagation,exocytosis and viability of L.interrogans were detected by laser confocal microscopy and counting method.MTT assay and flow cytometry were employed to detect the viability of L.interrogans-infected cells.Results:L.interrogans strain Lai invaded into both the human or mouse vascular endothelial cells and renal tubular epithelial cells by CAV-ITGB1-PI3K-MF pathway-dependent endocytosis but the human or mouse fibroblasts endocytose the spirochete through CAV-ITGB1-FAK-MF pathway.The vesicles of L.interrogans could recruit Rab5/Rabll and Sec15/Sec3 in turn to form RE and EC,respectively,and then recruit VAMP2 and SYN1 for exocytosis.However,the vesicles did not colocalize with LAMP1 of lysosomes in the whole process of leptospiral transcytosis.L.interrogans strain Lai traversed the monolayers of human or mouse vascular endothelial cells,renal tubular epithelial cells and fibroblasts by transcytosis through FAK-MF/MT pathway for exocytosis.The L.interrogans-infected human or mouse vascular endothelial cells,renal tubular epithelial cells and fibroblasts as well as the leptospires maintained the original viability.However,L.interrogans strain Lai was founded to propagate in mouse fibroblasts alone.Conclusion:L.interrogans invaded into human or mouse vascular endothelial cells and renal tubular epithelial cells or fibroblasts through CAV/ITGB1/PI3K/MF or CAV/ITGB1/FAK/MF pathway to form endocytic vesicles.The leptospiral vesicles can utilize ERS and VTS to form lysosome-non-fused RE and EC and then fuse with cell membrane through FAK-MF/MT pathway via SNARE complex for exocytosis,which causes the spirochete passing through small vessels and renal tubules.In the prosess of transcytosis,the viability of either L.interrogans or the infected cells is not affected,but the spirochete only propagates in mouse fibroblasts. |
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