Study On The Function And Mechanism Of Action Of Leptospiral Proteasome

Leptospirosis is a global distribution zoontic diseases when flood outbreak,which is one of the major infectious diseases in China’s key prevention and control.Leptospira interrogans is the causative agent of leptospirosis,but its pathogenic mechanism remains poorly understood.Since eukaryotic and prokaryotic proteins have been reported to be denatured or damaged in adverse environments and AAA+(ATPases Associated with diverse cellular Activities)proteases/proteasome are responsible for degrading these harmful proteins.We presume that infection is also an adverse environment to cause denaturation or damage of proteins in L.interrogans and leptospiral proteasome are benefit for survival of the spirochete in hosts.According to the results of bioinformatics analysis,leptospiral proteasome contains HslUV(ClpYQ),ClpAP and ClpXP.In the present study,we taken the HslUV AAA+ proteases as the key object of our study and the results showed that the recombinant or native HslU and HslV protein compounds from L.interrogans strain Lai with 1-8:1 proportion(r/nHslU1-8HslV1)only hydrolyzed the chymotrypsin-like substrates with 43.3 μmol·L-1 Km and 51.7 s-1 Kcat values,but this hydrolytic ability was blocked by the threonine protease inhibitors alone.The confocal microscopic and fluorospectrophotometric examination demonstrated that the denatured or damaged protein aggresomes in the spirochete during infection of J774A.1 macrophages were significantly increased,but the hslU and hslV gene-deleted mutant(△hslUV)produced more aggregative proteins.The iTRAQ and LC-MS/MS detection as well as KEGG pathway and GO analysis revealed the major harmful proteins involving in ribosomal structure,flagellar assembly,two-component signaling system and transmembrane transport.Compared to the wild-type strain,the mutant not only provided more dead leptospires,less leptospiral CFUs and lower growth ability during infection of the cells,but also displayed lower LD50,attenuated histopathological injury and decreased leptospiral loading in the lungs,liver,kidneys,peripheral blood and urine in hamsters.Taken together,our findings confirmed that HslU and HslV proteins of L.interrogans compose a chymotrypsin-like threonine protease-type proteasome HslUV and this protease complex contributes to the survival of the spirochete in hosts and the transmission of leptospirosis.