Responses Of Leptospira Interrogans To Host Innate Immunity:Modification,Increasing Quantity And Activity Of LPS

Background:Leptospirosis caused by pathogenic species of the genus Leptospira is an important zoonotic disease that occurs worldwide and is most commonly found in tropical or subtropical countries. Leptospirosis is also prevalent in China. The symptoms of human leptospirosis vary from mild to rapidly fatal. Clinical manifestations of the disease range from mild "flu-like" symptoms, moderate dengue-like symptoms such as high fever and severe headache as well as muscle pain to very severe symptoms such as pulmonary hemorrhage, jaundice and meningitis which frequently lead to respiratory failure and renal failure with high mortality rates. However, the pathogenetic mechanism is still unkown. But there is a clue that the clinical syndrome is similar to that of LPS-intoxication. There is a set of genes responsible for LPS synthesis and modification of Lipid A in leptospiral genome. What's more, there is report that polysaccharides of leptospiral LPS were changed after infection to rats by leptospires and LPS was detected in leptospiral uveitis in humans, which indicated that LPS is an active and virulent factor to cause leptospirosis.Content:Expression of LPS synthesis and lipid A modification genes, increase of quantities and activities of LPS, modification of lipid A, the effect of environmental factors on expression of lipid A synthesis and modification genes as well as the primary mechanism of regulation of lipid A synthesis and modification genes were detected in this research to determined the effect of Leptospiral LPS in the process of infection by leptospires.Methods:To better understand the interaction of pathogenic Leptospira and innate immunity, we employed microarray and real time RT-PCR to analyze the responses of L. interrogans to macrophage-derived cells. LAL assay was used to detect the activities of leptospiral LPS. KDO assay, Purpald assay, SDS-APGE-silver staining technique and HPLC-MS were performed to detect the quantity of LPS. MS was employed to detect the structure of different lipid A's. Real time RT-PCR was used to detect the effect of environmental factors on expression of lipid A synthesis and modification genes. Antibody block and knock-out techniques were used to determine the regulation of the up/down-regulated genes. Antibodyblock and ELISA assay were performed to detected the TLR recognition of lipid A's and the secretion of inflammatory cytokines.Results:13and11of34genes responsible for synthesis of leptospiral LPS and modification of lipid A were up/down-regulated after infecting macrophages J774A.1and THP-1respectively, of these genes, products of lpxB and lpxC are rate limiting enzymes, moreover, LpxDi, LpxD2and HtrB are responsible for addition of acetylated fatty acid chains to precursor lipid A. After infection to different macrophages, LAL activities of leptospiral LPS increased1.97and2.09times respectively than that of leptospires before infection, while the quantities of LPS of the post-infection leptospires were only1.02and1.06times more than that of pre-infection leptospires. MS assay shown that the lipid A of pre-infection leptospires(LepLipidA-EMJH, m/z=1921) contained one lauroleic acid, one myristoleic acid, two lauric acid and palmic acid chains, while, after infection, the leptospiral lipid A(LepLipidA-Infection, m/z=1949) changed in the structure of two lauric acid, teradecadienoic acid and palmic acid chains. Both lipid A's can introduce secretion of TNF-α IL-1β and IL-8by THP-1, but quantity of theses inflammatory cytokines introduced by LepLipidA-Infection were1.95,1.95and1.69times of those by LepLipidA-EMJH respectively. Comparing to LepLipidA-EMJH only recognized by TLR2of THP-1, LepLipidA-Infection was recognized by both TLR2and TLR4. Both lipid A can introduce secretion of similar quantities of TNF-α,IL-1β and IL-6by RAW264.7. Both lipid A's were recognized by TLR2and TLR4of this macrophage. Up/down-regulation of the genes including lpxC, lpxD1,lpxD2and htrB, as well as increase of quantity of LPS and modification of lipid A were mainly result from the low pH(6.0). Products of LA2222and LA3996regulated the expression of LpxC和LpxD1respectively, and product of LA2541regulated the expression of LpxD2和HtrB.Conclusion:Major genes of synthesis and modification of leptospiral lipid A were up/down-regulated after infecting macrophages. Up/down-regulation of the above genes lead to increase of LPS quantity and modification of lipid A, moreover, the the above-mentioned changes of LPS raised the activity of leptospiral LPS. Up/down-regulation of the genes including lpxC,lpxD1,lpxD2and htrB, as well as increase of quantity of LPS and modification of lipid A were mainly result from the low pH. As members of leptospiral two-component system, products of LA2222, LA2541and LA3996can sense low pH. Products of LA2222and LA2541regulated the expression of LpxC和LpxD1respectively, and products of LA3996regulated the expression of LpxD2and HtrB. There were differences of recognition of lipid A by Toll-like receptors and secretion of inflammatory cytokines induced by lipid A between human and mouse macrophages.