Bacterial Toxin-antitoxin Svstems ChpK/ChpI And MazF/MazE Play A Role In The Virulence Of Pathogenic Leptospiral Species

Background:The genus of Leptospira is divided into two groups:pathogenic and non-pathogenic leptospires. Non-pathogenic leptospires live in surface waters and soils and never induce any pathogenicity neither in humans nor in animals. Till nowaday several genospecies of pathogenic Leptospira species have been identified such as L. interrogans, L. borgpetersenii, L. kirschneri and L. weilii. 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 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.Leptospira interrogans serovar Icterohaemorrhagiae strain Lai is a gram negative obligate aerobe spirochete, of the pathogenic Leptospira species. Leptospirosis is a worldwide zoonosis caused by the spirochaetales of the genus Leptospira, especially the pathogenic serovars. It's an infectious disease, which has not been widely recognized although has emerged to become a major public health problem in a number of the developing countries. Currently, existing data reveal that little progress has been made in the domain of leptospirosis therapeutics. The recent discovery of the toxin-antitoxin modules in the pathogenic Leptospira species has called our attention since it has been reported that the bacterial toxin of the toxin-antitoxin system could be used as therapeutic tools for human disease.Objective:In this work, we investigated the chpIK/mazEF toxin-antitoxin systems of L. interrogans strain Lai and determined the function of toxic proteins ChpK/MazF in the toxin-antitoxin modules of Leptospira species and the cytotoxicity of ChpK/MazF proteins to host cells and therefore determined the implication of these toxins in the virulence of the pathogenic Leptospira species.Methods:In order to achieve our goals, by using the genomic DNA of pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as template, several PCRs were performed to amplify chpK; chpI; chpIK; mazE; mazF and mazEF genes. After that, prokaryotic expression systems of chpK; chpl; chpIK; mazE; mazF and mazEF were constructed. Expression of the target recombinant proteins rChpK; rChpI; rMazE and rMazF was detected by SDS-PAGE and expressed rChpK;.rChpI; rMazE and rMazF were extracted by Ni-NTA affinity chromatography. The activities of rChpK/rChpI and rMazE/rMazF to lyse the DNAs or RNAs from L. interrogans strain Lai and THP-1cells were then determined. Variation of transcription and expression of chpK/chpI and mazF/mazE genes of L. interrogans strain Lai before and after infection of THP-1cells was detected by real-time fluorescent quantitative RT-PCR and Western blot assay. The eukaryotic expression system of the chpK; chpl; chpIK; mazE; mazF and mazEF genes was then constructed for transfection of host cells and CCK-8agent was used to detect the effect of leptospiral rChpK/rChpI and rMazF/rMazE proteins action on host cells.Results:Our results here demonstrate that the leptospiral rMazF displayed RNase activity while rChpK displayed neither RNase activity nor DNA lysis. During infection of THP-1cells by L. interrogans strain Lai, the transcription and expression of chpK/chpI and mazF/mazE genes were up-regulated while rChpK and rMazF proteins were secreted by leptospires. The mass mortality was observed in transfected human embryonic kidney cells (HEK293cells) containing chpK and mazF genes. The recombinant toxic proteins rMazF and rChpK were detected in the THP-1cytosol by Laser confocal Microscope analysis after infection of the host cells by the pathogenic Leptospira interrogans strain Lai.Conclusion:Chpl/ChpK and MazE/MazF toxin-antitoxin systems play an important role during infection of host cells by pathogenic Leptospira interrogans strain Lai:toxic proteins rChpK and rMazF are secreted after infection of host cells and exert obvious cytotoxicity effect on the host cells. Toxic protein rMazF displayed RNase activity while rChpK displayed neither RNase activity nor DNA lysisWe conclude that Chpl/ChpK and MazE/MazF toxin-antitoxin systems play an important role in the virulence of pathogenic Leptospira species and rChpK and rMazF toxic proteins must be important virulence factors in pathogenic leptospira species. The toxicity mechanism of these toxins needs to be further studied.