Functional And Mechanism Research Of Metacaspases In Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is a major zoonosis to human and animal,which almost all kinds of karyocyte of warm-blooded animal and human beings can be infected.The apoptosis-like death of T.gondii was observed both in vitro and vivo,but the associated factors and mechanism were not clear.Metacaspases,a kind of cysteine peptidase that exhibited a catalytic cysteine-histidine dyad,were found in plant,fungi and protozoa.Function of Metacapases were apoptosis,cell-cycle regulation and protein degradation.A comprehensive search in T.gondii database(ToxoDB)revealed three genes encoding Metacaspase-like protein,which named TgMCA1,TgMCA2 and TgMCA3 respectively in our study.Using bioinformatics analysis,TgMCAl had 49%amino acid similarity to Yeast Metacaspase(YCA1)and revealed histidine and cysteine catalytic dyad.However,the conserved histidine or histidine adjacent to cysteine was absent in MCA3 or MCA2.We investigated the role of MCA1 and MCA2.TgMCAl localized in the cytoplasma of the intracellular parasites,and MCA1 in extracellular parasites translocated into the nucleus and was concentrated in the nucleus of the parasites.We also found TgMCA1 was co-localized with IMC1 of the daughter tachyzoits during endodyogeny.For futher study,TgMCA1 knockout strain(?mca1)and overexpressed strain(mcal-OE)were constructed in our research.The proliferation of Amcal was almost the same as that of Aku80 in vitro,but the growth of mcal-OE was significantly slower which cytokinesis of mcal-OE was postponed indicated by IFA.We hypothesized that MCA1 was involved in cell cycle of T.gondii.The extracellular MCA1 knockout strain showed stronger vitality compared to Aku80.TUNEL assay showed the apoptotic ratio of Amcal in extracellular for 4 hours is 20%,which was significantly lower than 37%of Aku80,and the mcal-OE in extrcellular buffer for 4 hours showed 54%apoptotic tachyzoits.We confirmed that TgMCAl had a role of apoptosis in T.gondii.To verify the hypothesis that MCAs regulated cell cycle of tachyzoits,we constructed TgMCA2 knockout strain(?mca2)as well as TgMCAl and TgMCA2 double knockout strain(?mca1?mca2).TgMCA2 was well distributed in the cytoplasm of the parasites when observing the TgMCA2 endogenous tachyzoits by IFA,but we didn't find its localization in nucleus or IMC1 till now.The growth of the parasites was much more slower when lack of MCA 1 and MCA2 silmultaneously,however,the growth of Amca2 were normal as Aku80 and Amcal.Virulence assay showed there's no siginificantly difference in mortality time and rate among Amcal,Amca2 and?ku80,and the mice in above groups were all dead at 8?11days post infection.37%mice of?mca 1 ?mca2 group(9/24)were survival which confirmed that the parasites had low pathogenicity to mice when lack both of MCA1 and MCA2.Endodyogeny were abnormal in most tachyzoits of AmcalAmca2,which showed multinucleus in the cytoplast of the tachyzoits and IMC1 of them were not formed as usual.We also observed the egress double knockout strain which showed low motility and abnormal shape of parasites.Western blot was carried out using total protein of the parasites extracted by surface active agent-deoxycholate,we found IMC1 of ?mca1?mca2 was sensitive to deoxycholate compared to Aku80,?mca1 and Amca2,which demonstrated that MCA1 and MCA2 were associated with IMC maturation.In this study,we found MCA1 and MCA2 had more than one important role in development of T.gondii.MCA1 was part of the pathway in T.gondii apoptosis,and its localization was different in extracellular and intracellular tachyzoits;MCA1 and MCA2 cooperated in the proliferation of the parasites,which were critical to IMC 1 maturity and contributed to T.gondii endodyogeny.The growth of the parasites was not affected when lack one of them.