Viruses Induced Metacaspase-Dependent Programmed Cell Death And Mitochondrial Function Impairment In Marine Emiliania Huxleyi

The autocatalytic PCD of phytoplankton is usually inducted by external nutrition,environmental stresses as well as virus infection.Metacaspases are evolutionarily distant homologs of caspases that are found outside the metazoan and are known to play key roles in PCD.The unicellular marine alga Emiliania huxleyi is a globally distributed eukaryotic phytoplankton with important ecological functions.E.huxleyi is surrounded by a calcified coccosphere,consisting of loosely intertwined calcite(CaCO3)platelets called coccoliths.The biological formation and physical deposition of CaCO3 is an important component of the oceanic carbon cycle.E.huxleyi forms massive annual spring blooms in the marine ecosystem that have been shown to be routinely terminated by lytic,giant,double-stranded,DNA-containing(dsDNA)coccolithoviruses.Lytic viral infection of E.huxleyi not only activates the PCD biochemical machinery,but also controls the algal community dynamics.They have played key roles in the regulation of ocean carbon cycle,sulfur cycle,and climate changing.Therefore,E.huxleyi-EhV has become the model system for studying the interactions between host-virus in the marine environment.However,very little is known about the molecular function and mechanisms mediating phytoplankton host-virus interactions and the associated regulation of cell fate.Here,the distribution,structure and functional characteristics of phytoplankton metacaspases were analyzed by using bioinformatics methods.Then,E.huxleyi BOF92 and its specific virus EhV-99B1 were taken as the research object to analyze the damage of mitochondrial structure and function induced by virus infection during programmed cell death process.Finally,we identified the interaction between metacaspase and EhVe-mir8 induced by EhV using the luciferase assay system,indicating that if microRNA was involved in the regulation of EhV-induced PCD process.The main results were showed as follows:(1)A total of 29 phytoplankton metacaspases were found in 11 phytoplankton species,which are grouped into three main types namely type Ⅰ,type Ⅱ and type Ⅲ based on their multiple alignment of amino acid sequences.Despite the lower amino acid sequence identity,they display high structural conservation in histidine-and cysteine-containing catalytic domain.The three-dimensional structures of E.huxleyi(EhMC)were very close to that of yeast metacaspase,suggesting that they might have similar functions.The results of the protein-protein interaction analysis revealed that metacaspases directly or indirectly interact with multiple cellular proteins,playing a critical role in cell apoptosis,environmental stress and energy metabolism.In addition,the phosphorylation modification of metacaspase may play a regulatory role in the PCD signal transduction of phytoplankton.(2)Cell samples were collected at different times after virus infection(0 h,24 h,45 h,and 60 h).The ultrastructure of the cells by TEM showed an enhanced damage along the extension of infection time,such as mitochondrial swelling,mitochondrial cristae disappearance and vacuoles forming.After 60 hours of infection,apoptotic bodies-like were formed and mitochondrial membranes were ruptured,mitochondrial ridges were severely damaged and vacuolated.Couple of mitochondrial functions were impaired in the late stage of viral infection.Mitochondrial membrane potential decreased,while the levels of ATP and cytochrome C gene expression increased significantly,indicating that mitochondrial mediation may be involved in the process of PCD induced by EhV.(3)The expression level of 9 EhMCs genes was up-regulated during the course of virus infection.Except for EhMC5,the expressions of other of 7 EhMCs were up-regulated significantly at the late stage of infection(P<0.01).Western blot analysis revealed the constitutive expression of 30-kDa and 42-kDa metacaspase proteins in control E.huxleyi cells after 45 h infection.EhV infection triggered a sharp increase in the intensity of the 42-kDa protein and these protein sizes correspond with the predicted molecular weights of several putatively annotated metacaspase proteins(EhMC7:27.3,EhMC8:41.2 and EhMC9:29.4 KDa)in the E.huxleyi 1516 genome.In conclusion,our results suggest that metacaspases play different roles in E.huxleyi,in which some metacaspases may possess caspase activity and are responsible for PCD execution and some vice versa.(4)Endogenous non-coding microRNA induced by EhV99B1 infection identified in our laboratory previous studies——The binding-sites of EhVe-miR8 as well as EhVe-miR9 and 9 metacaspases of host were precisely predicted by using miRanda software.The results showed the binding-site polymorphism between these miRNAs and their targets where miRNAs could bind target genes in 3’UTR,CDS or 5’UTR,subsequently.The wild type and mutant dual-luciferase recombinant plasmid including psiCHECK2-EhMC2-3’UTR were constructed base on psiCHECK2 dual-luciferase reporter system.These recombinant plasmids and miRNAs were co-transfected into 293T cell.The results showed that the relative luciferase activity of plasmid containing the 3’UTR of EhMC2 was significantly decreased by miR8,suggesting that EhMC2 probably was the target gene of miR8.From these result,it was concluded that the distribution and functional characteristics of phytoplankton metacaspase were investigated in this study.The experimental model used for the study was the E.huxleyi infected by specific lysis virus and the important role of metacaspase in the PCD process as well as the relationship with mitochondrial structure and functional damage.It was also initially explored that microRNA may be involved in the regulation of metacaspase expression during viral-induced PCD.The results showed that they played a significance role in the formation,development and termination of blooms in the ocean.