Effects Of ATG5 On Proliferation Of Toxoplasma Gondii In Vitro And Host Mitophagy

Toxoplasma gondii is an obligate intracellular parasitic protozoan which can infect many mammals and cause severe damage in pregnant and immunocompromised individuals.The double-membrane structure formed by autophagy can directly encapsulate T.gondii and be degraded after fusion with lysosomes.At the same time,various autophagy-related(ATG)proteins have also been shown to be involved in immune responses through noncanonical autophagy pathways.This project aims to investigate the effect of ATG5 on the proliferation and viability of T.gondii in vitro,and the regulation of ATG5 on mitophagy in host cells which infected by T.gondii.First,the role of ATG5 in dendritic cells(DCs)recognition of T.gondii was first verified.The mouse ATG5 gene was cloned and induced prokaryotic expression.Mouse bone marrow-derived DCs were isolated and used to infect T.gondii.The results showed that the proportion of CD11c~+CD83~+type of DCs in the ATG5 group was significantly higher than that of T.gondii group(P<0.05).It showed that the recombinant protein inhibited the maturation of DCs induced by T.gondii.Next,ATG5 knockdown and overexpressed Vero cell lines were constructed.Vero cells were infected with lentiviral vectors,and screened with puromycin to obtain stable cell lines.The knockdown efficiency of sh ATG5-2 was 79%,and overexpressed efficiency was12.62 times.Stable cell lines were then used to evaluate the effect of ATG5 on the efficiency of tachyzoite invasion.The growth of tachyzoites in each cell lines was observed and recorded by Wright-Giemsa staining.The percentage of infected cells in the total cells was counted under the light microscope(400×).It was initially shown that advancement of T.gondii invasion,proliferation and egress appeared in the overexpressed cells,and its delay appeared in the knockdown cells.The level of ATG5 was positively correlated with the proportion of T.gondii infected cells.The effect of host ATG5 on mitochondrial membrane potential of T.gondii was further explored.The tachyzoites were collected and labeled with JC-1 dye to detect the mitochondrial membrane potential.That is,the degree of membrane potential depolarization was the highest one in overexpression group,followed by control group,and the lowest in knockdown group.It suggested that host ATG5 could damage the mitochondrial function of T.gondii.Finally,the effects of ATG5 and T.gondii infection on host mitophagy were evaluated.Three type of cells were sampled after infection,and the transcription levels of the PINK1were detected by RT-q PCR.It found that the level of PINK1 first decreased,then increased,and then decreased,alternately,in the order of the invasion,proliferation,and egress of T.gondii.ATG5 overexpression group was not significantly different from the other two groups in the pre-infection period(0-36 h),but significantly higher than them after 48 h.It suggesting that the growth of T.gondii led to changes in the mitophagy of host cells,and this change was also regulated by host ATG5 in the late stage of infection.In summary,the host ATG5 promoted the rapid invasion of T.gondii,but caused the damage of its mitochondria,inhibiting recognition of DCs.The intracellular growth of T.gondii led to changes in the level of host mitochondrial autophagy,and this change was also regulated by ATG5 in the late stage of infection.