Research On Akt Pathway Activation And Autophagy Induced By Cryptosporidium Infection In HCT-8 Cell

Cryptosporidium parvum is an intracellular protozoan parasite that infects the mammal gastrointestinal epithelia cells and it is considered to be a major cause of diarrhea worldwide.Cryptosporidiosis outbreaks often caused by water contaminated with parasites via fecal-oral route.Unlike immunocompetent adults in whom cryptosporidiosis is usually self-limited,immunocompromised people,especially the patients afflicted with AIDS,the patients who need to take chemotherapy and immune-suppressing drugs in long term and malnourished children would be susceptible to chronic cryptosporidiosis which could be a life-threatening disease in this situation.Despite extensive researches and lots of medications test have been carried out,there is still no effective drug or available vaccine yet.So,in-depth study of the interaction between parasites and host cells,which is of great significance for elucidating the survive mechanism of C.parvum is still in need.EGFR signaling pathway and cell autophagy are important life activities of cells.Autophagy is a lysosome-dependent degradation pathway that is widely present in eukaryotic cells.On the one hand,autophagy is internalized and degraded to eliminate intracellular pathogens.On the other hand,intracellular pathogens can escape autophagy or utilize autophagy to proliferate.EGFR signaling pathway can regulate autophagy and other processes.However,there is no systematic study on the regulation of EGFR signaling pathway and autophagy in host cells by C.parvum.In this study,the effects of C.parvum on activation of host EGFR signaling pathway and autophagy and scavenging effects of autophagy on parasites were studied in order to determine whether C.parvum evades autophagy by activating EGFR signaling pathways.The main research contents and results are as follows:The role of EGFR-PI3K/Akt pathway activated by C.parvum HCT-8 cells treated with C.parvum were collected at different times and the phosphorylations of EGFR and Akt were detected by western blot.After the addition of EGFR or PI3 K inhibitors,western blot was used to examine the effect of inhibitors on the expression of EGFR and Akt induced by parasites.The amount of intracellular parasites was detected by RT-PCR after the addition of EGFR or Akt inhibitors.The results showed that C.parvum induced phosphorylation of EGFR and Akt in HCT-8 cells.EGFR inhibitor reduced the phosphorylation of EGFR and Akt in HCT-8 cells,and PI3 K inhibitor reduced Akt phosphorylation.EGFR inhibitor or Akt inhibitor decreased the number of intracellular parasites.The above results showed that C.parvum facilitated the proliferation of parasites through activation of the EGFR-Akt signaling pathway by PI3 K.Relationship between C.parvum and cell autophagy HCT-8 cells treated with C.parvum were harvested in different periods of time.The expressions of autophagy markers LC3 and Beclin1 were detected by western blot.After the addition of RNAi Beclin1,ATG7 and lysosome inhibitor respectively,the LC3 accumulations were measured.After adding autophagy inducer,autophagy inhibitor or lysosome inhibitor,RT-PCR method was used to detect the number of intracellular parasites.The results showed that the expressions of LC3 and Beclin1 were increased in HCT-8 cells induced by C.parvum,and the accumulation of LC3 was decreased after interference with Beclin1 or ATG7,while the accumulation of LC3 was increased with lysosome inhibitor.The autophagy inducer Rapamycin reduced the number of intracellular parasites and the autophagy inhibitor 3-MA and the lysosome inhibitor E64d+pep A increased the number of intracellular parasites.The above showed that C.parvum induced complete autophagy.Beclin1 and ATG7 participated in the formation of autophagy and autophagy played a certain role in the removal of parasite infection.Regulation of PI3K/Akt pathway in induced autophagy by C.parvum LC3 accumulation in HCT-8 cells induced by C.parvum was detect after addition of Akt inhibitor by indirect immunofluorescence assay.RT-PCR was used to detect the number of parasites under the conditions of Akt inhibitor and autophagy intervention.The results showed that Akt inhibitor increased the amount of LC3 accumulation.The number of intracellular parasites reduced with Akt inhibitor and autophagy inducer Rapamycin.The number of intracellular parasites increased with Akt inhibitor and autophagy inhibitor 3-MA or lysosome inhibitor E64 d and pep A.The above results showed that C.parvum was able to inhibit the autophagy by activating the PI3K/Akt pathway which was beneficial to the growth of parasites.In summary,this study found that C.parvum activates EGFR-PI3K/Akt signaling pathway and induces autophagy.On the one hand,parasites are eliminated through autophagy.On the other hand,parasites can inhibit the elimination of autophagy by activating the PI3K/Akt signaling pathway.These reveal a new mechanism for the interaction of C.parvum with host cells.