Effects Of Cryptosporidium Parvum Infection On Autophagy In HCT-8 Cells

Cryptosporidium parvum is a zoonotic parasitic protozoa,which mainly infect intestinal epithelial cells in host,destruct microvilli,cause intestinal dysfunction and diarrhea.The major transmission of Cryptosporidium parvum is waterborne transmission,human and animal can get Cryptosporidiosis by drinking water contained with Cryptosporidium parvum oocysts.People with lower immune function or immunodeficiency can be easily infected,such as the elderly,children and HIV patients.Cryptosporidium parvum can cause persistent diarrhea in HIV patients,easily lead to dehydration,shock,and even death.At present,there is no effective way for Cryptosporidiosis prevention.The reason is that the interaction between Cryptosporidium parvum and host cells is not clear.Studies have shown that autophagy can help cells to eliminate invading pathogens,and maintain cell health.Toxoplasma gondii can induce autophagy in the host cells,it can make use of the energy generated by autophagy to proliferate,and enhance the persistent infection of Toxoplasma gondii.Cryptosporidium and Toxoplasma belong to intracellular parasitic protozoa,but how’s the influence on autophagy when Cryptosporidium parvum invade host cells,and what’s the relationship between Cryptosporidium parvum infection and autophagy is unclear.The aim of this study was to investigate the effects on autophagy in host cells infected with Cryptosporidium parvum,and to explore the relationship between autophagy and Cryptosporidium parvum infection.Detection of autophagy in HCT-8 cells induced by Cryptosporidium parvum.HCT-8 cells were selected as the infection model of Cryptosporidium parvum,and investigating the effects on autophagy in HCT-8 cells infected with Cryptosporidium parvum by Western blot,indirect immunofluorescence,EGFP-LC3 transfection experiment and transmission electron microscope.The experiments as follows:1.Western blot: We extracted the total protein of HCT-8 cells from different culture time,and carried out Western blot test to detect the changes of autophagy marker protein LC3,Beclin-1.The results showed that LC3 I and LC3 II increased gradually after co-culture with Cryptosporidium parvum sporozoites and HCT-8 cells,indicating that there was a significant phenomenon of autophagy in cells;Beclin-1 accumulated the highest at 8h,indicating that the autophagy intensity was the strongest at 8h.2.Indirect immunofluorescence: After 8h of co-culture with Cryptosporidium parvum sporozoites and HCT-8 cells,the cells were washed and fixed for indirect immunofluorescence test,and the changes of LC3 protein were observed under confocal laser scanning microscopy.The results showed that the LC3 fluorescent spots in the cells were punctate or clustered,indicating that there was autophagy in cells.3.Transfection and detection of EGFP-LC3: HCT-8 cells transfected with EGFP-LC3 plasmid were co-cultured with Cryptosporidium parvum sporozoites,then the cells collected from different culture time were fixed,stained with DAPI,and the dynamic changes of LC3 protein were observed under confocal laser scanning microscopy.The results showed that the number of EGFP-LC3 spots increased gradually with the time increasing by co-culture with HCT-8 cells and Cryptosporidium parvum sporozoites,indicating that the intensity of autophagy increased gradually.4.Ransmission electron microscope: After 8h of co-culture,the cells were collected and washed,and the ultrathin sections were made.Searching and observing the microscopic structure of Cryptosporidium parvum sporozoites and autophagic vacuoles under transmission electron microscope.The results showed that the Cryptosporidium parvum sporozoites could invade the HCT-8 cells to form a parasitic vacuole,and it’s the first time to observe the autophagic vacuoles aroud the parasitic vacuole,indicating that Cryptosporidium parvum can induce autophagy in HCT-8 cells by invasion.Detection of pathway proteins related with autophagy in HCT-8 cells induced by Cryptosporidium parvum.After 8h of co-culture with Cryptosporidium parvum sporozoites and HCT-8 cells,the cells were collected,washed,and extracted total protein to conduct Western blot test,then detecting the changes of PI3 K,AKT,MTOR,AMPK and ULK1.The results were as follows: compared with the control group,PI3 K,AKT and m TOR were down-regulation in the experimental group,when there were no significant changes in AMPK and ULK1,indicating that Cryptosporidium parvum can induce autophagy in HCT-8 cells by the PI3K-AKT-m TOR pathway.Effects of Ra and 3-Ma treatment on the infection of Cryptosporidium parvum in HCT-8 cells.HCT-8 cells were treated with autophagy inducer rapamycin(Ra)and autophagy inhibitor 3-Ma,then making co-culture with Cryptosporidium parvum sporozoites and HCT-8 cell,establishing the fluorescence quantitative standard curve of Cryptosporidium parvum,and using fluorescence quantitative PCR to detect infection status of Cryptosporidium parvum in HCT-8 cells.The results were as follows: The infection of Cryptosporidium parvum were decreased when HCT-8 cells were treated with autophagy inducer Ra;however,the infection of Cryptosporidium parvum were enhanced significantly when HCT-8 cells were treated with autophagy inhibitor 3-Ma.In summary,Cryptosporidium parvum can induce autophagy in HCT-8 cells,and PI3K-AKT-m TOR pathway may be needed in the process;the infection of Cryptosporidium parvum for cells can be inhibited when cells were treated with autophagy inducer Ra,while the cells treated with 3-Ma had an enhanced effect on the infection of Cryptosporidium parvum.