| Cryptosporidium parvum (C. parvum) is a species of apicomplexan cryptosporidium that caninfect the intestinal epithelial cells. The cryptosporidiosis caused by drinking infected water can bea deadly threat for the immunocompromised patients, such as those with AIDS or organ transplantpatients. Currently, there is no significant medicine or vaccine for cryptosporidiosis and it is veryimportant to identify the mechanisms of C. parvum recognition and response in order to preventand cure this disease. Studies have shown that Toll-like receptors (TLRs) of host cells play a keyrole in the identification of parasite and the interactions between parasites and host cells. Whileinfected by parasites, TLRs and NF-κB will be activated and the production of cytokines will beup-regulated. However, C. parvum is a kind of parasites which mainy infect the intestinalepithelial cells, the recognition mechanism of TLRs in human intestinal epithelial cells or mousemacrophages for C. parvum infection were unknown. The aim of this study is to research theidentification mechanisms of host cells’TLRs after the infection of C. parvum.Up-regulated TLRs in RAW264.7cells and Caco-2cells after stimulated by C. parvum.C. parvum sporozoites were used to stimulate Caco-2cells and RAW264.7cells in our research,the activation of TLRs were detected by RT-qPCR2h later, activation of Nuelear faetor kappa B(NF-κB) were detected by Western blot2h later and expresssion of TNF-α were measured byELISA12h later. The results showed up-regulation of TLR2and TLR4expression, NF-κBactivation and TNF-α expression in Caco-2cells, however, not only TLR2and TLR4but TLR11and TLR12expression were up-regulated in RAW264.7cells.Up-regulated TLRs in RAW264.7cells after stimulated by secretory antigen from C.parvum. To ascertain whether the molecules from secretory antigens of C.parvum can causeTLR11and TLR12activation, cracked antigens and secretory antigens of C. parvum sporozoiteswere used for stimulation of RAW264.7cells, TLRs and NF-κB activation and TNF-α expression were detected after the stimulation. The results showed TLR2, TLR4, TLR11and TLR12up-regulation in the cracked antigens stimulation and only TLR11up-regulation in the secretoryantigens stimulation, up-regulation of NF-κB activation and TNF-α expression were found in bothstimulation. These results indicated that there are molecules in secretory antigens which can leadto the activation of TLR11, and molecules which can lead to the activation of TLR12exist incracked antigens Secretory antigens from Toxoplasma gondii can activate TLR11and TLR12because of profilin. By searching the C. parvum genome, profilin protein was found in C. parvum,what is more, it is a secreted antigen molecules and has high homology (63%) to profilin fromToxoplasma gondii.Up-regulated TLRs in RAW264.7cells after stimulated by CpProfilin protein. Forfurther analyses of the roles of TLRs in responses to CpProfilin, a prokaryotic expression systemfor CpProfilin expression was built. After the stimulation of purified CpProfilin, significantup-regulation of TLR11were detected while no significant changes in other TLRs were found,NF-κB activation and TNF-α expression were also significant. Additionally, CpProfilin inducesIL-12production in TLR11-transfected HEK293cells.In conclution, the results demonstrated that TLR2and TLR4mediate the recognition andresponse of Caco-2cells to C. parvum, TLR2, TLR4, TLR11and TLR12mediate the recognitionand response of RAW264.7cells to C. parvum, what is more, CpProfilin lead to the up-regulationof TLR11only. |
