Secretion And Distribution Of Rhoptry Protein 16 During Toxoplasma Gondii Invasion Into Host Cells

Part1 BackgroundToxoplasma gondii is an obligate intracellular protozoan, a globally distributed Apicomplexan, more than one third of the world’s population are latently infected by the parasites. The infection is mainly acquired by ingestion of uncooked or raw meat(mainly pork and lamb)/water containing tissue cysts or water/food contaminated with oocysts excreted in the faeces of infected cats. As an opportunistic parasite, most human infections of T.gondii are asymptomatic; however, in immunocompromised individuals, such as those suffering from AIDS chemotherapy,or organ transplantation, the infection can cause severe pathology like disseminated congenital infections and neurological complications and even death. Infection acquired during pregnancy may cause severe damage to the fetus, like miscarry, premature-birth, stillbirth. T. gondii infects nearly all animals and most birds,cause loss to the livestock industry.Internalisation of apicomplexan zoites into host cells is composed of a very conserved scheme as follows:an initial contract between zoite and the host cell triggers a recognition event which starts the entire process sequentially; host-cell entry is immediately followed by progressive internalization at the site of apical contact-which proceeds from anterior to posterior, eventually closing the vacuole behind the parasites. The entry usually takes about 5-10s. The host cell plus the vacuole, once inside the cell, the parasite no Longer moves.Host-cell invasion by apicomplexan parasites involves the successive secretion of three different secretory organelles, namely micronemes, rhoptries and dense granules. Micronemes proteins are apparently used for host-cell recognition, binding and possibly motility; rhoptry proteins are involved in parasitophorous vacuole (PV) formation; and dense granule proteins are responsible for remodelling the vacuole into a metabolically active compartment. Many kinds of rhoptry proteins are secreted into the host cell cytosol or in the forming PVduring parasite’s entry to co-opt the host cell for growth and survival, such as ROP2, ROP4, ROP5, ROP7 and ROP18.Generally, rhoptry proteins are classified into ROP1 and ROP2 families. As a representative member in ROP1 family, ROP1 promotes the process of Toxoplasma gondii infection in early stage. At all stages of parasite growth ROP1 was found within the parasite’s body but rarely within the peduncle of rhoptries, even in rhoptry’s other parts appeared to be empty. After host cell invasion, ROP1 was immediately found associated with the parasitophorous vacuole membrane(PVM). Hours after invasion, the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was notably decreased.During acute T.gondii infection, tachyzoites progress through the lytic cycle including host cell infection, replication within the host cell and egress into the extracellular environment for a new host cell invasion. Among those proteins, the polymorphic protein, ROP16 is released from rhoptries and injected into the host cell quickly with the help of its own nuclear localization signal, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways. ROP16 directly induced phosphorylation of STAT3/STAT6, and interfered host cell signal transduction-consequently disturbed the growth, differentiation and apoptosis of host cells. ROP16 is highly polymorphic between type Ⅰ (or Ⅲ) and Ⅱ parasites. After Toxoplasma gondii infection, host macrophages, dendritic cells, and neutrophils released IL-12 and other cytokines, induced secondary immune response or direct clearance of parasites. It has been reported that can quickly activate the STAT3 signaling, and then inhibit the release of pro-inflammatory factors such as IL-12 in the host cell infected by type Ⅰ strains, but this phenomenon is not found in the infecton of type Ⅱ strains.Functions of ROP16 are different in different strains, then what about the transcriptional level and protein expression level of ROP16 in the parasites during invasion? This research detected the different transcriptional level of ROP16 in different strains with Real-time PCR and the translational level by western blotting with the polyclonal antibody of Tg-ROP16 and Tg-Tublulin; indirect immunofluorescence assay (IFA) was conducted to detect the secretion of ROP16 and the localization in the host cell during invasion by different T.gondii strains These studies layed a foundation for deeply detecting the function of ROP16 from different strains on invasion and the pathogenic mechanism of Toxoplasm gondii invasion.Part 2 ObjectiveHost-cell invasion by Apicomplexan parasites is a subsequence of attachment to host cell membrane, membrane surrounding the vacuole (parasitophorus vacuole membrane, PVM) is originally formed from the host plasma membrane as the parasite invades, parasites invade into host cell cytosol, then absorb nutrition from host cell to complete its own growth, manipulation, egressed from lysed host cell and then invade next cell. Apical complex of Toxoplasma gondii plays a great importance upon the process which can secret proteins namely microneme protein, rhoptry protein and granule protein. Among those proteins, ROPs plays a key role in infection.This research is aimed to explore whether the expression of ROP16 in different strains during its infection are in the same level, and handling the distribution of ROP16 in host cell after Toxoplasma gondii infection.Part3 Methods1. Preparation of antibody against Toxoplasma gondii rhoptry protein 16As Tg-ROP16 gene contains no intron, the ROP16 coding sequence was amplified by polymerase chain reaction (PCR) with the genomic DNA of Tg RH strain, then subcloned into the plasmid of pET-32a(+). The expression of recombinant TgROP16 was induced by IPTG in the E.coli containing pET-32a(+)-TgROP16 plasmid. Recombinant protein TgROP16 was purified and used to immune the rabbit for twice.2. Preparation of antibody against Toxoplasma gondii TubulinThe Tubulin coding sequence was amplified by PCR with the cDNA of Tg RH strain, then subcloned into the plasmid of pET-32a(+).The expression of recombinant Tubulin was induced by IPTG in the E.coli containing pET-32a(+)-TgTubulin plasmid. Recombinant protein TgROP16 was purified and used to immune the rabbit for twice.3. Detection of ROP16 transcriptional level in different types of T. gondii with Real-time PCR.Tachyzoites of RH or Pru strains were collected for total RNA extraction, and the cDNA was preparaed by reverse transcription with the total RNAs. Real-time PCR was performed with ROP16 cDNA and the Toxoplasma gondii GAPDH was used as endogenous reference, copy number-cycle threshold curve for the two genes was built and gene ROP16 from cDNA, copy number-cycle threshold curve for the two genes was built.4. Detection of ROP16 expression level in different types of T.gondii tachyzoites by western blotting with the equivalent total protein.Total proteins of the tachyzoites of the two types of T.gondii were collected. After lysis of the tachyzoites, the concentration of the total protein was measured with BCA assay. SDS-PAGE was performed to separate the proteins and the proteins on the gel was tranferred to a PVDF membrane and prepared for western blotting. The first antibody of polyclonal antibody against Tg-ROP16 was used at a 1:200 dilution and the polyclonal antibody against Tg-tubulin was used at a 1:1000 dilution, and added to the the corresponding blot respectively, rocking gently at 4℃ for overnight. The blots were washed with PBST for three times. The second antibody of goat anti-rabbit IgG (HRP)was used at a 1:1000 dilution, and added to the blot, incubated for 2 h at RT. After three times of wash with PBST, the blots were used for chemiluminescence, to detect the target bands.5. Examination of the secretion and distribution of T.gondii ROP16 from different types during infection by indirect immunofluorescence assay (IFA)Under sterile conditions, coverslips were placed into 6-well plate, HFFs were seeded into three wells, and grown to a monolayer of 100 confluency in 37℃,5% CO2 incubator. A well of cell without infection was used as negative control. Another two wells were infected with RH or Pru tachyzoites respectively at a multiplicity of infection(MOI) of 10 parasites for 4h. The cells were fixed with 4% formaldehyde for 30min and permeabilised with Triton X-100 for 10min at room temperature(RT) and then blocked with 1%BSA PBS-Tween for 1h at RT.The cells were incubated with the polyclonal antibody against Toxoplasma gondii ROP16 at a 1:200 dilution for overnight at 4℃ at slow rocker. After three times of washing with PBST, the cells were incubated by the secondary antibody(green) of goat anti-rabbit (FITC) IgG at 1:100 dilution for 1h at RT. After threes times of washing, DAPI was used to stain the cell nuclei at a concentration of 10nM. The coverslips were rinsed with ddH2O after thee times of washing with PBST, and mounted. The localization of ROP16 in the parasite or the host cell was observed and photographed under a fluorescence microscope.Part4 Results1. The recombinant protein of Tg-ROP16 were successfully expressed and purified, and specific rabbit-derived polyclonal antibody was obtained at a titer of 1:256002. Real-time PCR confirmed that the relative transcription of ROP16 in Pru strain(7.786±0.206) was 7 times higher than in RH strain(1.000±0.110);3. Western blotting confirmed that translation level of ROP16 was higher in RH strain than Pru in strain;4. The IF A result showed that ROP16 was localized on the apical complex of the unrecruited tachyzoite of Toxoplasma gondii but not the recruited tachyzoite in the host cell. ROP16 was probably secreted out of the parasite during infection.Part5 ConclusionThis research successfully prepared and purified rabbit-derived polyclonal antibody against Tg-ROP16 and Tg-tubulin with a reasonable sensitivity and specificity. Real-time PCR confirmed that the relative transcription of ROP16 in Pru strain was 7 times more than in RH strain(p<0.05); Much more TgROP16 was detected in RH strain than Pru strain by western blotting. Before T. gondii infection, TgROP16 was localized on the apical complex of T. gondii; while during infection, TgROP16 was secreted out of the parasite.