Fachbereich Veterinärmedizin



    The role of Heat Shock Protein 90 HSP90 in the transformation of Theileria annulata infected cells and the parasite stage differentiation (2015)

    Mohammed, Sara Basher Taha (WE 13)
    Berlin: Mensch und Buch Verlag, 2015 — XI, 134 Seiten
    ISBN: 978-3-86387-632-6
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000100298
    Institut für Parasitologie und Tropenveterinärmedizin

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35, 22, 23
    14163 Berlin
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    Abstract / Zusammenfassung

    Theileria annulata is an obligate intracellular parasite that causes great health problems in cattle and domestic buffalo in tropical and subtropical areas. The parasite is transmitted transstadially by several species of Hyalomma ticks.
    T. annulata has a unique ability to transform and to induce a permanent proliferation of bovine leukocytes in a similar manner as tumor cells do. Among others, this transformation results in clonal expansion and metastasis of the infected cells in various organs of the affected animal. The mechanism (s) underlying the transformation process is still not entirely clear.
    However, in the last years, it has been found that a number of genes are involved in the regulation of cell proliferation and / or apoptosis such as NF-κB, PI-3k/Akt and Heat shock proteins (HSPs). Moreover, HSP90 has been shown to be involved in the host cell proliferation/apoptosis as well as in stage differentiation of parasites.
    Accordingly, this work was designed:
    i) To investigate the functional role of HSP90 in the maintenance of the cell viability and proliferation ii) To analyse the differential expression profile of HSP90 in T. annulata Ankara 288-infected cells under stress conditions iii) To investigate the role of HSP90 during the developmental stages of the parasite.
    In a number of studies, geldanamycin (GA) was used to inhibit the HSP90 function in cell proliferation and survival. In the present work, I also used this drug to dissect a possible impact of the HSP90 on the host-parasite interaction in Theileria-transformed bovine cells.
    As a first step, the effect of HSP90 inhibition by GA on the cell viability was examined at 37°C by Trypan blue test. The percentages of dead cells were determined and was less than 20% in the cells incubated with GA (0.5 μM or 1μM) for 1 day, while this percentage increased up to 66% after 2 days. The treatment with GA ≥ 5μM showed more than 60% of dead cells after 1 day and increased up to 100% after 2 days. The flow cytometric assays for apoptosis was carried out on one hand, to verify the previous result and on the other hand, to investigate the effect of GA (0.001μM-0.5μM) at 37°C and 41°C. The analysis revealed that GA induced significant apoptosis in T. annulata Ankara 288- infected cells in a dose- and time- dependent manner. The effect of GA depended also on the incubation temperature, where low concentrations of GA could significantly induce apoptosis at 41°C.
    These findings were confirmed by the immunofluorescence staining of host cell p53 as a great accumulation of p53 was observed in the host cell nucleus after treatment with GA.
    The cell proliferation analysis by flow cytometry showed a significant reduction in the cell proliferation after treatment with GA ≥ 0. 05 μM for 3 days, while after 6 days incubation at 37°C only GA ≥ 0.25 μM has a similar effect.
    The effect of HSP90 inhibition on the parasite differentiation in T. annulata Ankara 288- infected cells was evaluated by using two different methods. The first method was immunofluorescence staining using specific antibodies against TamS1 and TamR1, which can detect those cells in which schizonts differentiate to merozoites. The qRT-PCR was applied as another method to evaluate the expression level of TamR1 gene during the parasite differentiation. Both methods revealed that the number of the cells undergoing merogony increased in T. annulata Ankara 288-infected cells after GA treatment regardless of the incubation temperature.
    The regulation of bovine HSP90 genes (BHSP90-alpha and BHSP90-beta) and T. annulata HSP90 genes (TaHSP90-Chr1 and TaHSP90-Chr4) in T. annulata Ankara 288-infected cells was evaluated by qRT-PCR. The qRT-PCR analysis showed that the mRNA expression levels of BHSP90-alpha and BHSP90-beta were significantly up-regulated after treatment with GA in a dose- and time dependent manner. In agreement with a previous result, the expression levels of TaHSP90-Chr1 mRNA and TaHSP90-Chr4 mRNA were slightly up-regulated after GA treatment at 37°C. An interesting finding was the significant decrease in the expression level of TaHSM90-Chr1 and TaHSP90-Chr4 in the cells incubated with GA at 41°C. This variance raised an important requirement for tools to distinguish between T. annulata HSP90 and bovine HSP90.
    As mentioned above, reference sequences of two isoforms of T. annulata HSP90 were found and named TaHSP90-Chr1 and TaHSP90-Chr4. Characterization of TaHSP90-Chr1 and TaHSP90-Chr4 gene and its protein forms was carried out using molecular biological methods and bioinformatic analysis. Phylogenetic tree analysis indicated that TaHSP90-Chr1 and TaHSP90-Chr4 are more closely related to HSP90 from Theileria species and B .bovis than to HSP90 from human or bovine. Both TaHSP90-Chr1 and TaHSP90-Chr4 showed most of the characteristics reported for HSP90 e.g. the presence of ATP sequence, the presence of the functional HSP domain and the existence of a signal sequence located in the N-terminal sequence of the TaHSP90-Chr1 and TaHSP90-Chr4.
    Antigenic peptides were deduced from the sequence of TaHSP90-Chr1 and TaHSP90-Chr4 using the predication server Predicting Antigenic Peptides (http://imed.med.ucm.es /Tools/antigenic.pl). The selected peptide was synthesized and used to immunize two rabbits. However, the anti- T. annulata HSP90 antiserum failed to recognize T. annulata HSP90 (native protein).
    Regarding bovine HSP90, immunoblotting staining carried out using anti-human HSP 90α/β (HSP 90α/β (F-8): sc-13119, Santa Cruz) showed the ability of anti-human HSP 90α/β to detect bovine HSP90 in the cell lysates. Moreover, the immunofluorescence staining of T.annulata Ankara 288-infected cells showed the specificity of this antibody.
    Novobiocin was used as an alternative method to distinguish between the role of bovine HSP90 and T. annulata HSP90. Although, novobiocin was utilized as a specific inhibitor of bovine HSP90, the novobiocin treatment resulted in death of the parasites also. Whether this was a direct effect on the parasite or rather on the host cell is not clear.
    Nevertheless, the results of this study showed that the inactivation of HSP90 in T. annulata Ankara 288-infected cells leads to cell cycle arrest, apoptosis and differentiation of the schizonts to merozoites.