Identification of hsp90 in Cryptococcus neoformans and examination of the effect of Mycograb® on hsp90 gene expression in cells exposed to various stress factors
Abstract number: 1733_1448
Al-Akeel R., Curry A., Hoyes E., Nooney L., Matthews R., Burnie J.
Cryptococcus neoformans is the causative agent of cryptococcosis, a chronic and life-threatening infection common in AIDS patients. Mycograb® is a recombinant antibody derived against the LKVIRKNIV epitope of candidal heat shock protein 90. Hsp90 is an important molecular chaperone in all eukaryote cells. Its interaction with groups of proteins involved in cellular regulatory processes helps to maintain their active conformation. Little is known about the function of Hsp90 in C. neoformans in protection against cryptococcosis. A monoclonal antibody specific for Hsp90 was used to detect Hsp90 in C. neoformans cells grown in sub-inhibitory concentrations of amphotericin B and to identify the effects of amphotericin B on Hsp90 expression. We have examined the expression of the hsp90 gene in C. neoformans and the ability of Mycograb® to inhibit its expression under various stress factors, including heat shock and treatment with various antifungal agents. Cells were exposed to sub-inhibitory concentrations of anti-fungal agents alone and plus three different concentrations of Mycograb®. The expression of Hsp90 in presence of these antifungal agents and the effect of Mycograb® on its expression was examined. Total RNA was isolated from stressed cells and analysed by RT-PCR. Immuno-electron microscopy showed that Hsp90 was mainly located on the outer membrane surface of the cells. The electrophoretic outer membrane protein patterns indicated 14 major protein bands within a molecular weight range varying from 19 to 188 kDa. All isolates displayed similar protein patterns irrespective of the test conditions although band intensity was clearer when cells were grown in the presence of amphotericin B than those were not. Western blotting with anti-Hsp90 monoclonal antibody revealed that the 90kDa was present in the outer membrane fraction of disrupted cells. Results of RT-PCR showed that the treatment of C. neoformans with sub-inhibitory concentrations of amphotericin B and fluconazole induced Hsp90 mRNA expression, while treatment with caspofungin did not. Hsp90 gene expression was inhibited by different concentrations of Mycograb®. The presence of Hsp90 in the capsule of C. neoformans supports the idea of using this protein as a target for new antifungal or antibody therapy. Inhibition of Hsp90 mRNA gives the potential to investigate the use of Mycograb® in the treatment of cryptococcosis.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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