[Using of antibodies against Microsporia Hsp70 family proteins for analysis of secretome of intracellular parasites].

Microsporidia is a large group of fungi-related unicellular parasites with obligate intracellular lifestyle. Unlike other protozoan intracellular parasites (Kinetoplastida and Apicomplexa), most microsporidian species develop in direct contact with the host cell cytoplasm. This fact, acquisition of unique transporters to exploit host metabolic system (alongside the strong minimization of own machinery) and predicted repertoire of microsporidia secretome altogether suggest an active role of parasite proteins in the control of infected cell. Lack of information about secretome of microsporidia intracellular stages is largely due to the methodological difficulties of working with the obligate intracellular parasites. An important problem of such study is the contamination of preparations of host cell cytoplasm by inner (nonsecreted) parasite proteins. Even the homogenization of infected tissue in mild conditions and removal of parasite cells by low-speed centrifugation may result in their partial disruption. We expressed the fragments of three Hsp70 family chaperones from the microsporidium Paranosema (Antonospora) locustae in bacteria Escherichia coli. Immunoblotting with proteins of microsporidia intracellular stages and infected host tissue (locust fat bodies) demonstrated that antibodies against recombinant polypeptides may be used to monitor the integrity of parasite cells during homogenization of infected host tissue and subsequent removal of parasites by centrifugation.