Investigations into microsporidian methionine aminopeptidase type 2: a therapeutic target for microsporidiosis

Abstract

Abstract. The Microsporidia have been reported to cause a wide range of clinical diseases particularly in patients that are immunosuppressed. They can infect virtually any organ system and cases of gastrointestinal infection, encephalitis, ocular infection, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles such as albendazole are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin, ovalicin and their analogues have been demonstrated to have antimicrosporidial activity in vitro and in animal models of microsporidiosis. Fumagillin has also been demonstrated to have efficacy in human infections due to E. bieneusi. Fumagillin is an irreversible inhibitor of methionine aminopeptidase type 2 (MetAP2). Homology cloning employing the polymerase chain reaction was used to identify the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae and E. bieneusi. The full-length MetAP2 coding sequence was obtained for all of the Encephalitozoonidae. Recombinant E. cuniculi MetAP2 was produced in baculovirus and purified using chromatographic techniques. The in vitro activity and effect of the inhibitors bestatin and TNP-470 on this recombinant microsporidian MetAP2 was characterized. An in silico model of E. cuniculi MetAP2 was developed based on crystallographic data on human MetAP2. These reagents provide new tools for the development of in vitro assay systems to screen candidate compounds for use as new therapeutic agents for the treatment of microsporidiosis.

System for Expression of Microsporidian Methionine Amino Peptidase Type 2 (MetAP2) in the Yeast Saccharomyces cerevisiae

“…Saccharomyces cerevisiae cells deficient in MetAP1 (⌬map1) are killed by ovalicin, but yeast cells deficient in MetAP2 (⌬map2) are not (17,29). Deletion of both MetAP1 and MetAP2 is lethal in yeast (17,34). These data confirm that fumagillin selectively targets MetAP2 and not MetAP1.…”

“…Independent colonies from both of the plates were inoculated into SC-Ura medium for growth. Cell lysates were subjected to immunoblot analysis to investigate whether EcMetAP2 protein was expressed in yeast, using mouse polyclonal antibodies raised against recombinant EcMetAP2 (34). A minimum of four colonies was examined for each transformation, and a representative figure is shown in Fig.…”

“…MetAP2 gained a lot of attention recently as an excellent therapeutic target not only in treatment of various parasite infections but also in anticancer therapy (26). Data suggest that the cellular target for fumagillin and its analogs in microsporidia is a microsporidian MetAP2 homologue and that this is an essential enzyme for these organisms (34).…”

“…We have reported the identification and characterization of the MetAP2 gene and its protein product from the humanpathogenic microsporidian E. cuniculi (34). In an attempt to design a simple and rapid approach to screen for novel E. cuniculi MetAP2 (EcMetAP2) inhibitors, we devised a complementary approach which is independent of recombinant protein or parasite culture and instead dependent on the expression of EcMetAP2 in the yeast S. cerevisiae.…”

Efficacy of Gamma Interferon and Specific Antibody for Treatment of Microsporidiosis Caused byEncephalitozoon cuniculiin SCID Mice

“…In such cases, conventional chemotherapy (albendazole and fumagillin) must remain the first choice for treatment. Recently, therapeutic development studies have focused on compounds that target microsporidian polyamines (polyamine analogues), methionine aminopeptidase 2 (fumagillin analogues), chitin (nikkomycins), and topoisomerases (fluoroquinolones) (1,4,27).…”