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

Abstract

Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against Enterocytozoon bieneusi, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including E. bieneusi, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered Saccharomyces cerevisiae to be dependent on Encephalitozoon cuniculi MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracilselectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.

Long-Term Prophylactic Antibiotic Treatment: Effects on Survival, Immunocompetence and Reproduction Success ofParasemia plantaginis(Lepidoptera: Erebidae)

“…2011 ). MetAP2 is an essential enzyme in microsporidia and thus its inhibition by fumagillin kills microsporidian cells ( Upadhya et al. 2006 ).…”

Parasitic diarrheal disease: drug development and targets

“…Fumagillin along with its analogs act on the methionine aminopeptidase type 2(MetAP2) by non-competitive inhibition, i.e., by irreversibly blocking the active site in E. cuniculi and it seems to be the potent inhibitor (Molina et al, 2002 ). Analysis of EcMetAP2 demonstrated conservation of the key residues associated with the active site of this class of enzymes including Asp251, Asp262, His331, Glu364, and Glu459 (involved in coordination of metal binding); His231 (to which fumagillin covalently binds); and Phe219, Leu328, Ile338, His339, Asp378, Tyr444, Leu447 (involved in binding substrates into the active site; Upadhya et al, 2006 ). The aspartic proteases are a family of protease enzymes that use two highly conserved aspartic acid residues in the active site for catalytic cleavage of their peptide substrates (Pozio and Morales, 2005 ).…”

Molecular cloning and characterization of a gene encoding methionine aminopeptidase 2 of Schistosoma japonicum

“…MAP2 regulates protein synthesis by inhibiting phosphorylation of the a-subunit of eukaryotic initiation factor-2α and thus enhances the rate of global protein synthesis (Upadhya et al 2006). The functions of MAP2 are evolutionarily conserved and essential, as demonstrated by the lethal phenotype of knockouts in both bacteria and yeast (Lee et al 2004).…”