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 uracil-selectable 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.

FIG. 1.

Immunoblot analysis of the expression of EcMetAP2 protein in yeast. Fifty micrograms of protein from yeast cell lysate was loaded per lane, and the immunoblot was probed with anti- rEcMetAP2 antibody. Lane 1, lysate from cells containing pCM190 vector alone; lane 2, lysate from cells containing pCM190:EcMetAP2.

FIG. 2.

Heterologous expression of EcMetAP2 in yeast can be modulated by the addition of doxycycline to the medium. Tenfold serial dilutions of the wild type (S228C), isogenic Δmap1 strains containing pCM190 and pRS425Tet vectors, and the corresponding test strains of Δmap1 map2 background containing pCM190:EcMetAP2 and pRS425Tet:HuMetAP2 were spotted onto YPD medium in the absence (A) or in the presence (B) of 10 μg/ml of doxycycline (Dox).

FIG. 3.

Yeast MAP2 deletion strategy and confirmatory PCR. (A) Schematic representation of the yeast MAP2 deletion strategy and the position of the diagnostic primers. The diagram is not drawn to scale. (B) Diagnostic PCR was performed with P1, P2, and P3 primers, using genomic DNA as the template (lanes 2 to 4). Lane 1, DNA ladder; lane 2, wild-type S228C; lane 3, cells transformed with pCM190:EcMetAP2; lane 4, genomic DNA from Δmap1 map2 cells expressing a functional copy of EcMetAP2 protein.

FIG. 4.

Immunoblots demonstrating the expression of human MetAP2 from the TetO-CYC1 hybrid promoter in the vector pRS425Tet:HuMetAP2. Whole-cell lysates from Δmap1 cells which were transformed with either pRS425Tet:HuMetAP2 (lane 1) or pRS425Tet (lane 2) are shown. Fifty micrograms of total protein was loaded in each lane, and the immunoblot was probed with mouse anti-human MetAP2 antibody. The specific signal corresponding to the HuMetAP2 protein is shown by an arrow.

FIG. 5.

Expression of mutant EcMetAP2 in yeast. Yeast cell lysates from Δmap1 cells containing pCM190:EcMetAP2 (lane 1), pRS425Tet (lane 2), and pRS425Tet:mutEcMetAP2 (lane 3) are shown. Immunoblots were probed with mouse antibody against rEcMetAP2.

FIG. 6.

Validation of drug screening against E. cuniculi and human MetAP2s, using yeast as the surrogate system. Tenfold serial dilutions of the wild type and isogenic yeast strains containing vector alone or vectors containing either E. cuniculi or human MetAP2 were spotted onto YPD medium containing fumagillin (1 and 5 nM), ovalicin (0.5 and 1 nM), or TNP-470 (250 nM), the known inhibitors of MetAP2. Photographs were taken after the plates were incubated at 30°C for 4 to 5 days.