Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites

Philip M Frasse,Justin J Miller,Alexander J Polino,Ebrahim Soleimani,Jian-She Zhu,David L Jakeman,Joseph M Jez,Daniel E Goldberg,Audrey R Odom John

doi:10.1016/j.jbc.2021.101550

Abstract

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.

Highlights

Malaria remains an enormous global health burden for much of the world, resulting in over 200 million infections and 400,000 deaths every year, the majority of which are in children under the age of five[1]
Loss of HAD5 leads to growth arrest in asexually replicating parasites, marked by defects in egress and reinvasion
This growth defect can be rescued by media supplementation with D-mannose, indicating that disruption of mannose metabolism is the primary mechanism of death in HAD5KD parasites; a physiologically relevant concentration of 50 μM D-mannose is unable to significantly rescue growth, bolstering the case for this pathway as a therapeutic target

Summary

INTRODUCTION

Malaria remains an enormous global health burden for much of the world, resulting in over 200 million infections and 400,000 deaths every year, the majority of which are in children under the age of five[1]. GPI anchors are an essential component of all life stages of Plasmodium falciparum In intraerythrocytic parasites, these glycolipid anchors tether several essential proteins to the parasite plasma membrane prior to red blood cell egress and reinvasion[16]. When expression of HAD5 is reduced with an intermediate concentration of aTc (3 nM) that still permits modest asexual growth, knockdown parasites yielded a marked shift in half-maximal effective concentration (EC50) (Fig. 3D, 3E) These results further implicate HAD5 in the production of GPI anchors in P. falciparum. MSP1 must be targeted and anchored through GPIs and proteolytically processed in order for schizont-stage parasites to egress from the erythrocyte, and the MSP1 complex is critical for binding and reinvading new red blood cells[19, 64]. The combination of lowresolution of the HAD5 crystal structure and computational docking precludes more detailed analysis, the resulting model suggests that interactions between the hydroxyaminomethyl group of D9 and the variable active site loop likely account for HAD5 selectivity; additional studies are needed to further validate this model

DISCUSSION

EXPERIMENTAL PROCEDURES Parasite Strains and Culturing

Findings

FUNDING AND ADDITIONAL INFORMATION