A paternal lactate dehydrogenase critically enhances male gametogenesis and malaria transmission

Abstract

Malaria blood stage parasite development relies on glycolysis to generate ATP, which requires pyruvate to lactate conversion by an essential lactate dehydrogenase enzyme (LDH1). Conversely, parasites developing in the mosquito employ mitochondrial chemiosmosis for ATP production. The source of ATP during transition from vertebrate to insect is less clear; gametes form in the mosquito midgut lumen within minutes of gametocyte ingestion, and while female gametes possess a mitochondrion, this organelle is absent from male gametes (microgametes). Here, we investigate a second LDH enzyme (LDH2) found exclusively in male gametocytes and microgametes. Knockout of Plasmodium berghei LDH2 expression reduces the number and size of exflagellation centres and radically diminishes oocyst development in Anopheles stephensi mosquitoes. Our data indicate that LDH2 supplements LDH1 activity to facilitate the cytokinesis step of male gametogenesis, while LDH1 alone is sufficient for motility of free-swimming microgametes. Our results point to a key role for glycolytic ATP production in microgamete formation and function and identify LDH activity as a potential malaria transmission-blocking drug target.

Keywords: Gametogenesis; Glycolysis; Lactate dehydrogenase; Plasmodium; Transmission-blocking drugs.

Fig. 1

Plasmodium encodes a second lactate dehydrogenase (LDH). (A) Part multiple sequence alignment of LDH and malate dehydrogenase (MDH) from Plasmodium, Toxoplasma and human, spanning the substrate binding loops (red). PfLDH1, P. falciparum LDH1 (PF3D7_1324900); PvLDH1, P. vivax LDH1 (PVP01_1229700); PkLDH1, P. knowlesi LDH1 (PKNH_1203900); PbLDH1, P. berghei LDH1 (PBANKA_1340100); TgLDH1, T. gondii LDH1 (TGME49_232350); TgLDH2, T. gondii LDH2 (TGME49_291040); PfLDH2, P. falciparum LDH2 (PF3D7_1325200); PvLDH2, P. vivax LDH2 (PVP01_1229400); PkLDH2, P. knowlesi LDH2 (PKNH_1203600); PbLDH2, P. berghei LDH2 (PBANKA_1340400); PfMDH, P. falciparum MDH (PF3D7_0618500); PvMDH, P. vivax MDH (PVPO1_1131000); PkMDH, P. knowlesi MDH (PKNH_1131900); PbMDH, P. berghei MDH (PBANKA_1117700); TgMDH, T. gondii MDH (TGME49_318430); HsLDH, Homo sapiens lactate dehydrogenase A (KAI4070372). (B) Phylogeny of the sequences shown in (A). Sequences with LDH1-type (blue), LDH2-type (red) and MDH-type (yellow) substrate binding loops are highlighted. Bootstrap values (n = 1000) are indicated at nodes. Rooted on HsLDH as outgroup. (C) Pairwise structure alignment of P. falciparum LDH1 (PDB: 1T2D, maroon) and the AlphaFold-predicted structure of P. falciparum LDH2 (Q8IE66, blue). Amino acid identity is 43% across 302 aligned residues, root mean square deviation is 1.03. Substrate binding loops are highlighted in red (LDH1) and yellow (LDH2).

Fig. 2

LDH2 is expressed in male gametocytes and microgametes. (A) Schematic diagram of the ldh2 alleles in parental (wildtype) parasites and in the transgenic lines LDH2/GFP and LDH2-KO. The ldh2 gene is shown in grey with coding sequence (wide bars) and 5′ and 3′ untranslated regions (narrow bars). Also shown are the gfp module, the selectable marker (hdhfr), and positions of primers P1-P3 used for diagnostic PCR amplification. (B) PCR with primers P1 and P3 diagnostic for integration of the modified ldh2 alleles into the ldh2 locus, or with primers P1 and P2 diagnostic for absence of the unmodified parental ldh2 allele. See Materials and Methods section for primer sequences. Corresponding uncropped DNA agarose gels are shown in Supplementary Fig. S3. (C) Live fluorescence confocal images of exflagellating LDH2/GFP and LDH2-KO microgametocytes with emerging microgametes (arrows). Scale bar 5 μm.

Fig. 3

LDH2 promotes oocyst development in Anopheles stephensi. (A) Oocyst loads in mosquitoes infected with parasite lines LDH2/GFP and LDH2-KO via natural gametocyte feeds. Each scatter plot represents pooled mosquitoes from three independent experiments (n = 60). Horizontal lines mark mean oocyst numbers. Pie charts show percentage of mosquitoes with at least one oocyst (prevalence of infection). Only blood-fed mosquitoes were included. Statistical significance (p values, Mann-Whitney) is indicated (ns, not significant). (B) Oocyst loads in mosquitoes infected with LDH2/GFP and LDH2-KO via direct ookinete feeds (n = 20).

Fig. 4

LDH2 promotes male gametogenesis. (A) Relative exflagellation levels of LDH2/GFP and LDH2-KO parasite lines (normalised for male gametocytemia, LDH2/GFP exflagellation set at 100%). Each bar represents mean ± sem of three independent experiments. (B) Proportion of different size exflagellation centres in LDH2/GFP and LDH2-KO parasites. Each bar represents mean ± sem of three independent experiments, each scoring at least 100 exflagellation events. Statistical significance (p values, unpaired t-test) is indicated (ns, not significant).