Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility

Abstract

Background: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.

Methods: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.

Results: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.

Conclusions: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

Figure 1

PbHT localizes to the male gamete surface. The P. berghei HT-myc line was labelled with an α-tubulin antibody (red), an anti-c-myc antibody (green) and stained with DAPI (blue). (A) 3-D projection of a male gametocyte in the process of exflagellation. Scale bar 5 μm. (B) Enlarged inset from (A) displaying the distribution PbHT-myc and α-tubulin in sequential Z stacks (0.1 μm). Scale bar 1 μm. PbHT-myc is abundant and localized to the periphery of male gametes.

Figure 2

Male motility is suppressed by glycolytic inhibitors. The proportion of male gametes exhibiting each class of flagellar beat was determined in the presence of the glucose analogues 2-deoxy-D-glucose (not processed after phosphorylation by hexokinase) (A) and CM3361 (competitive inhibitor of glucose transport) (B) in the presence or absence of excess glucose (10 mM). (C, D) Patterns of motility were further categorized into fast beat, slow beat and immotile. (E) Values of flagella wave frequencies were measured with different concentrations of CM3361. Frequencies of waves were not altered by the presence of the inhibitor.

Figure 3

A model of the Plasmodium flagellum and proteins comprising it. (A) A cross section of a typical axoneme is shown (as observed in Plasmodium and other eukaryotic species). Proteins that were present in the male gametocyte (MGY) or male gamete (MG) proteomes are shown and their sequence count is given. It is noteworthy that the sequence counts originate from different datasets from distinct experimental set-ups [10]; they are thus indicative of presence of these proteins in the two datasets but are not meant as quantitative information on protein abundance in these cells types. Typical components of an axoneme are clearly identified. (B) Longitudinal section of an axoneme and basal body and the proteins identified in the male proteomes that are putatively associated with these structures. (C) Proteins that were identified in the male gamete proteomic analysis but that could not be attributed to a specific compartment of the flagellum, these proteins possess bio-informatic prediction that suggests they could play a role in flagellar biology. CPP = conserved Plasmodium protein.