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

Talman AM, Prieto JH, Marques S, Ubaida-Mohien C, Lawniczak M, Wass MN, Xu T, Frank R, Ecker A, Stanway RS, Krishna S, Sternberg MJ, Christophides GK, Graham DR, Dinglasan RR, Yates JR, Sinden RE - Malar. J. (2014)

Bottom Line: 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.It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College, London, UK. arthur.talman@yale.edu.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4150949&req=5

Fig1: 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.

Mentions: To examine energy metabolism in male gametes, the dataset was scanned for metabolic enzymes. Strikingly, all 11 enzymes of the glycolytic pathway and the hexose transporter (PbHT) were present (Table 2). PbHT is the only predicted Plasmodium hexose transporter and is expressed ubiquitously in the life cycle [63]. The presence of all molecules participating in glucose fermentation, as well as the result of the GO enrichment test for glycolysis (see Additional file 4) led to the hypothesis that male gametes rely exclusively on glucose metabolism for energy production. To test this hypothesis, a transgenic P. berghei line expressing a double c-myc tag on the carboxyl-terminus of the endogenous PbHT was generated [see Additional file 1]. PbHT-myc was labelled and imaged on/in male gametes/gametocytes. Because male gametes are very narrow (0.28 μm), it is challenging to distinguish between cytoplasmic and surface labelling. Confocal microscopy was thus used to enhance the resolution of the signal on the exflagellating microgametocyte (Figure 1A). One can distinguish the surface-association of PbHT in Figure 1B, which displays sequential Z-stacks of PbHT labelling. This observation strongly but not unambiguously suggests that PbHT is a component of the male gamete plasma membrane.Table 2


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

Talman AM, Prieto JH, Marques S, Ubaida-Mohien C, Lawniczak M, Wass MN, Xu T, Frank R, Ecker A, Stanway RS, Krishna S, Sternberg MJ, Christophides GK, Graham DR, Dinglasan RR, Yates JR, Sinden RE - Malar. J. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150949&req=5

Fig1: 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.
Mentions: To examine energy metabolism in male gametes, the dataset was scanned for metabolic enzymes. Strikingly, all 11 enzymes of the glycolytic pathway and the hexose transporter (PbHT) were present (Table 2). PbHT is the only predicted Plasmodium hexose transporter and is expressed ubiquitously in the life cycle [63]. The presence of all molecules participating in glucose fermentation, as well as the result of the GO enrichment test for glycolysis (see Additional file 4) led to the hypothesis that male gametes rely exclusively on glucose metabolism for energy production. To test this hypothesis, a transgenic P. berghei line expressing a double c-myc tag on the carboxyl-terminus of the endogenous PbHT was generated [see Additional file 1]. PbHT-myc was labelled and imaged on/in male gametes/gametocytes. Because male gametes are very narrow (0.28 μm), it is challenging to distinguish between cytoplasmic and surface labelling. Confocal microscopy was thus used to enhance the resolution of the signal on the exflagellating microgametocyte (Figure 1A). One can distinguish the surface-association of PbHT in Figure 1B, which displays sequential Z-stacks of PbHT labelling. This observation strongly but not unambiguously suggests that PbHT is a component of the male gamete plasma membrane.Table 2

Bottom Line: 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.It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College, London, UK. arthur.talman@yale.edu.

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.

Show MeSH
Related in: MedlinePlus