Identification and preliminary characterization of Plasmodium falciparum proteins secreted upon gamete formation

Abstract

Malaria long-term elimination depends on parasite transmission control. Plasmodium sexual stage maturation in the mosquito, including egress from the host erythrocyte, is one of the prime targets for transmission-blocking interventions. This work aims to identify candidate molecules potentially involved in gamete emergence from the host erythrocyte, as novel transmission blocking targets. We analyzed by quantitative mass spectrometry the proteins released/secreted by purified Plasmodium falciparum gametocytes upon induction of gametogenesis. The proteome obtained showed a good overlap (74%) with the one previously characterized in similar conditions from gametocytes of the rodent malaria parasite P. berghei. Four candidates were selected based on comparative analysis of their abundance values in released vs total gametocyte proteome. We also characterized the P. falciparum orthologue of the microgamete surface protein (MiGS), a marker of male gametocyte secretory vesicles in murine models of malaria. The findings of this study reveal that all the selected candidate proteins are expressed in both genders and localize to vesicle-like structures that respond to gametogenesis stimuli. This result, together with the fact that the selected proteins are released during gamete emergence in both Plasmodium species, makes them interesting candidates for future functional studies to investigate their potential role in the gametogenesis process.

Figure 1

(A) Experimental design for characterization of proteins released by activated P. falciparum gametocytes. Enriched stage V gametocytes were induced to form gametes by a drop in temperature in induction medium. Culture supernatants were collected by centrifugation and proteins released during gametogenesis were aceton-precipitated and subjected to mass spectrometry analysis. (B) the overlap between the proteins identified by mass spectrometry in each technical replicate (R1–R3) is shown as a Venn diagram; (C) the reproducibility assessment of proteomic data is schematized. The distributions of Top3 values of proteins identified in 2 out of 3 replicates (R1–R3) are represented as box plots (T-test probabilities P = 0.94). Pearson’s correlation values calculated for each pair of replicates are reported in the Table (ANOVA P < 10–3). (D) Functional analysis based on GO annotations available in PlasmoDB is shown as a histogram.

Figure 2

Statistical analysis of the proteome released by activated gametocytes. (A) The log2 distribution of the ratio between the mean abundance values of released proteome R and those of the same protein dataset in total gametocytes G (R/G) is shown as a bar chart; black line represents the corresponding fit Gaussian function. Orange and green bars highlight values exceeding the mean (m = 0.8) plus and minus one standard deviation (sd = 2.1) respectively. (B) G (red line) and R (black line) abundance profiles are ranked according to descending log2 ratio values. The orange bar below the plot corresponds to Top3 ratio values higher than the mean of at least one standard deviation; the green bar to ratio values lower than the mean of at least one standard deviation; the grey bar to values around the mean. Abundances of known egress markers (DPAP2, SUB2, MDV1, GEST G377 and PSOP12), selected egress-related candidates (boxed) and the contaminant G25/27 are indicated. (C) Plot of R vs G highlights protein abundance values around the mean (gray dots) and, values exceeding one standard deviation from the mean (orange and green dots). The correlation values R (ANOVA P ≤ 10–3, alpha = 0.05) and the linear coefficient (a) of the regression lines (dotted) are indicated.

Figure 3

Subcellular localization before and after gametocyte activation. Immuno-localization of mature P. falciparum gametocytes before and after activation, by using specific immune sera against four proteins selected from the activated gametocytes secretome and PfMiGS, ortholog of a murine male-specific protein. Pfg377, a female-specific OB marker and alpha-Tubulin, staining male gamete forming flagella, were used to distinguish between genders.