Critical role for a stage-specific actin in male exflagellation of the malaria parasite

Abstract

SummaryMale gametogenesis occurs directly after uptake of malaria parasites by the mosquito vector and leads to the release of eight nucleated flagellar gametes. Here, we report that one of the two parasite actin isoforms, named actin II, is essential for this process. Disruption of actin II in Plasmodium berghei resulted in viable asexual blood stages, but male gametogenesis was specifically inhibited. Upon activation, male gametocyte DNA was replicated normally and axonemes assembled, but egress from the host cell was inhibited, and axoneme motility abolished. The major actin isoform, actin I, displayed dual localization to the cytoplasm and the nucleus in male gametocytes. After activation actin I was found to be restricted to the cytoplasm. In actII(-) mutant parasites, this re-localization was abolished and actin I remained in both cellular compartments. These findings reveal vital and pleiotropic functions for the actin II isoform in male gametogenesis of the malaria parasite.

Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

“…Hence, the vector effectively functioned as a Gametocyte conversion rates, male:female ratios, and exflagellation rates of act2 − ::mCherry with GFPcon, 6GR, and 6G parasites, demonstrating that neither gametocyte conversion rate nor male:female ratios were affected. In contrast, male exflagellation of act2 − ::mCherry was completely abrogated, reproducing the act2 − phenotype [13]. (E) Live imaging of mixed blood stage parasites under exflagellation conditions between 10 and 20 min.…”

“…Ring and trophozoite stage parasites showed virtually undetectable levels of mCherry expression. Activated female and male were easily distinguished by the amount of DNA (1n versus 8n) as well as by the level of mCherry expression, which is considerably higher in males [13]. As expected [13], no exflagellation of the bright red male parasites was recorded.…”

“…First, we used a b3D-based knockout vector that targets P. berghei actin 2 (PbACT2) through ends-out recombination (Fig. S5A) [13]. The vector was effectively integrated in both 6rec and Bergreen as demonstrated by integration-specific PCR amplifications (Fig.…”

“…Parasitaemia was determined by quantification of daily Giemsa-stained thin blood smears. Gametocyte conversion rates, male:female ratios, and exflagellation rates were determined at day 3 after intravenous injection of 10 7 blood stage parasites into naive recipient NMRI mice [13]. Ookinete cultures were conducted in RPMI 1640 medium with lglutamine and 25 mM HEPES supplemented with 100 mM sodium bicarbonate, 100 M hypoxanthine, 10% fetal calf serum, 50 M xanthurenic acid, and 125 U ml −1 penicillin/streptomycin.…”

The development of malaria parasites in the mosquito midgut

“…Further, the sexual stage‐specific actin isoform actin 2, which was originally reported to play a role in male gametogenesis of P . berghei , is additionally important for ookinete formation (Deligianni et al ., 2011; Andreadaki et al ., 2014). Also, deletion of three ookinete‐specific alveolins, IMC1a, b and h, resulted in reduced mechanical strength of this midgut stage (Volkmann et al ., 2012) (Table 1).…”