PbSR is synthesized in macrogametocytes and involved in formation of the malaria crystalloids

Abstract

Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts.

Fig. 1

Generation and molecular analysis of genetically modified parasite lines. A. Schematic diagram of the different versions of PbSR expressed by the parasite lines used. 1, wild-type parental parasite; 2, PbSR/EGFP; 3, mCherry/PbSR/EGFP; 4, ΔSRCR/EGFP; 5, ΔPbSR/EGFP. Indicated are the lipoxygenase homology domain (LH2), the Limulus clotting factor C, Coch-5b2, Lgl1 domain (LCCL); the scavenger receptor cysteine-rich domain (SRCR), the pentaxin domain (PTX), the green fluorescent protein module (GFP) and the red fluorescent protein module (RFP). B. Schematic diagram of the corresponding wild-type and genetically modified pbsr loci on genomic DNA. Numbers 1–5 correspond to the parasite lines shown in (A). Indicated are positions of the SpeI restriction sites (vertical arrows), and expected SpeI restriction fragments (dotted lines) with sizes shown in kb. C. Southern blot analysis of SpeI-digested parasite genomic DNA. Lanes 1–5 correspond to the parasite lines shown in (A). Indicated are the sizes of the pbsr- and tgdhfr-specific fragments.

Fig. 2

Life stage expression of PbSR by Western blot analysis. A. Purified gametocytes from wild-type (lane 1), PbSR/EGFP (lane 2) and mCherry/PbSR/EGFP (lanes 3 and 4) parasites under reducing (lanes 1–3) and non-reducing (lane 4) conditions. Blot was developed with anti-RFP (top) or anti-GFP (bottom) antibodies. B. Western blot analysis of different mCherry/PbSR/EGFP life stages: purified asexual blood stages (lane 1); purified gametocytes (lane 2); purified ookinetes (lane 3); two heavily infected midguts at 5 days post infection (lane 4); two heavily infected midguts at 11 days post infection (lane 5); purified midgut sporozoites (lane 6); purified salivary gland sporozoites (lane 7). Blot was developed with anti-RFP (top) or anti-CS (bottom) antibodies.

Fig. 3

Cellular localization of PbSR by fluorescence confocal microscopy. A. Female gametocyte just emerged from the host erythrocyte, whose membrane is still attached (arrow). B. Group of three mature ookinetes. C. Very young oocyst/rounded-up ookinete situated on the basal side of a midgut at 2 days post mosquito infection.

Fig. 4

PbSR is targeted to the crystalloid organelle of the ookinete. A. Image of an ookinete showing the colocalization of the fluorescent spots with malaria pigment (arrows). B. Transmission electron microscopic image of a whole ookinete section, and a subsection thereof at higher magnification (boxed) showing the crystalloid (cr) surrounded by vacuoles containing malaria pigment (p). C. Immunogold labelling (arrow) of the ookinete crystalloid in parasite line mCherry/PbSR/EGFP. This picture again clearly shows how the crystalloid is surrounded by pigment (p). D. Immunogold electron micrograph of a wild-type ookinete, showing absence of labelling.

Fig. 5

PbSR loss-of-function phenotype. A. Bright-field image of a sporulating ΔSRCR/EGFP oocyst at 15 days post infection. B. Midgut-associated sporozoites released from ΔSRCR/EGFP oocysts. C. Immunofluorescence image of a ΔSRCR/EGFP sporozoite with anti-CS antibodies (3D11), showing surface localization and trailing. D. Typical localization of PbSR in a ΔSRCR/EGFP ookinete, clearly showing the absence of focal spots.

Fig. 6

Oocyst development in vitro. A. Giemsa-stained sporulating oocysts of parasite lines PbSR/EGFP and ΔSRCR/EGFP. Scale bars represent 10 μm. B. Time-course of oocyst sporulation for PbSR/EGFP (dotted line) and ΔSRCR/EGFP (solid line). Each count was performed in triplicate and data points represents mean ± SD.