Dysregulated gene expression in oocysts of Plasmodium berghei LAP mutants

Abstract

Malaria parasite oocysts generate sporozoites by a process termed sporogony. Essential for successful sporogony of Plasmodium berghei in Anopheles stephensi mosquitoes is a complex of six LCCL lectin domain adhesive-like proteins (LAPs). LAP null mutant oocysts undergo growth and mitosis but fail to form sporozoites. At a cytological level, LAP null mutant oocyst development is indistinguishable from its wildtype counterparts for the first week, supporting the hypothesis that LAP null mutant oocysts develop normally before cytokinesis. We show here that LAP1 null mutant oocysts display highly reduced expression of sporozoite proteins and their transcription factors. Our findings indicate that events leading up to the cytokinesis defect in LAP null mutants occur early in oocyst development.

Keywords: LCCL; Malaria transmission; Oocyst; Plasmodium berghei; Sporogony.

Graphical abstract

Fig. 1

CSP expression in oocyst populations of LAP mutants assessed by western blot. Ten oocyst-infected midguts at 11 days post-infection were dissected and pooled. Samples were heated in reducing SDS-PAGE sample buffer at 70 °C for 10 min and proteins fractionated by electrophoresis through NuPage 4–12% Bis-Tris precast gels (Invitrogen) followed by transfer to PVDF membrane (Invitrogen). The gel was loaded with the equivalent of 2 (lanes 1, 3 and 5) or 4 (lanes 2, 4 and 6) oocyst-infected guts. The blot was developed using monoclonal antibody against CSP (3D11) as primary antibody, and goat anti mouse IgG conjugated to horseradish peroxidase (Invitrogen 81-6520) as secondary antibody, followed by chemiluminescence signal detection (ECL western blotting substrate, Pierce). Laboratory animal work was carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 implementing European Directive 2010/63 for the protection of animals used for experimental purposes and was approved by the London School of Hygiene & Tropical Medicine ethical review committee and United Kingdom Home Office. Experiments were conducted in 6–8 weeks old female CD1 mice, specific pathogen free and maintained in filter cages. Animal welfare was assessed daily and animals were humanely killed upon reaching experimental or clinical endpoints. Mice were infected with parasites by intraperitoneal injection. Intra-erythrocytic parasitemia was monitored regularly by collecting of a small volume of blood from a superficial tail vein. Drugs were administered by intraperitoneal injection or where possible were supplied in drinking water. Parasitized blood was harvested by cardiac bleed under general anaesthesia without recovery.

Fig. 2

Sporozoite gene expression in LAP mutants assessed by RT-qPCR. Bar chart showing fold changes in messenger RNA levels of csp, imc1a, trap, spect, a2-sp and ap2-sp2 genes relative to cytoplasmic hsp90 and hsp70 in oocyst populations of parasite lines LAP4/GFP, LAP1/GFP (wildtype reference) and LAP1-KO at 7 and 9 days post-infection of Anopheles stephensi mosquitoes. Lowest values were set to 1. Error bars denote standard deviations from three replicates. See Table 1 for gene IDs and primer properties.