Fluorescent tagging ofPlasmodiumcircumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts

Abstract

The circumsporozoite protein, CSP is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Antibodies against CSP can stop infection prior to the first round of parasite replication in the liver. CSP consists of different domains and is proteolytically cleaved prior to hepatocyte invasion. Part of CSP has been developed into a licensed vaccine against malaria. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we generated a series of parasites expressing full-length versions of CSP as fusion proteins with the green fluorescent protein. This enabled the investigation of sporozoite formation in living oocysts and revealed a dominant negative function of some GFP-CSP fusions during sporozoite egress.

Using qPCR to compare the detection ofPlasmodium vivaxoocysts and sporozoites inAnopheles farautimosquitoes between two DNA extraction methods

“…To overcome the limitations of microscopy a number of assays have been developed to enable high throughput detection of parasites in the mosquito gut and salivary glands. These assays include ELISA to detect the circumsporozoite protein (CSP) in mosquito lysates (CSP-ELISA) [9-11], bioluminescence assays to detect transgenic parasites with the green fluorescence protein (GFP) [12-14], near-infrared spectroscopy (NIRS) to detect parasites within mosquitoes [15, 16], enhanced chemiluminescent slot blot (ECL-SB) for detecting Pf CSP in mosquito samples [17, 18] and molecular detection of Plasmodium DNA [19-21]. Although the CSP-ELISA is relatively robust and cost effective it is only semi quantitative [9-11].…”

“…Bioluminescence GFP assays allow for high-through-put but it cannot be used with wild parasites. [12-14] NIRS has been successfully used to detect P. falciparum parasites in lab reared mosquitoes with relatively high accuracy but it is still semi quantitative. [15, 16] ECL-SB assays can potentially be used to screen large numbers of mosquitoes for oocysts with high sensitivity and specificity.…”