Plasmodium berghei LAPs form an extended protein complex that facilitates crystalloid targeting and biogenesis

Annie Z Tremp,Sadia Saeed,Vikram Sharma,Edwin Lasonder,Johannes T Dessens

doi:10.1016/j.jprot.2020.103925

Abstract

Passage of malaria parasites through mosquitoes involves multiple developmental transitions, from gametocytes that are ingested with the blood meal, through to sporozoites that are transmitted by insect bite to the host. During the transformation from gametocyte to oocyst, the parasite forms a unique transient organelle named the crystalloid, which is involved in sporozoite formation. In Plasmodium berghei, a complex of six LCCL domain-containing proteins (LAPs) reside in the crystalloid and are required for its biogenesis. However, little else is known about the molecular mechanisms that underlie the crystalloid's role in sporogony. In this study, we have used transgenic parasites stably expressing LAP3 fused to GFP, combined with GFP affinity pulldown and high accuracy mass spectrometry, to identify an extended LAP interactome of some fifty proteins. We show that many of these are targeted to the crystalloid, including members of two protein families with CPW-WPC and pleckstrin homology-like domains, respectively. Our findings indicate that the LAPs are part of an intricate protein complex, the formation of which facilitates both crystalloid targeting and biogenesis. SignificanceReducing malaria parasite transmission by mosquitoes is a key component of malaria eradication and control strategies. This study sheds important new light on the molecular composition of the crystalloid, an enigmatic parasite organelle that is essential for sporozoite formation and transmission from the insect to the vertebrate host. Our findings provide new mechanistic insight into how proteins are delivered to the crystalloid, and indicate that the molecular mechanisms that underlie crystalloid function are complex, involving several protein families unique to Plasmodium and closely related organisms. The new crystalloid proteins identified will form a useful starting point for studies aimed at unravelling how the crystalloid organelle facilitates sporogony and transmission.

Highlights

Despite a significant fall in incidence rates in the last decade, malaria remains the most serious parasitic infection in humans
We previously studied molecular interactions between the six LCCL domain-containing proteins (LAPs) using a series of genetically modified parasite lines stably expressing LAPs individually fused to green fluorescent protein (GFP), combined with GFP affinity purification and label free quantitative mass spectrometry (AP-MS) [7]
To identify other proteins interacting with the LAP complex and discover potential new crystalloid proteins, we made assumptions that such proteins would behave like LAPs 4–6 in GFP pulldown experiments, and would have similar features to LAPs 4–6 with regards to their expression and subcellular trafficking in the parasite

Summary

Introduction

Despite a significant fall in incidence rates in the last decade, malaria remains the most serious parasitic infection in humans. Malaria parasite transmission is initiated when Anopheles female mosquitoes ingest blood-borne gametocytes during blood feeding on a parasite-infected host. This sets off a rapid process of gamete formation and fertilization in the insect midgut. Oocysts grow and divide by a process called sporogony, generating thousands of haploid sporozoites. These make their way to the insect's salivary glands to be transmitted to new hosts by mosquito bite. A small percentage of intraerythrocytic parasites transform into gametocytes to complete the life cycle

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