Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach

Krishan Kumar,J Kathleen Moch,David L Narum,Dan Sturdevant,Julian C Rayner,Scott J Geromanos,Vijayaraj Nagarajan,Michael J Nold,Thomas D Otto,Raul Herrera,Prakash Srinivasan,Karine Reiter,J David Haynes,R Burke Squires,Stephen F Porcella

doi:10.1038/s41598-017-17505-9

Abstract

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10–300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion.

Highlights

Merozoites have been a focus of malaria vaccine research and development for nearly a half a century
These early studies with P. knowlesi, and subsequent studies involving P. falciparum without purified invasive merozoites, and other apicomplexan parasites have facilitated our current understanding of erythrocyte invasion by merozoites as a well-orchestrated and rapid process that relies on precise signaling and multiple receptor-ligand interactions[19,20]
Live cell imaging of long-lived merozoite (LLM) invasion indicated that the invasion kinetics were similar to what was previously reported for other P. falciparum strains (Fig. 1D)

Summary

Introduction

Merozoites have been a focus of malaria vaccine research and development for nearly a half a century. In the 1970s, it was discovered that the merozoites of P. knowlesi, a simian malaria parasite, could be purified using cell-sieving techniques and the resultant merozoites were invasive with an observed half-life of approximately twenty minutes[15,16] This discovery aided in a number of significant observations involving ultra-structural analyses of the merozoite and mechanistic studies of erythrocyte invasion (see review Bannister et al.[17]). To identify novel proteins likely involved in invasion as well as improve our basic understanding of erythrocyte invasion, an integrated omics approach was used to study a γ-irradiated parasite line that was selected for long-lived merozoite (LLM) phenotype and that was amenable to cell-sieve purification for the isolation of viable merozoites as compared to the parental wild-type (WT) parasite line. Of interest was an observation of an apparent delayed processing of two merozoite proteins in the merozoite surface protein complex of the LLM compared to WT merozoites, which highlights the importance of protein stability and its relationship to erythrocyte invasion

Objectives

Methods

Results

Conclusion