First Imaging Mass Spectrometry analysis of Plasmodium berghei infections in the mosquito host.

By Berin Boughton

University of Melbourne.

 

Malaria (Plasmodium sp.) is one of the most serious causes of morbidity and mortality in tropical countries in the world. The World Health Organization estimates malaria causes approximately 240 million bouts of illness and 0.86 million deaths per year. Currently, little is known of the metabolism of the parasite as it passes through the mosquito vector and of the metabolic requirements of its host during development. Thus an examination of host-parasite metabolism will provide new insights into developmental stages within the mosquito. Current techniques for in situ examination are limited, and new methods are required to examine underlying lipid metabolism in complex biological systems. Here we report the development of an imaging mass spectrometry method to examine malaria oocyst. Methods Plasmodium berghei ANKA (GFP labelled) infected mosquitoes were harvested at 12 days post infection and the midguts dissected. The complement of host and parasite lipids were extracted from pools of 50 excised midguts of control and infected mosquitoes. Separation of lipids was conducted by Thin Layer Chromatography using optimised separation conditions then MALDI matrix (DHB) was applied using a TM Sprayer prior to MALDI Fourier Transform MS (FTMS) analysis. Whole mosquitoes harvested for sectioning were immobilized then then placed between two pieces of boiled albumin (egg white) and frozen. Frozen egg white blocks were sectioned and sections mounted by freeze-thaw. A novel MALDI matrix 1,8-Bis-(1-pyrrolidinyl)naphthalene (BPYN) was applied using a TM Sprayer then sections were analysed by MALDI-FTMS. Preliminary Data Using Thin Layer Chromatography (TLC) coupled to Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-MS) we tentatively identified a phosphatidylethanolamine (PE(38:5)) as a marker of the infection status. Subsequent tissue imaging using MALDI-FTMS and classification using Receiver Operating Characteristic (ROC) curve analysis of control and infected mosquitoes tissue sections confirmed the PE(38:5) to be collocated with the site of infection within the midgut. The identity of the lipid species was confirmed using tandem MS to be PE(18:0/20:5). A total of 28 species were found to classify infection status with a number of polyunsaturated fatty acids corresponding to eicosapentaenoic acid (EPA) and arachidonic acid, implicated in both parasite development and host immune response, present in high abundance in infected tissues sections.

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