Identification of Plasmodium falciparum proteoforms from liver stage models

Abstract

Background: Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. Methods:In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. Results:These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. Conclusions:This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.

In vitro models for human malaria: targeting the liver stage

“…A similar labeling pattern is observed for the cytoplasmic macrophage migration inhibitory factor (MIF) [44,48,85]. The heat shock protein 70 (HSP70) also localizes to the cytoplasm and has been the most widely used marker in identifying liver-stage forms through microscopy, including hypnozoites [40,41,43,44,46,48,51,54,58,86,[95][96][97][98]. The HSP60 is the only Plasmodium-specific marker available for the mitochondria [44,85,94], while the acyl carrier protein (ACP) is a marker for the apicoplast, an essential organelle in fatty acid and isoprenoid synthesis, and is present in all liver stages, including the hypnozoite [41,44,48,85].…”

“…The CSP protein is the most widely used antigen with presence on infective sporozoites and expression retained by all exoerythrocytic stages [37,40,42,45,49,54,[85][86][87]. Following sporozoite invasion of a hepatocyte, the parasite establishes a PVM, the membrane that separates the parasite from the host cells, as it is required to hijack the host to mediate nutrient exchange and other processes to facilitate successful development.…”

“…The whole cycle was completed in the study when reticulocytes added 7 days postinfection showed the presence of asexual stages after 14 days of subsequent culturing [53]. A following study in 2013 showed that PHHs seeded on LabTek® chamber slides infected with P. falciparum NF54 had better infection rates than HC-04 cells [21-fold higher in quantitative real-time reverse transcriptase PCR (qRT-PCR)] [51], which later enabled a proteomics study [54].…”

Amyloid β Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer’s Disease

“…DDA .raw files were processed using a cloud-based search on TDPortal (http://galaxy.kelleher.northwestern.edu, Code Set 3.0.2) against a Uniprot human proteome database 46,47 (Taxon ID, 9606 Proteome ID, UP000005640) with curated histones (June 2020). Two different search types were performed.…”