Proteomic Analysis of Plasmodium Merosomes: The Link between Liver and Blood Stages in Malaria

Abstract

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.

Keywords: PEXEL; exoerythrocytic stage; liver stages; malaria; merosomes; proteomics; sporozoites.

Figure 1.

Overview of the core P. berghei merosome proteome. (A) Immunofluorescence analysis of merosome samples used for proteomics. Top: Staining with antibodies specific for MSP1 identifies hepatic merozoites within merosomes and confirms merosomes are intact. Middle and bottom: Staining with antibodies specific for the PV marker UIS4 demonstrates the vacuole is absent or fragmented in merosomes. DNA stained with Hoechst. Scale bar 10 μm. (B) Overview of the P. berghei merosome proteome. Genomes refer to the number of hepatic merozoite genomes estimated by quantitative PCR. PSMs refers to peptide spectrum matches. Total IDs are proteins identified by at least one peptide. IDs ≥ 2 unique are proteins identified by at least two unique peptides. (C) Correlation among biological replicates. iBAQ values were log₂ transformed to generate scatter plots showing the correlation among biological replicates. The Pearson correlation between replicates is indicated in each plot. Data shown are for proteins with >2 unique peptides and plots were generated using Perseus version 1.6.0.2078. (D) Concordance between protein IDs with ≥2 unique peptides between replicates. We define the core merosome proteome as the 1188 proteins identified by ≥2 unique peptides in all three biological replicates.

Figure 2.

Overlap between the core merosome proteome and published liver and blood stage proteomes. Venn diagram depicting the overlap between our core merosome proteome, the P. yoelii liver stage proteome, and the combined proteomes of four Plasmodium blood stage schizont or merozoite proteomes.^– Proteins in the published proteomes were converted to their syntenic P. berghei orthologs for this comparison. The 73 proteins not overlapping with liver and blood stage proteomes were further analyzed. Proteins previously described in other life cycle stages or part of multigene families were removed, and the remaining 28 proteins may be unique to merosomes. These proteins are presented in Table 2.

Figure 3.

The absence of merozoite surface protein 4/5 in merosomes is associated with alternative splicing of the mRNA transcript. (A) Diagram of the P. berghei MSP4/5 gene structure, spliced mRNA, and unspliced mRNA. Unspliced mRNA contains a premature stop codon (red line). Primers used to quantify total MSP4/5 mRNA (P1 + P2) and detect splice variants (P3 + P4) are shown. (B) Quantitative reverse transcriptase PCR demonstrates relative abundance of total MSP4/5 mRNA is comparable in blood stage schizonts and merosomes. Relative abundance calculated by comparison to HSP70. Relative abundance of MSP4/5 was normalized to 1.0 for schizonts. Error bars show mean ± standard deviation. (C) Nonquantitative reverse transcriptase PCR demonstrates MSP4/5 is alternatively spliced in schizonts and merosomes. Abbreviations: Schiz, schizonts; Meros, merosomes; +RT, with reverse transcriptase; − RT, without reverse transcriptase.

Figure 4.

Localization of the PHIST protein in late liver stage parasites and merosomes. (A) Immunofluorescence staining of late liver stage parasites at 60 h postinfection indicates the PHIST protein (PBANKA_1145400) partially colocalizes with the parasitophorous vacuole marker UIS4. Colocalized pixels in white. DNA stained with Hoechst. (B,C) Immunofluorescence staining of merosomes and free merozoites at 65 h postinfection demonstrates the PHIST protein associates with individual liver stage merozoites. No UIS4 staining was detected in the mature merosome or free hepatic merozoite shown.