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Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease.

ABSTRACT: Compartmentalization of proteins into subcellular organelles in eukaryotic cells is a fundamental mechanism of regulating complex cellular functions. Many proteins of Plasmodium falciparum merozoites involved in invasion are compartmentalized into apical organelles. We have identified a new merozoite organelle that contains P. falciparum rhomboid-1 (PfROM1), a protease that cleaves the transmembrane regions of proteins involved in invasion. By immunoconfocal microscopy, PfROM1 was localized to a single, thread-like structure on one side of the merozoites that appears to be in close proximity to the subpellicular microtubules. PfROM1 was not found associated with micronemes, rhoptries, or dense granules, the three identified secretory organelles of invasion. Release of merozoites from schizonts resulted in the movement of PfROM1 from the lateral asymmetric localization to the merozoite apical pole and the posterior pole. We have named this single thread-like organelle in merozoites, the mononeme.

SUBMITTER: Singh S

PROVIDER: S-EPMC2148419 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease.

Singh Subhash S Plassmeyer Matthew M Gaur Deepak D Miller Louis H LH

Proceedings of the National Academy of Sciences of the United States of America 20071128 50

Compartmentalization of proteins into subcellular organelles in eukaryotic cells is a fundamental mechanism of regulating complex cellular functions. Many proteins of Plasmodium falciparum merozoites involved in invasion are compartmentalized into apical organelles. We have identified a new merozoite organelle that contains P. falciparum rhomboid-1 (PfROM1), a protease that cleaves the transmembrane regions of proteins involved in invasion. By immunoconfocal microscopy, PfROM1 was localized to a ...[more]

PMID: 18048320

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Project description:Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 μm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 μm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 μm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.

Project description:BackgroundPlasmodium vivax infection remains a major public health problem, especially along the Thailand border regions. We examined the genetic diversity of this parasite by analyzing single-nucleotide polymorphisms (SNPs) of the P. vivax rhomboid-like protease 1 gene (Pvrom1) in parasites collected from western (Tak province, Thai-Myanmar border) and eastern (Chanthaburi province, Thai-Cambodia border) regions.MethodsData were collected by a cross-sectional survey, consisting of 47 and 45 P. vivax-infected filter paper-spotted blood samples from the western and eastern regions of Thailand, respectively during September 2013 to May 2014. Extracted DNA was examined for presence of P. vivax using Plasmodium species-specific nested PCR. Pvrom1 gene was PCR amplified, sequenced and the SNP diversity was analyzed using F-STAT, DnaSP, MEGA and LIAN programs.ResultsComparison of sequences of the 92 Pvrom1 831-base open reading frames with that of a reference sequence (GenBank acc. no. XM001615211) revealed 17 samples with a total of 8 polymorphic sites, consisting of singleton (exon 3, nt 645) and parsimony informative (exon 1, nt 22 and 39; exon 3, nt 336, 537 and 656; and exon 4, nt 719 and 748) sites, which resulted in six different deduced Pvrom1 variants. Non-synonymous to synonymous substitutions ratio estimated by the DnaSP program was 1.65 indicating positive selection, but the Z-tests of selection showed no significant deviations from neutrality for Pvrom1 samples from western region of Thailand. In addition McDonald Kreitman test (MK) showed not significant, and Fst values are not different between the two regions and the regions combined. Interestingly, only Pvrom1 exon 2 was the most conserved sequences among the four exons.ConclusionsThe relatively high degree of Pvrom1 polymorphism suggests that the protein is important for parasite survival in face of changes in both insect vector and human populations. These polymorphisms could serve as a sensitive marker for studying plasmodial genetic diversity. The significance of Pvrom1 conserved exon 2 sequence remains to be investigated.

Dataset Information

Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease.

Publications

Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease.

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