Whole-Genome Analysis of Plasmodium spp. Utilizing a New Agilent Technologies DNA Microarray Platform
The application of DNA microarray technologies to malaria genomics has been widely used but has been limited by sample availability and technical variability. To address these issues, we present a microarray hybridization protocol that has been optimized for use with two new Agilent Technologies DNA microarrays for Plasmodium falciparum and P. berghei. Using the most recent genome sequences available for each species, we have designed ∼14,000 oligonucleotide probes representing ∼5,600 transcripts for each species. Included in each array design are numerous probes that allow for the identification of parasite developmental stages, common Plasmodium molecular markers used in genetic manipulation, and manufacturer probes that control for array consistency and quality. Overall, the Agilent Plasmodium spp. array designs and hybridization methodology provides a sensitive, easy-to-use, high-quality, cost-effective alternative to other currently available microarray platforms.
Available from: Geoffrey I Mcfadden
- "The genome of Plasmodium falciparum is a chimera of two eukaryotes and two prokaryotes currently housed on 14 nuclear chromosomes, a 6 kb reduced mitochondrial genome, and a 35 kb apicoplast genome (Gardner et al. 2002). Publication of the P. falciparum strain 3D7 genome in 2002 allowed parasitologists to accelerate the task of assigning function to the ~5,600 genes (Painter et al. 2013). Postgenomic studies indicate that only ~8% of P. falciparum genes are involved in metabolism compared to 17% of the ~6,000 genes in Saccharomyces cerevisiae (Goffeau et al. 1996). "
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ABSTRACT: Malaria plagues one out of every 30 humans and contributes to almost a million deaths, and the problem could worsen. Our current therapeutic options are compromised by emerging resistance by the parasite to our front line drugs. It is thus imperative to better understand the basic biology of the parasite and develop novel drugs to stem this disease. The most facile approach to analyse a gene's function is to remove it from the genome or inhibit its activity. Although genetic manipulation of the human malaria parasite P. falciparum is a relatively standard procedure, there is no optimal method to perturb genes essential to the intra-erythrocytic development cycle (IDC) – the part of the life cycle that produces the clinical manifestation of malaria. This is a severe impediment to progress because the phenotype we wish to study is exactly the one that is so elusive. In the absence of any utilitarian way to conditionally delete essential genes, we are prevented from investigating the parasite's most vulnerable points. This review aims to focus on the development of tools identifying essential genes of P. falciparum and our ability to elicit phenotypic mutation.This article is protected by copyright. All rights reserved.
Journal of Eukaryotic Microbiology 09/2014; 61(6). DOI:10.1111/jeu.12176 · 3.22 Impact Factor
Available from: Sebastian Rusch
- "Parasite RNA was isolated at four specific time points (6–14 hpi, 16–24 hpi, 26–34 hpi & 36–44 hpi) from synchronized SEMP1-DD parasites cultured with and without Shield. The Cy5-labelled SEMP1-DD cRNA was mixed with an equal amount of Cy3-labelled 3D7 reference cRNA (mixed stages) and hybridized onto an Agilent P. falciparum Microarray slide (AMADID #037237) containing oligonucleotides for all P. falciparum genes . We did not detect any significant transcriptional changes in SEMP1-depleted parasites using Significance Analysis for Microarrays . "
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ABSTRACT: Survival and virulence of the human malaria parasite Plasmodium falciparum during the blood stage of infection critically depend on extensive host cell refurbishments mediated through export of numerous parasite proteins into the host cell. The parasite-derived membranous structures called Maurer's clefts (MC) play an important role in protein trafficking from the parasite to the red blood cell membrane. However, their specific function has yet to be determined. We identified and characterized a new MC membrane protein, termed small exported membrane protein 1 (SEMP1). Upon invasion it is exported into the RBC cytosol where it inserts into the MCs before it is partly translocated to the RBC membrane. Using conventional and conditional loss-of-function approaches we showed that SEMP1 is not essential for parasite survival, gametocytogenesis, or PfEMP1 export under culture conditions. Co-IP experiments identified several potential interaction partners, including REX1 and other membrane-associated proteins that were confirmed to co-localize with SEMP1 at MCs. Transcriptome analysis further showed that expression of a number of exported parasite proteins was up-regulated in SEMP1-depleted parasites. By using Co-IP and transcriptome analysis for functional characterization of an exported parasite protein we provide a new starting point for further detailed dissection and characterisation of MC-associated protein complexes.
PLoS ONE 07/2014; 9(7):e103272. DOI:10.1371/journal.pone.0103272 · 3.23 Impact Factor
Available from: Damián Pérez-Mazliah
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ABSTRACT: CD4(+) T-cells have been shown to play a central role in immune control of infection with Plasmodium parasites. At the erythrocytic stage of infection, IFN-γ production by CD4(+) T-cells and CD4(+) T-cell help for the B-cell response are required for control and elimination of infected red blood cells. CD4(+) T-cells are also important for controlling Plasmodium pre-erythrocytic stages through the activation of parasite-specific CD8(+) T-cells. However, excessive inflammatory responses triggered by the infection have been shown to drive pathology. Early classical experiments demonstrated a biphasic CD4(+) T-cell response against erythrocytic stages in mice, in which T helper (Th)1 and antibody-helper CD4(+) T-cells appear sequentially during a primary infection. While IFN-γ-producing Th1 cells do play a role in controlling acute infections, and they contribute to acute erythrocytic-stage pathology, it became apparent that a classical Th2 response producing IL-4 is not a critical feature of the CD4(+) T-cell response during the chronic phase of infection. Rather, effective CD4(+) T-cell help for B-cells, which can occur in the absence of IL-4, is required to control chronic parasitemia. IL-10, important to counterbalance inflammation and associated with protection from inflammatory-mediated severe malaria in both humans and experimental models, was originally considered be produced by CD4(+) Th2 cells during infection. We review the interpretations of CD4(+) T-cell responses during Plasmodium infection, proposed under the original Th1/Th2 paradigm, in light of more recent advances, including the identification of multifunctional T-cells such as Th1 cells co-expressing IFN-γ and IL-10, the identification of follicular helper T-cells (Tfh) as the predominant CD4(+) T helper subset for B-cells, and the recognition of inherent plasticity in the fates of different CD4(+) T-cells.
Frontiers in Immunology 01/2014; 5:671. DOI:10.3389/fimmu.2014.00671
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