Molecular detection of human Plasmodium species using a multiplex real time PCR

International audienceMolecular detection methods have revealed higher sensitivity and specificity than conventional microscopy or rapid diagnostic tests for malaria diagnosis. In this study, we implemented, evaluated and validated according to the ISO 15,189 requirements, a multiplex real-time PCR assay to detect and identify the five human malaria parasites. DNA samples were extracted from whole blood or dried blood spots drawn from patients. Based on the External Quality Assessment (whole blood), this method shows 100% sensitivity and specificity. This PCR detected P. vivax up to 0.25 p/µl, P. falciparum and P. knowlesi up to 0.5 p/µl, P. ovale up to 1 p/µl and P. malariae up to 5 p/µl of blood. From blood spots (extraction from four punches), it detected P. vivax at 5 p/µl, P. falciparum , P. ovale and P. knowlesi at 20 p/µl and P. malariae at 125 p/µl. In conclusion, this quantitative PCR shows excellent performance, is easy to use and DNA saver. It is especially useful to actively screen large population groups and identify the five human malaria parasites in a context of low malaria transmission

Correction: BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.

[This corrects the article DOI: 10.1371/journal.ppat.1004654.]

Interaction between the flagellar pocket collar and the hook complex <i>via</i> a novel microtubule-binding protein in <i>Trypanosoma brucei</i>

<div><p><i>Trypanosoma brucei</i> belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body <i>via</i> the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of <i>in vitro</i> and <i>in vivo</i> approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.</p></div

BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC

Bhalin, an essential cytoskeleton-associated protein of Trypanosoma bruceiTrypanosoma\ brucei linking TbBILBO1 of the flagellar pocket collar with the hook complex

International audienceBackground: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites

BILBO1—FPC4 interaction involves their N-terminal and C-terminal domains respectively.

<p>(A) BILBO1-FPC4 yeast two-hybrid interaction test. Left panel: Schematic overview of BILBO1 and FPC4 domains and of the combinations tested by Y2H. The EF-hand calcium binding sites of BILBO1 are represented in green and the coiled-coil domains in BILBO1 and FPC4 are represented in blue. The FPC4 B1BD is represented in violet. Right panel: The interactions tested were probed on–histidine selective medium (-His) and on growth control medium (control) (n = 3). Positive control involved p53 and T-antigen, whereas negative control involved Lamin and T-antigen. (B) Heterologous expression and co-expression in U-2 OS cells of FPC4 and FPC4 truncations fused to a C-terminal GFP tag, and of BILBO1. BILBO1 (a), FPC4 (b), FPC4-ΔB1BD (c) and FPC4-B1BD (d) were expressed alone and cells were probed with anti-BILBO1 (red) and anti-GFP (green). In e, f, g, cells were expressing BILBO1 + FPC4, BILBO1 + FPC4-ΔB1BD, and BILBO1 + FPC4-B1BD respectively and were also probed with anti-BILBO1 (red) and anti-GFP (green). Cells were extracted before labelling, except in (d) to show the cytoplasmic localisation of FPC4-B1BD, which is not visible on extracted cells. The transfections were performed more than three independent times. Nuclei were DAPI stained (blue). Scale bar represents 10 μm.</p

FPC4 binds to the remnant MtQ complex of isolated flagella.

<p>(A) Co-labelling of myc-tagged proteins (green) and tubulin (red) on isolated flagella from WT cells (a), and cells over-expressing myc-FPC4 (b) or myc-FPC4-ΔB1BD (c). White arrowheads are pointing to the zoom areas. Scale bars 5 μm. (B) Immuno-gold labelling of myc-FPC4-ΔB1BD (anti-myc, 10 nm gold beads) and tubulin (15 nm gold beads) on isolated flagella. In the zoom image (b), the arrows highlight the MtQ decorated with anti-tubulin and anti-myc labelling that appears close to the MtQ. In the zoom image (c), the black arrowheads highlight the FPC structure decorated by the anti-myc antibody. All the experiments were performed more than three independent times.</p

FPC4 is a hook complex and an FPC protein.

<p>(A) (a) Immunolabelling of endogenous FPC4 on cytoskeleton extracted cells from <i>Tb</i>427 29–13 cells using the rat anti-FPC4 antibody. (b) Cytoskeletons from the cell line expressing endogenously myc-tagged FPC4 probed with anti-myc. (c and d) Cytoskeletons from the cell line overexpressing myc-FPC4 using anti-myc. Co-labelling with anti-BILBO1 (a, b, c) or anti-MORN1 (d). (B) Anti-myc immuno-electron microscopy on flagella isolated from myc-FPC4 expressing cells (a), and the respective enlargements (b, c and d). (C) FPC4 immunolabelling using anti-FPC4 on CK extracted <i>Tb</i>427 90–13 BSF cells. (D) Co-labelling on cytoskeleton extracted <i>Tb</i>427 29–13 cells using anti-FPC4 and anti-MORN1 (a-d), and rat anti-FPC4 and anti-BILBO1 (e-h). (E) BILBO1^<i>RNAi</i> was induced for 36 h and cells were detergent extracted and probed with rat anti-FPC4 (green) and anti-MORN1 (red). The white arrowheads indicate the area that is enlarged in the zoom image and highlight the labelling of FPC4 and MORN1 within the new detached flagellum (a) and at the base of the axoneme of the new detached flagellum (b). Kinetoplasts and nuclei were DAPI stained in A, C, D and E. Scale bars in A, C, D, and E represent 5 μm. In A c and d where the cells were induced for 24 h. All the experiments were performed more than three independent times.</p

Immuno-localisation of endogenously tagged 10xTY1-FPC4, BILBO1 and MORN1.

<p>(A) Wide-field microscopy observation of the triple labelling of BILBO1, MORN1 and FPC4 on cytoskeleton from a SmOxP427 cell line expressing endogenously 10xTY1 tagged FPC4. (B) Immuno-electron microscopy on flagella labelled with anti-TY1 for FPC4 (5 nm gold beads), anti-BILBO1 (15 nm gold beads), and anti-MORN1 (15 nm gold beads). (C) STED confocal microscopy views of the co-labelling of BILBO1, MORN1 and FPC4 on cytoskeletons from a SmOxP427 cell line expressing endogenously 10xTY1 tagged FPC4 (a-c). The asterisks indicate the kinetoplast position relative to the structure. The snapshots of the 3D reconstructions show the close proximity of BILBO1 (green), FPC4 (red) and MORN1 (cyan). (D) Normalised intensities Plot corresponding to the section indicated by the white lines (a, b) indicate the co-localisation of the proteins. In A and B, scale bars represent 1 μm.</p

The overexpression of FPC4-ΔB1BD induced morphological phenotypes.

<p>(A) Co-localisation of GFP-FPC4 (anti-GFP, green) and BILBO1 (anti-BILBO1, red) by immunofluorescence on cytoskeletons. The cells were induced to express GFP-FPC4 for 48 h. (B) Transmission electron micrograph of a thin section of embedded cells that were over-expressing GFP-FPC4 (72 h of induction). Black arrowheads highlight the electron dense structure observed in these cells resembling the filament observed by immunofluorescence. (C) Co-localisation of myc-FPC4-ΔB1BD (anti-myc, green) and BILBO1 (red), MORN1 (red) and FAZ (red) by immunofluorescence on cytoskeletons. The cells were induced for 48 h. Several phenotypes were observed: filament connection between 2 FPCs (a, c, e), epimastigote-like cells (b), and localisation of myc-FPC4-ΔB1BD together with MORN1 at the distal end of the cell body (d). Abnormal cells still exhibit FAZ structures that were positively labelled with L3B2 (f). (D) Immuno-electron microscopy localisation of gold particles of myc-FPC4-ΔB1BD (anti-myc, 15 nm gold) and BILBO1 (anti-BILBO1, 10 nm gold) on isolated flagella from cells expressing myc-FPC4-ΔB1BD. (E) Transmission electron microscopy of thin sections of embedded cells over-expressing myc-FPC4-ΔB1BD showing an epimastigote-like phenotype. (F) Counts of cells showing different phenotypes related to kinetoplast positioning. Normal phenotype (brown - 1K1N, 2K1N, and 2K2N cells where cell morphology was normal), abnormal (yellow—Zoids and multinucleated), and abnormal (green—misplaced kinetoplast) for WT and non-induced and induced (24, 48, and 96 h) myc-FPC4-ΔB1BD over-expressing cells. The misplaced kinetoplast category was then divided into subcategories (48 h induction). Kinetoplasts and nuclei were DAPI stained and scale bars represent 5 μm in A and C. Initial immuno-localisations were performed on two individual clones.</p