IFT25 is required for the construction of the trypanosome flagellum.

Intraflagellar transport (IFT), the movement of protein complexes responsible for the assembly of cilia and flagella, is remarkably conserved from protists to humans. However, two IFT components (IFT25 and IFT27) are missing from multiple unrelated eukaryotic species. In mouse, IFT25 (also known as HSPB11) and IFT27 are not required for assembly of several cilia with the noticeable exception of the flagellum of spermatozoa. Here, we show that the Trypanosoma brucei IFT25 protein is a proper component of the IFT-B complex and displays typical IFT trafficking. By performing bimolecular fluorescence complementation assays, we reveal that IFT25 and IFT27 interact within the flagellum in live cells during the IFT process. IFT25-depleted cells construct tiny disorganised flagella that accumulate IFT-B proteins (with the exception of IFT27, the binding partner of IFT25) but not IFT-A proteins. This phenotype is comparable to the one following depletion of IFT27 and shows that IFT25 and IFT27 constitute a specific module that is necessary for proper IFT and flagellum construction in trypanosomes. Possible reasons why IFT25 and IFT27 would be required for only some types of cilia are discussed.

Sensing of red blood cells with decreased membrane deformability by the human spleen.

The current paradigm in the pathogenesis of several hemolytic red blood cell disorders is that reduced cellular deformability is a key determinant of splenic sequestration of affected red cells. Three distinct features regulate cellular deformability: membrane deformability, surface area-to-volume ratio (cell sphericity), and cytoplasmic viscosity. By perfusing normal human spleens ex vivo, we had previously showed that red cells with increased sphericity are rapidly sequestered by the spleen. Here, we assessed the retention kinetics of red cells with decreased membrane deformability but without marked shape changes. A controlled decrease in membrane deformability (increased membrane rigidity) was induced by treating normal red cells with increasing concentrations of diamide. Following perfusion, diamide-treated red blood cells (RBCs) were rapidly retained in the spleen with a mean clearance half-time of 5.9 minutes (range, 4.0-13.0). Splenic clearance correlated positively with increased membrane rigidity (r = 0.93; P < .0001). To determine to what extent this increased retention was related to mechanical blockade in the spleen, diamide-treated red cells were filtered through microsphere layers that mimic the mechanical sensing of red cells by the spleen. Diamide-treated red cells were retained in the microsphilters (median, 7.5%; range, 0%-38.6%), although to a lesser extent compared with the spleen (median, 44.1%; range, 7.3%-64.0%; P < .0001). Taken together, these results have implications for understanding the sensitivity of the human spleen to sequester red cells with altered cellular deformability due to various cellular alterations and for explaining clinical heterogeneity of RBC membrane disorders.

The Cyclical Development of Trypanosoma vivax in the Tsetse Fly Involves an Asymmetric Division.

Trypanosoma vivax is the most prevalent trypanosome species in African cattle. It is thought to be transmitted by tsetse flies after cyclical development restricted to the vector mouthparts. Here, we investigated the kinetics of T. vivax development in Glossina morsitans morsitans by serial dissections over 1 week to reveal differentiation and proliferation stages. After 3 days, stable numbers of attached epimastigotes were seen proliferating by symmetric division in the cibarium and proboscis, consistent with colonization and maintenance of a parasite population for the remaining lifespan of the tsetse fly. Strikingly, some asymmetrically dividing cells were also observed in proportions compatible with a continuous production of pre- metacyclic trypomastigotes. The involvement of this asymmetric division in T. vivax metacyclogenesis is discussed and compared to other trypanosomatids.

The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

Scanning transmission electron microscopy through-focal tilt-series on biological specimens.

Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.

The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport.

The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.DOI: http://dx.doi.org/10.7554/eLife.02419.001.

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

Forward motility is essential for trypanosome infection in the tsetse fly.

African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.

Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body.

The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

Surface area loss and increased sphericity account for the splenic entrapment of subpopulations of Plasmodium falciparum ring-infected erythrocytes.

Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs' morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3-21.9%), 9.6% (7.3-12.2%) and 3.7% (0-8.4), respectively. Microsphilters retained 100%, ∼50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15-18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).

Microsphiltration: a microsphere matrix to explore erythrocyte deformability.

The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.

Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells.

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen.

Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.

Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties.

Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.

Artesunate tolerance in transgenic Plasmodium falciparum parasites overexpressing a tryptophan-rich protein.

Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins.

The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro.

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-µm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.

Anopheles gambiae PRS1 modulates Plasmodium development at both midgut and salivary gland steps.

BACKGROUND: Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches. METHODOLOGY/PRINCIPAL FINDINGS: PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues. CONCLUSIONS/SIGNIFICANCE: While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.

Serial Analysis of Gene Expression in Plasmodium berghei salivary gland sporozoites.

BACKGROUND: The invasion of Anopheles salivary glands by Plasmodium sporozoites is an essential step for transmission of the parasite to the vertebrate host. Salivary gland sporozoites undergo a developmental programme to express genes required for their journey from the site of the mosquito bite to the liver and subsequent invasion of, and development within, hepatocytes. A Serial Analysis of Gene Expression was performed on Anopheles gambiae salivary glands infected or not with Plasmodium berghei and we report here the analysis of the Plasmodium sporozoite transcriptome. RESULTS: Annotation of 530 tag sequences homologous to Plasmodium berghei genomic sequences identified 123 genes expressed in salivary gland sporozoites and these genes were classified according to their transcript abundance. A subset of these genes was further studied by quantitative PCR to determine their expression profiles. This revealed that sporozoites modulate their RNA amounts not only between the midgut and salivary glands, but also during their storage within the latter. Among the 123 genes, the expression of 66 is described for the first time in sporozoites of rodent Plasmodium species. CONCLUSION: These novel sporozoite expressed genes, especially those expressed at high levels in salivary gland sporozoites, are likely to play a role in Plasmodium infectivity in the mammalian host.