3D FIB-SEM structural insights into the architecture of sub-pellicular microtubules of Trypanosoma cruzi epimastigotes.

BACKGROUND INFORMATION: Trypanosomatidae, which includes eukaryotic species agents of diseases like leishmaniasis, sleeping sickness, and Chagas disease, have special structures and organelles not found in mammalian cells. They present a layer of microtubules, known as subpellicular microtubules (SPMT), located underneath the plasma membrane and responsible for preserving cell morphology, cell polarity, the position of single copy organelles, and morphological changes that occur throughout the protozoan life cycle. Even though a lot of knowledge about the SPMT is available, we still do not know exactly how each microtubule in the system is organized in three dimensions. Here, we use focused ion beam scanning electron microscopy (FIB-SEM) to analyze the tridimensional organization of epimastigotes SPMT. RESULTS: The high-resolution 3D analyses revealed that certain microtubules of the SPMT end more prematurely than the neighboring ones. CONCLUSIONS: These microtubules could (1) be shorter or (2) have the same length as the neighboring ones, assuming that those end up earlier at their other end, might be treadmilling/catastrophe events that have not yet been described in trypanosomatids.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent.

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

Morphogenesis Dynamics in Leishmania Differentiation.

Leishmania, the causative agent of leishmaniasis, is an obligatory intracellular parasite that cycles between phagolysosome of mammalian macrophages, where it resides as round intracellular amastigotes, and the midgut of female sandflies, where it resides as extracellular elongated promastigotes. This protozoan parasite cytoskeleton is composed of stable and abundant subpellicular microtubules (SPMT). This study aims to determine the kinetics of developmental morphogenesis and assess whether microtubules remodelling is involved in this process. Using image-streaming technology, we observed that rounding of promastigotes during differentiation into amastigotes was initiated promptly after exposure to the differentiation signal. Stabilizing microtubules with taxol sped rounding, but later killed differentiating parasites if taxol was not removed. Microtubule destabilizers such as vinblastine had no effect on the rate of rounding, nor on the viability of differentiating parasites. In the reverse process, elongation is initiated after a delay of 7.5 and completed 72 h after exposure to the amastigote to the promastigote differentiation signal. During the delay, parasites became highly sensitive to treatment with microtubule destabilizers. The addition of vinblastine during the first 7.5 h halted differentiation and killed parasites. Between 8 and 24 h, parasites gradually became resistant to vinblastine and, in parallel, started to elongate. In contrast, taxol had no effect on parasite elongation, nor on the viability of these cells. In a parallel study, we showed that the Leishmania-specific protein kinase A (PKA) holoenzyme containing the LdPKAR3-C3 complex is essential for promastigote elongation. Mutant promastigotes lacking either of these proteins are round, but maintain their flagella. Here, we observed that during differentiation into amastigotes, these mutants round at the same rate as the wild type, but never exceed the WT density of round amastigotes. In the reverse process, these mutants undergo the same initial delay and then elongate at the same rate as the WT. They stop elongating when they reach 20% of elongated cells in mature promastigotes. Our analysis indicates that while promastigote rounding into amastigotes did not require microtubule remodelling, morphogenesis of round amastigotes into elongated promastigotes required microtubule rearrangement before elongation was initiated. This is the first study that investigates the dynamics of microtubules during parasite development.

Revisiting the Role of Toxoplasma gondii ERK7 in the Maintenance and Stability of the Apical Complex.

Toxoplasma gondii extracellular signal-regulated kinase 7 (ERK7) is known to contribute to the integrity of the apical complex and to participate in the final step of conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade, or egress from host cells. In contrast to a previous report, we show here that the depletion of ERK7 phenocopies the depletion of the apical cap protein AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring (APR), the disorganization of the basket of subpellicular microtubules (SPMTs), and a severe impairment in microneme secretion. Ultrastructure expansion microscopy (U-ExM), coupled to N-hydroxysuccinimide ester (NHS-ester) staining on intracellular parasites, offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7-depleted parasites confirmed the disappearance of known apical complex proteins, including markers of the apical polar ring and a new apical cap named AC11. Concomitantly, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in the exocytosis of the organelles. AC9-depleted parasites were included as controls and exhibited an increase in inner membrane complex proteins, with two new proteins assigned to this compartment, namely, IMC33 and IMC34. IMPORTANCE The conoid is an enigmatic, dynamic organelle positioned at the apical tip of the coccidian subgroup of the Apicomplexa, close to the apical polar ring (APR) from which the subpellicular microtubules (SPMTs) emerge and through which the secretory organelles (micronemes and rhoptries) reach the plasma membrane for exocytosis. In Toxoplasma gondii, the conoid protrudes concomitantly with microneme secretion, during egress, motility, and invasion. The conditional depletion of the apical cap structural protein AC9 or AC10 leads to a disorganization of SPMTs as well as the loss of the APR and conoid, resulting in a microneme secretion defect and a block in motility, invasion, and egress. We show here that the depletion of the kinase ERK7 phenocopies AC9 and AC10 mutants. The combination of ultrastructure expansion microscopy and NHS-ester staining revealed that ERK7-depleted parasites exhibit a dilated apical plasma membrane and an altered positioning of the rhoptries, while electron microscopy images unambiguously highlight the loss of the APR.