Richard J. Wheeler

The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology

The cell cycle is central to understanding fundamental biology of Leishmania, a group of human‐infective protozoan parasites. Leishmania have two main life cycle morphologies: the intracellular amastigote in the mammalian host and the promastigote in the fly. We have produced the first comprehensive and quantitative description of a Leishmania promastigote cell cycle taking a morphometric approach to position any cell within the cell cycle based on its length and DNA content. We describe timings of cell cycle phases and rates of morphological changes; kinetoplast and nucleus S phase, division and position, cell body growth and morphology changes, flagellum growth and basal body duplication. We have shown that Leishmania mexicana undergoes large changes in morphology through the cell cycle and that the wide range of morphologies present in cultures during exponential growth represent different cell cycle stages. We also show promastigote flagellum growth occurs over multiple cell cycles. There are clear implications for the mechanisms of flagellum length regulation, life cycle stage differentiation and trypanosomatid division in general. This data set therefore provides a platform which will be of use for post‐genomic analyses of Leishmania cell biology in relation to differentiation and infection.

Antigen Potency and Maximal Efficacy Reveal a Mechanism of Efficient T Cell Activation

Efficient T cell activation depends on the rate with which T cell receptors and antigens bind and unbind, rather than simply their equilibrium affinity. Modeling T Cell Activation The efficiency of T cell activation depends on the binding parameters of the interaction between T cell receptors (TCRs) and peptide-bound major histocompatibility complexes (pMHCs). Two models propose different explanations for the relationship between TCR-pMHC binding and T cell functionality. The “affinity model” suggests that the number of TCR-pMHC complexes at equilibrium, which is governed by the dissociation constant KD, is the main determinant, whereas the “productive hit rate model” suggests that T cell responses depend on productive TCR-pMHC interaction times, which depend on the off-rate, koff. Dushek et al. performed mathematical modeling to show that both models predicted a correlation between KD and antigen potency (as measured by the EC50), but that only the productive hit rate model predicted a correlation between the maximal efficiency of antigen-induced responses and koff. Predictions from the productive hit rate model were validated in experiments with T cell clones and a panel of pMHC variants. Improved understanding of TCR-pMHC binding has implications to enhance responsiveness in various immunotherapies. T cell activation, a critical event in adaptive immune responses, depends on productive interactions between T cell receptors (TCRs) and antigens presented as peptide-bound major histocompatibility complexes (pMHCs). Activated T cells lyse infected cells, secrete cytokines, and perform other effector functions with various efficiencies, which depend on the binding parameters of the TCR-pMHC complex. The mechanism through which binding parameters are translated to the efficiency of T cell activation, however, remains controversial. The “affinity model” suggests that the dissociation constant (KD) of the TCR-pMHC complex determines the response, whereas the “productive hit rate model” suggests that the off-rate (koff) is critical. Here, we used mathematical modeling to show that antigen potency, as determined by the EC50 (half-maximal effective concentration), which is used to support KD-based models, could not discriminate between the affinity and the productive hit rate models. Both models predicted a correlation between EC50 and KD, but only the productive hit rate model predicted a correlation between maximal efficacy (Emax), the maximal T cell response induced by pMHC, and koff. We confirmed the predictions made by the productive hit rate model in experiments with cytotoxic T cell clones and a panel of pMHC variants. Thus, we propose that the activity of an antigen is determined by both its potency (EC50) and maximal efficacy (Emax).

A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids

One of the first steps in understanding a proteins function is to determine its localization; however, the methods for localizing proteins in some systems have not kept pace with the developments in other fields, creating a bottleneck in the analysis of the large datasets that are generated in the post-genomic era. To address this, we developed tools for tagging proteins in trypanosomatids. We made a plasmid that, when coupled with long primer PCR, can be used to produce transgenes at their endogenous loci encoding proteins tagged at either terminus or within the protein coding sequence. This system can also be used to generate deletion mutants to investigate the function of different protein domains. We show that the length of homology required for successful integration precluded long primer PCR tagging in Leishmania mexicana. Hence, we developed plasmids and a fusion PCR approach to create gene tagging amplicons with sufficiently long homologous regions for targeted integration, suitable for use in trypanosomatids with less efficient homologous recombination than Trypanosoma brucei. Importantly, we have automated the primer design, developed universal PCR conditions and optimized the workflow to make this system reliable, efficient and scalable such that whole genome tagging is now an achievable goal.

Cytokinesis in Trypanosoma brucei differs between bloodstream and tsetse trypomastigote forms: implications for microtubule-based morphogenesis and mutant analysis.

Trypanosomes use a microtubule‐focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early‐branching eukaryote Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge‐like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α‐tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis‐positioning during cell division and cytokinesis defects.

The trypanolytic factor-mechanism, impacts and applications.

The Trypanosoma brucei subspecies T. brucei brucei is non-human infective due to susceptibility to lysis by trypanolytic factor (TLF) in human serum. Reviewed here are the advances which have revealed apolipoprotein L1 (ApoL1), found in high density lipoprotein, as the lysis-inducing component of TLF, the means of uptake via haptoglobin-related protein receptor and the mechanism of resistance in T. b. rhodesiense via its serum resistance-associated (SRA) protein. The first practical steps to application of these discoveries are now in progress; transgenic animals expressing either baboon or minimally truncated human ApoL1 show resistance to both T. b. brucei and T. b. rhodesiense. This has major implications for treatment and prevention of human and animal African trypanosomiasis.

The Limits on Trypanosomatid Morphological Diversity

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.

TrypTag.org: A Trypanosome Genome-wide Protein Localisation Resource

TrypTag is a major resource which will contain the localisation of every protein encoded in the Trypanosoma brucei genome. Localisations of over 2000 proteins are already available via http://tryptag.org. This will be a transformative resource for enabling sophisticated analysis of conserved eukaryotic and parasite specific cell biology.

Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella

Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.

Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid

Significance Kinetoplastids are a group of protists with unique morphology and molecular features. Many have developed a parasitic lifestyle and are economically and medically important causative agents of serious crop, animal, and human diseases. Evolutionarily, Paratrypanosoma confusum sits between parasitic trypanosomatids and free-living bodonids and therefore is uniquely informative for study of the emergence of parasitism. It is morphologically very flexible, as it forms three distinct life stages that can be studied separately. Particularly interesting is the haptomonad stage in which it rebuilds its flagellum into an extensive adhesive plaque. As an adaptation to parasitism, Paratrypanosoma lost a plethora of enzymes involved in breakdown of macromolecules and the capacity of receptor-mediated endocytosis but has gained surface proteins and membrane transporters to obtain nutrients from the host. Paratrypanosoma confusum is a monoxenous kinetoplastid flagellate that constitutes the most basal branch of the highly diverse parasitic trypanosomatids, which include human pathogens Trypanosoma and Leishmania. This makes Paratrypanosoma uniquely informative for the evolution of obligatory parasitism from free-living lifestyle and the evolution of human parasitism in some trypanosomatid lineages. It has typical promastigote morphology but also forms surface-attached haptomonads and amastigotes. Haptomonads form by attachment to a surface via a large bulge at the base of the flagellum, which is then remodeled into a thin attachment pad associated with flagellum shortening. Promastigotes and haptomonads multiply by binary division, and the progeny of a haptomonad can either remain attached or grow a flagellum and resume swimming. Whole genome sequencing and transcriptome profiling, in combination with analysis of the cell ultrastructure, reveal how the cell surface and metabolism are adapted to parasitism and how characteristic cytoskeletal features are conserved. Our data demonstrate that surface attachment by the flagellum and the flagellar pocket, a Leishmania-like flagellum attachment zone, and a Trypanosoma cruzi-like cytostome are ancestral features, while evolution of extant trypanosomatids, including the human parasites, is associated with genome streamlining and diversification of membrane proteins.

Use of chiral cell shape to ensure highly directional swimming in trypanosomes

Swimming cells typically move along a helical path or undergo longitudinal rotation as they swim, arising from chiral asymmetry in hydrodynamic drag or propulsion bending the swimming path into a helix. Helical paths are beneficial for some forms of chemotaxis, but why asymmetric shape is so prevalent when a symmetric shape would also allow highly directional swimming is unclear. Here, I analyse the swimming of the insect life cycle stages of two human parasites; Trypanosoma brucei and Leishmania mexicana. This showed quantitatively how chirality in T. brucei cell shape confers highly directional swimming. High speed videomicrographs showed that T. brucei, L. mexicana and a T. brucei RNAi morphology mutant have a range of shape asymmetries, from wild-type T. brucei (highly chiral) to L. mexicana (near-axial symmetry). The chiral cells underwent longitudinal rotation while swimming, with more rapid longitudinal rotation correlating with swimming path directionality. Simulation indicated hydrodynamic drag on the chiral cell shape caused rotation, and the predicted geometry of the resulting swimming path matched the directionality of the observed swimming paths. This simulation of swimming path geometry showed that highly chiral cell shape is a robust mechanism through which microscale swimmers can achieve highly directional swimming at low Reynolds number. It is insensitive to random variation in shape or propulsion (biological noise). Highly symmetric cell shape can give highly directional swimming but is at risk of giving futile circular swimming paths in the presence of biological noise. This suggests the chiral T. brucei cell shape (associated with the lateral attachment of the flagellum) may be an adaptation associated with the bloodstream-inhabiting lifestyle of this parasite for robust highly directional swimming. It also provides a plausible general explanation for why swimming cells tend to have strong asymmetries in cell shape or propulsion.