Simulating the complex cell design of Trypanosoma brucei and its motility

The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and predict a flagellar course we have not been able to measure in experiments so far

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating

Visualising blood flagellates infections in transparent zebrafish

Trypanosomes of the Trypanosoma genus are blood flagellates, and important causative agents of diseases of humans, livestock and cold-blooded species. Numerous in vitro studies and infection studies in mice contributed enormously to the insights into the biology of trypanosomes, their interaction with and evasion of the host immune system, as well as into various aspects related to vaccine failure and (uncontrolled) inflammation. A tight regulation of the early innate immune response to trypanosome infections was shown to be critical to obtain a balance between parasite control and inflammation-associated pathology. Trypanosome morphology was observed to be essential for their motility, the adaptation to their host’s environment and pathogenesis. One of the best-studied non-mammalian trypanosomes is Trypanosoma carassii, which presents many morphological similarities to mammalian trypanosomes. T. carassii is regularly observed co-infecting fish with Trypanoplasma spp such as T. borreli. Currently, few or no in vitro studies have been performed to unravel the swimming behaviour and host-pathogen interaction of Trypanoplasma species. For both trypanosomes and trypanoplasma, in vivo studies to visualise the parasite motility and host immune response have not been reported so far.     In this thesis we describe for the first time blood flagellate infections in vivo in the natural environment of a vertebrate host (zebrafish). We did this by studying the parasite motility in vitro and in vivo and the kinetics of innate immune responses in vivo. The T. carassii and T. borreli zebrafish infection models are promising complementary models to existing (mammalian) animal models, and can contribute to fundamental mechanistic insights into host-parasite interactions

Investigation of conserved Flagellum proteins in Trypanosoma brucei

The single celled protozoan parasite Trypanosoma brucei is an excellent model organism to study eukaryotic cilia and flagella as it has a single flagellum that remains assembled throughout the cell cycle. The new flagellum assembles in a known position relative to the old flagellum, therefore creating a model system of identifiable organelle generations. In additional to a sequenced genome, there are many reverse genetics tools developed for T. brucei which makes the functional analysis of proteins possible. More than 300 proteins have been identified as components of the T. brucei flagellum but functional analysis of the majority of these proteins has not been carried out to date. This project used a bioinformatics approach to identify potential flagellum proteins in T. brucei that were also conserved in Homo sapiens, thereby identifying potential ciliopathy candidates. Candidate proteins were confirmed as flagellum components through endogenous localisation techniques and co-localisation studies. Functional analysis was performed using inducible RNAi cell lines. Light and electron microscopy techniques were used for phenotypic analysis. Through bioinformatics analysis a novel family of coiled-coil TPH domain-containing proteins were identified that are highly conserved in flagellated eukaryotes. There are three TPH domain-containing proteins conserved in T. brucei that all have a role in flagellum length control and cell morphogenesis. In all three cases protein ablation has a detrimental effect on cellular motility. This work provides further understanding into the complexities of flagellum biogenesis in T. brucei and the downstream effects on cell motility and morphogenesis

Visualizing trypanosomes in a vertebrate host reveals novel swimming behaviours, adaptations and attachment mechanisms.

Trypanosomes are important disease agents of humans, livestock and cold-blooded species, including fish. The cellular morphology of trypanosomes is central to their motility, adaptation to the host's environments and pathogenesis. However, visualizing the behaviour of trypanosomes resident in a live vertebrate host has remained unexplored. In this study, we describe an infection model of zebrafish (Danio rerio) with Trypanosoma carassii. By combining high spatio-temporal resolution microscopy with the transparency of live zebrafish, we describe in detail the swimming behaviour of trypanosomes in blood and tissues of a vertebrate host. Besides the conventional tumbling and directional swimming, T. carassii can change direction through a 'whip-like' motion or by swimming backward. Further, the posterior end can act as an anchoring site in vivo. To our knowledge, this is the first report of a vertebrate infection model that allows detailed imaging of trypanosome swimming behaviour in vivo in a natural host environment

Functional characterization of Mucin-Associated Surface Protein (MASP) in the human parasite Trypanosoma cruzi

MASPs are members of a multigenic family recently identified during the sequencing of the T. cruzi CL Brener genome. This family contains around 1,400 members, consisting of approximately 6% of the whole coding genes. Highly conserved N- and C-terminal domains, which encode a signal peptide and GPI-anchor addition site respectively, and a hypervariable central region, characterize MASPs. Members of this family are predominantly expressed in the infective trypomastigote form. We hypothesized that members of the T. cruzi MASP protein family play a major role in the interaction of the parasite with the host cell. In order to investigate a putative role for T. cruzi MASP at the host-pathogen interface, we have used MASP as a bait protein against the human proteome using a high-throughput platform that we have recently established for identifying protein-protein interactions between pathogens and theirs hosts. Yeast two-hybrid screens identified human SNAPIN as one of two major MASP interacting proteins. SNAPIN is a member of the SNARE protein complex, which may have a role in a calcium-dependent exocytosis. The MASP-SNAPIN interaction was further validated using in vivo co-Affinity Purification and in vitro pull-down assays. Immunofluorescence assays showed human SNAPIN is recruited to the parasite surface during invasion. Co-localization experiments indicated that SNAPIN is associated with the late endosomes and lysosomes. Supporting our initial hypothesis, SNAPIN depletion using siRNA oligomers in HeLa cells and snapin-/- in Mouse Embryonic Fibroblast (MEF) cells significantly inhibited T. cruzi invasion, suggesting a role for SNAPIN in this process. Lysosomes in snapin-/- MEF cells displayed aberrant morphology and distribution and the parasites did not recruit host lysosomes efficiently when compared to wild-type cells. This was likely due to an impaired calcium-dependent lysosome exocytosis in snapin-/- MEF cells. SNAPIN was translocated to the plasma membrane upon calcium influx induced by a calcium ionophore (Ionomycin), resulting in the exposure of the luminal domain of SNAPIN to the extracelluar space. Leishmania tarentolae transgenic strains expressing two different MASP proteins were shown to trigger intracellular calcium transients in HeLa cells, presumably by injuring the cell membrane. We propose that T. cruzi MASP plays a role in wounding the plasma membrane of the host cell, which in turn elicits a transient intracellular calcium flux and leads to the translocation of lysosome-associated SNAPIN to the plasma membrane. Human SNAPIN, through its exposed luminal domain would then provide an anchor for the entry to the parasite into the cell. The mechanism of T. cruzi MASP evoked calcium influx in the host cell membrane remains under investigation

Design, Mathematical Modelling, Construction and Testing of Synthetic Gene Network Oscillators to Establish Roseobacter Clade Bacteria and the Protozoan Trypanosoma brucei as Synthetic Biology Chassis.

The aim of this project is to establish Roseobacter marine bacteria and Trypanosoma brucei (T. brucei) protozoa as synthetic biology chassis. This work addresses the gap within synthetic biology resulting from the limited choice of host cells available for use in practice. This was done by developing synthetic bacterial and trypanosomal genetic regulatory networks (GRNs) which function as an oscillator as well as by developing the necessary protocols and set-ups to allow for the analysis of GRN dynamics within the host. Roseobacter clade bacteria are naturally found in diverse oceanic habitats and have an important ecological role in balancing global carbon levels. This makes Roseobacter an ideal chassis for future efforts to apply synthetic biology to bioremediation and geo-engineering challenges. The aim of this investigation was to establish straight-forward molecular biology procedures in Roseobacter bacteria followed by characterisation and modelling of an E. coli oscillator in Roseobacter. Results showed that Roseobacter synthetic biology is non-trivial. Protozoa are exploited as host cells for industrial production of biotherapeutics due to fast doubling times and host proteins’ mammalian-like post-translational glycosylation. As an established model organism for studying protozoa, T. brucei provided a test case for establishing synthetic biology in this phylum for the first time. T. brucei is highly divergent from eukaryotes commonly used in synthetic biology and possesses a sophisticated genomic machinery to evade host immune systems. The establishment of standard synthetic biology approaches in mathematical modelling and gene network design in T. brucei will underpin application of synthetic biology to enhance the industrial capability of the protozoa as a chassis and to probe its pathobiology. This investigation involved design and assembly of a Goodwin oscillator, followed by characterisation and modelling of the network and the development of a novel experimental set-up for live-cell imaging of single motile trypanosomes. Results showed that T. brucei is a promising novel synthetic biology chassi

Investigation of the early immune events in African trypanosome infections

African trypanosomes, the causative agent of sleeping sickness in humans, and nagana in cattle, are typically transmitted by the bite of an infected tsetse fly. The nature of the mammalian innate immune response during and immediately after the bite of an infected tsetse fly remains poorly understood. Previous studies characterising the events occurring in the skin post-infected tsetse fly bite have mainly focussed on the development of the chancre, which occurs from day 5 post-infection. Additionally, most immunopathological studies on trypanosomes have used intravenous or intraperitoneal injections of blood stage parasites, therefore bypassing relevant inoculation routes (tsetse fly), site (skin), and parasite life cycle stages (metacyclics). It is known that following tsetse fly bites, trypanosomes leave the skin via the host lymphatic system in order to initiate a blood stage infection. However, how the host responds to this challenge and how the parasite negotiates the anatomy of the host immune system remains unclear. In the present study, I have built on existing intravital microscopy tools to visualise T. b. brucei infections in the dermis and lymphatics of an infected mouse ear after transmission. I have also characterised by flow cytometry, taqman low density arrays and depletion studies the magnitude and kinetics of the early innate immune response in the skin, as well as the functional role of neutrophils, by examining infections in the context of the natural route of infection- the bite of a tsetse fly. Neutrophils were identified to be the predominant responders at the bite site, the neutrophil response was rapid, and they were recruited independent of the infection status of the tsetse flies. Taqman low-density arrays, which measured expression levels of inflammation-associated genes, suggested that neutrophil recruitment was mediated by CXCL1/CXCL2 release in the skin following mechanical trauma by the tsetse fly, in addition to the release of pro-inflammatory cytokines- IL-1β and IL-6. Following the identification of neutrophils by flow cytometry, I then applied intravital microscopy to visualise influx of neutrophils, which was rapid, directed at the bite site, and did not form dynamic clusters. To further test the functional role of neutrophils very early in infection, neutrophils were depleted using a monoclonal antibody and mice infected via tsetse fly bites. Neutrophil depleted mice had no effect on pathogenesis in vivo. Using Prox-1 mOrange reporter mice, I also examined the interaction of bloodstream trypanosomes with lymphatic vessels in the skin in the period immediately following inoculation using intravital imaging. I imaged metacyclic trypanosomes in situ and demonstrated that they had significantly higher velocity in the extravascular matrix compared to bloodstream forms. Additionally, my data showed bloodstream parasites actively migrating towards lymphatic vessels, and intra- lymphatic T. b. brucei were also observed, enabling comparison of trypanosome motility in the extravascular matrix and lymphatic vessels; in lymph vessels trypanosomes were moving in a more directional and rapid manner. This work revealed the early cellular and molecular responses to T. b. brucei infection and investigated interactions of parasites with the anatomy and cells of the host immune system. These studies demonstrate that furthering our understanding of the very early events in trypanosome infections is essential to understand how a systemic trypanosome infection is established