Motility of flagellated bacteria has been a topic of increasing scientific interest over the past decades, attracting the attention of mathematicians, physicists, biologists and engineers alike. Bacteria and other micro-organisms cause substantial damage through biofilm growth on submerged interfaces in water cooling systems, ship hulls and medical implants. This gives social and economic motivations for learning about how micro-organisms swim and behave in different environments. Fluid flows on such small scales are dominated by viscosity and therefore behave differently from the inertia-dominated flows that we are more familiar with, making bacterial motility a physically intriguing phenomenon to study as well. We use the boundary element method (BEM) to simulate the motion of singly flagellated bacteria in a viscous, Newtonian fluid. One of our main objectives is to investigate the influence of external surfaces on swimming behaviour. We show that the precise shape of the cell body and flagellum can be important for determining boundary behaviour, in particular, whether bacteria are attracted or repelled from surfaces. Furthermore, we investigate the types of motion that may arise between two parallel plates and in rectangular channels of fluid and show how these relate to the plane boundary interactions. As an extension to original models of flagellar propulsion in bacteria that assume a rotation of the rigid helical flagellum about an axis fixed relative to the cell body, we consider flexibility of the bacterial hook connecting the aforementioned parts of the swimmer. This is motivated by evidence that the hook is much more flexible than the rest of the flagellum, which we therefore treat as a rigid structure. Elastic dynamics of the hook are modelled using the equations for a Kirchhoff rod. In some regimes, the dynamics are well described by a rigid hook model but we find the possibility of additional modes of behaviour.

There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.

Les cils et les flagelles sont des organites cellulaires très conservés qui assurent des fonctions essentielles. Chez l'Homme, les défauts d'assemblage des cils et des flagelles conduisent à de multiples pathologies, les ciliopathies. Afin de comprendre comment se forment et fonctionnent les cils, j'ai analysé la fonction de deux nouveaux gènes identifiés comme cible des facteurs de transcription de ciliogenèse RFX. Tout d'abord je me suis focalisée sur le gène CCDC151, évolutivement conservé dans les espèces possédant des cils motiles. J'ai pu montrer que CCDC151 est impliquée dans le transport dépendant de l'IFT des bras de dynéine chez les animaux et qu'elle est nécessaire à la perception sensorielle chez la drosophile. Par ailleurs, j'ai également montré que cette protéine possède des fonctions cellulaires additionnelles puisqu'elle est requise pour l'orientation correcte des plans de division cellulaire et qu'elle est impliquée dans la régulation de la taille du cil primaire chez les mammifères. Je me suis ensuite intéressée au gène LRRC48 également conservée dans les espèces possédant des cils motiles. Cette protéine est nécessaire à la motilité des flagelles de spermatozoïdes et des cils des neurones sensoriels en 9+0 et dans la réponse auditive chez la drosophile. De plus LRRC48 est indispensable au développement des vertébrés puisque son absence chez le poisson zèbre conduit à l'hydrocéphalie, des kystes rénaux et des défauts de motilité des cils. Elle est également essentielle à la biogenèse de l'oreille dans cet organisme.En conclusion, il s'agit de deux nouveaux acteurs de la ciliogenèse potentiellement impliqués dans les pathologies ciliaires chez l'Homme / Cilia are highly conserved structures found from protozoa to mammals where they play essential physiological and developmental functions and cilia dysfunction leads to various syndromes in humans known as ciliopathies. To understand cilia formation and function, I performed functional analysis of two new target genes of the RFX ciliogenic transcription factors. First, I focused on CCDC151 that is evolutionary conserved in motile ciliated species. I showed that CCDC151 is involved in the control of IFT-dependent dynein arm assembly in animals and required for geotaxis behavior of adult flies. In zebrafish, depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts, two phenotypes resulting from impaired ciliary beating. However, I also showed that CCDC151 is also implicated in other cellular functions in vertebrates as it is involved in proper orientation of cell divisions and implicated in the regulation of primary cilium length in mammalian cells. In a second part, I studied LRRC48 that is also conserved in species with motile cilia. I showed that this protein is essential for motility of flagellar spermatozoids and for motility of the 9+0 sensory cilia as well as in the auditory response in drosophila. In zebrafish, morpholinos induced depletion of this protein leads to hydrocephaly, kidney cysts, inner ear abnormalities and cilia motility defects. Moreover this protein is also required for inner ear biogenesis in the model. In conclusion, these two genes are essential for ciliogenesis and they are new candidate genes potentially implicated in human ciliary diseases

Bacterial motility, and in particular repulsion or attraction towards specific chemicals, has been a subject of investigation for over 100 years, resulting in detailed understanding of bacterial chemotaxis and the corresponding sensory network in many bacterial species including Escherichia coli. E. Coli swims by rotating a bundle of flagellar filaments, each powered by an individual rotary motor located in the cell membrane. When all motors rotate counter-clockwise (CCW), a stable bundle forms and propels the cell forward. When one or more motors switch to clock-wise (CW) rotation, their respective filaments fall out of the bundle, leading to the cell changing orientation. Upon switching back to CCW, the bundle reforms and propels the cell in a new direction. Chemotaxis is performed by the bacterium through prolonging runs by suppressing CW rotation when moving towards nutrients and facilitating reorientation by increasing CW bias when close to a source of a harmful substance. Chemicals are sensed through interaction with membrane bound chemosensors. These proteins can interact with a very specific set of chemicals and the concentrations they are able to sense are in the range between 10-⁶ and 10-² M. However, experiments have shown that the osmotic pressure exerted by large (> 10-¹ M) concentrations of solutes, which have no specificity for binding to chemosensors (e.g. sucrose), is able to send a signal down the chemotactic network. Additionally, clearing of bacterial density away from sources of high osmolarity has been previously observed in experiments with agar plates. This behaviour has been termed osmotaxis. The aim of this doctoral thesis work is to understand how different environmental cues influence the tactic response and ultimately, combine at the network output to direct bacterial swimming. As tactic responses to chemical stimuli have been extensively studied, I focus purely on the response to non-specific osmotic stimuli, using sucrose to elevate osmolarity. I monitor the chemotactic network output, the rotation of a single bacterial flagellar motor, using Back Focal Plane Interferometry over a variety of osmotic conditions. Additionally, in collaboration with Vincent Martinez, I studied the effect of elevated osmolality on swimming speed of large (104) bacterial populations, using differential dynamic microscopy (DDM). I have found that sudden increases in media osmolarity lead to changes of both motor speed and motor clockwise bias, which is the fraction of time it spends rotating clockwise. Changes in CW Bias proceed in two phases. Initially, after elevating the osmolarity, CW Bias drops to zero, indicating that the motor is exclusively in the ‘cell run’ mode. This phase lasts from 2-5 minutes depending on the magnitude of the change in solute concentration. What follows then is a distinct second phase where the CW Bias is elevated with respect to the initial levels and this phase lasts longer than 15-20 minutes. In comparison, for defined chemical stimuli, the motor output resets after several seconds, a behaviour termed perfect adaptation. For changes of 100 mOsm/kg and 200 mOsm/kg in magnitude the motors speed up, often by as much as a factor of two, before experiencing a gradual slow down. Despite the slow down, motors still rotate faster 15-20 minutes after the change in osmolarity, than they did before. For changes of 400 mOsm/Kg in magnitude the motors decrease sharply in speed, coming to a near halt, recovering after 5 minutes and eventually, on average, speeding up. DDM studies of free swimming bacteria have shown that elevated osmolality leads to higher swimming speeds, in agreement with single motor data. Using theoretical models of bacterial swimming from the literature, it is discussed how this motor output, although different to what is expected for chemotaxis, is able to drive bacteria away from regions of space with high osmolalities. Additionally, I have started extending the work done with sucrose, to another solute often used to elevate osmolality, sodium chloride. While sucrose is outer membrane impermeable, NaCl can cross the outer membrane into the periplasmic space. Another layer of complexity is that NaCl has some specificty for the chemoreceptors. The preliminary results are shown and qualitatively agree with those obtain with sucrose.

Près de 15% des couples sont confrontés à des problèmes d'infertilité. Dans près de la moitié des cas, une composante masculine est retrouvée, avec souvent une anomalie des paramètres du spermogramme montrant une diminution de la qualité du sperme. L'étiologie de la grande majorité des infertilités masculines reste inconnue et une origine génétique est probablement responsable d'une proportion importante des troubles de la spermatogénèse. Ce travail comporte deux parties: dans la 1ère partie, l'analyse d'une large cohorte de patients (n=87), nous a permis d'identifier deux nouvelles mutations du gène AURKC. La mutation [c.36-2A>G] a été identifiée uniquement à l'état hétérozygote chez deux frères et le 2ème variant identifié [p.Y248*]: est une mutation récurrente retrouvée chez 11 patients non apparenté d'origine maghrébine et européenne. La 2ème partie de notre étude a été réalisée sur 20 patients infertiles présentant un phénotype homogène d'anomalies flagellaire de type flagelles courts, absents et de calibre irrégulier associé a une asthénozoospermie. Nous avons appliqué la stratégie d'homozygotie par filiation qui a permis de mettre en évidence deux régions d'homozygoties communes: la 1ère région, située sur le chromosome 3, est commune à 9/20 patients et la 2ème sur le chromosome 20 commune à 13/20 patients. Trois gènes candidats présents dans ces régions ont été sélectionnés : les gènes KIF9, SPAG4 et DNAH1. Le séquençage du gène DNAH1 a permis de mettre en évidence des mutations de type faux-sens [c.3877G>A], run-on [c.12796 T>C] et d'épissage [c.5094+1G>A] [c.11958-1G>A]. L'absence de la protéine DNAH1 a pu être mise en évidence par immunomarquage sur les spermatozoïdes d'un patients porteur de la mutation [c.11958-1G>A] et confirme la dégradation du transcrit muté par NMD également observé. Les analyses par microscopie électronique sur les spermatozoïdes d'un patient de la cohorte ont permis de mettre en évidence des anomalies de la structure de l'axonème. Cette étude précise le diagnostic d'infertilité masculine et élargit les connaissances sur les gènes impliquées dans la spermatogenèse. / About 15% of couples are confronted with infertility problems. In half of the cases, a male factor component is found, often with abnormal semen parameters. The etiology of the large majority of male infertility remains unknown and genetic origin is probably responsible of a significant proportion of spermatogenesis disorders. This work comprises two parts: in the first part, the analysis of a large cohort of patients (n = 87), allowed us to identify two new mutations in AURKC gene. A splice site mutation [c.36-2A> G] was identified in only two brothers and the second variant identified [p.Y248*] is a recurrent mutation found in 11 unrelated patients. The second part of our study was carried out on 20 infertile patients with flagellar abnormalities associated with asthenozoospermia. We have applied the strategy of homozygosity by descent who has bring out two regions of homozygosity: the first region, located on chromosome 3, is common for 9/20 patients and the second one, located on chromosome 20, is common for 13/20 patients. Three candidate genes present in these regions were selected: KIF9, SPAG4 and DNAH1. Sequencing of DNAH1 gene has bring out three type of mutations: missense mutation [c.3877G> A], run-on mutation [c.12796 T> C] and splice site mutation [c.5094 +1 G> A] [c.11958-1G> A]. The absence of dnah1 protein has been shown by immunostaining of spermatozoa of a patient carrier the mutation [c.11958-1G> A] and confirms the degradation of the mutated transcript by NMD. An electron microscopic analysis of spermatozoa of one patient of the cohort reveals axoneme abnormalities. This study clarifies the diagnosis of male infertility and broadens the knowledge of the genes involved in spermatogenesis.

Le parasite Trypanosoma brucei est retrouvé en Afrique sub-Saharienne et est responsable de la maladie du sommeil chez l’homme et de la Nagana chez les animaux. Il cause de graves problèmes sanitaires et économiques car il affecte le bétail. La vaccination est impossible à cause de la variation antigénique. Les traitements actuels sont difficiles à mettre en place avec des effets secondaires importants. Il est donc urgent de trouver de nouvelles cibles thérapeutiques afin de développer de nouveaux médicaments. T. brucei possède un flagelle unique qui émerge de la cellule par une structure appelée la poche flagellaire (FP). Cette FP est une invagination de la membrane plasmique. Elle est nécessaire à la survie du parasite car c’est le seul site d’endo- et d’exocytose. Au cou de la FP on trouve le collier de la poche flagellaire (FPC) en forme d’anneau. Le FPC est composé de nombreuses protéines dont BILBO1 qui est nécessaire à la biogenèse de la FP et du FPC. De nombreux partenaires de BILBO1 ont été identifiés. Dans cette thèse, je caractérise deux d’entre eux : FPC5, une kinésine putative et FPC9, une synaptotagmine putative. J’ai pu montrer que FPC5 est localisée aux corps basaux mais aussi au FPC. Cette protéine n’est pas essentielle à la survie des parasites bien que des phénotypes de croissance et de ségrégation de la FP apparaissent après induction de l’ARNi. Nous ne sommes pas parvenus à prouver sa fonctionnalité, cependant j’ai pu montrer que son domaine moteur est capable de lier les microtubules. FPC9 est trouvée au niveau de la zone de transition du flagelle. L’ARNi contre cette protéine n’étant pas effectif, nous ne pouvons pas conclure quant à sa fonction dans la cellule. / Trypanosoma brucei is a parasite found in sub-Saharan Africa and is responsible for sleeping sickness in humans and Nagana in animals. It is the source of serious health and economic problems because it kills livestock. Vaccination is not possible because of antigenic variation and current treatments are difficult to implement or have toxic side effects. For these reasons it is urgent to find new therapeutic targets in order to develop effective treatments. T. brucei has a single copy flagellum that emerges from the cytoplasm through a unique structure called the Flagellar Pocket (FP). This pocket is an invagination of the pellicular membrane and because it is the sole site of endo- and exocytosis, it is essential for parasite survival. At the neck of the FP there is a cytoskeletal structure: the Flagellar Pocket Collar (FPC) that forms a “ring” around the flagellum. The FPC consists of numerous proteins, including the first to be identified - BILBO1, which is necessary for FP and FPC biogenesis. A number of potential BILBO1 partners were identified. In this thesis I characterize two of these proteins: FPC5, a putative kinesin and FPC9, a putative synaptotagmin. I show that FPC5 localizes mainly in the basal body area, but also at the FPC. This protein is not essential for parasite survival although reduced FP segregation and growth phenotypes appear after RNAi induction. We are not able to prove its functionality, however I could show its motor domain is able to bind microtubules. FPC9 is found in the transition zone of the flagellum. However RNAi knockdown against this protein was not efficient, so we are currently unable to define a function for this protein.

The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer. While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host. This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa. Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided. Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23. Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration. / Doctor of Philosophy / Bacteria are small autonomous single-celled organisms capable of existing and thriving in highly diverse environments. Motility is achieved by these organisms in various ways, but the most common approach is to produce one or more corkscrew-shaped propeller systems known as flagella that are constructed upon and anchored within the wall of the bacterial cell. Rotation of these propellers relies on power converters known as stators to transform the flow of ions down self-produced gradients into useful rotational energy. This process can be likened to the way that the stored energy of water behind a dam can be harnessed and used to power hydroelectric generators. While the core components of flagellar motors are well conserved and understood among distantly related bacteria, billions of years of evolution and refinement of additional structures have allowed bacteria to accommodate swimming in diverse habitats with e.g. low nutrient availability or high viscosity. Here we describe the discovery and characterization of additional components in the flagellar motor system of the soil-dwelling bacterium Sinorhizobium meliloti to navigate soil environments. We report the first identification of a flagellar motor that requires two copies of a pervasive flagellar motor protein known as FliL and have named the more distinct version of the protein MotF. We found that FliL is required for the power converter components to install into the motor and that MotF is necessary to activate them. Next, we identify another motor component, Orf23, that is dispensible for motility but appears to be required to achieve maximum swimming velocity and may serve to shift the motor into a "higher gear". We find that disruption of a regulator of cell wall modification systems leads to defects in motility that are only restored when Orf23 is removed or when the power converter is modified. Ideas are proposed for how FliL, MotF, and Orf23 are integrated into the motor and may contribute to stator function. An advanced understanding of the mechanisms governing flagellar motor structure and function will provide avenues for the improvement of bacteria-based agricultural improvements, development of optimized bacteria-mediated drug delivery systems, bioremediation techniques, and more.

Les parasites de la classe des Kinetoplastidae, comprenant notamment les trypanosomes et les leishmanies, sont responsables pour plusieurs maladies d’importance socio-économique et de santé publique. La maladie du sommeil, la maladie de Chagas et la leishmaniose, classées comme maladies tropicales négligées (NTD) par l’Organisation mondiale de la santé (OMS) et la Surra, reportée par l’Organisation pour l’alimentation et l’agriculture, des Nations Unies (FAO). La Trypanosomiase Animale Africain sub-saharienne entraîne la mort de 3 millions bovins par an accompagné d'une perte annuelle de l'économie de 4,5 milliards de dollars américains. La leishmaniose cutanée, une maladie zoonose, présente 1,5 millions de nouveaux cas chaque année.Trypanosoma brucei (T. brucei) est un ancien eucaryote, utilisé comme organisme modèle dans le laboratoire pour l’étude des cils et des flagelles. Le remodelage du cytosquelette des trypanosomes est essentiel pour la morphologie cellulaire, le positionnement et la division des organites. L’étude des protéines essentielles du cytosquelette permet de mieux comprendre les processus cellulaires. Ces protéines pourraient également constituer des cibles potentielles pour des traitements thérapeutiques. Les trypanosomes échappent au système immunitaire de l’hôte en modifiant périodiquement les antigènes de présent à leur surface. En effet ces antigènes de surface sont endocytés, ainsi que les anticorps de l’hôte qui y sont attachés, au niveau d’une structure appelée la poche flagellaire (FP). TbBILBO1 est une protéine structurelle du collier de la poche flagellaire (FPC), essentielle à la biogenèse du FPC et à la survie du parasite. En raison du rôle majeur de la protéine TbBILBO1 dans le parasite, des partenaires de TbBILBO1 ont été recherchés.Dans ce travail, j’ai pu caractériser une nouvelle protéine essentielle du cytoskelette, la protéine FPC6, partenaire de TbBILBO1, qui se situe au niveau du complexe FPC/Complexe du Hook de T. brucei. L’ARN interférence de FPC6 conduit à une mort rapide des formes sanguines des trypanosomes, accompagnée d’un blocage de l’endocytose. Ensuite, j’ai produit un nanobody (Nb48), dirigé contre TbBILBO1, dans le système d’expression bactérien. Je l’ai également exprimé dans les lignées de trypanosomes. Le Nb48 reconnait TbBILBO1 sur les trypanosomes fixés par immunofluorescence et dans les extraits totaux de protéines dénaturées. L’analyse par résonance plasmonique de surface (SPR) a confirmé une haute affinité du Nb48 pour TbBILBO1. L’expression de Nb48 dans le parasite T. brucei en tant qu’intrabody demontrant que ce nanobody pouvait être exprimé de manière fonctionnelle, capable de reconnaitre spécifiquement sa cible protéique, TbBILBO1, intra-cellulaire et de bloquer sa fonction conduit à un effet trypanocide rapide. Ces études ouvrant ainsi la voie pour de nouvelles utilisations potentielles thérapeutiques dans le traitement des trypanosomiases. / Kinetoplastid parasites, including trypanosomes and leishmania, are responsible for several diseases of socio-economic and public health importance worldwide. These include the Neglected Tropical Diseases: Sleeping Sickness, Chagas disease and Leishmaniasis, as classified by the World Health Organisation (WHO) and the global wasting disease of animals, Surra, as reported by the Food and Agricultural Organisation of the United Nations (FAO). Animal African Trypanosomiais (AAT) causes the death of 3 million cattle per year in sub-Saharan Africa, with an annual loss of 4.5 billion US dollars to the African economy. Cutaneaous leishmaniasis is a zoonotic disease, with 1.5 million new cases reported globally each year.Trypanosoma brucei is an ancient, early diverging eukaryote, used as a model organism in the laboratory for studying eukaryotic cilia and flagella. Remodelling of the trypanosome cytoskeleton is essential for cell morphology, organelle positioning and division. Study of essential proteins of the cytoskeleton provides insight into intracellular processes and could provide potential targets for therapeutic interventions. Trypanosomes evade the host immune system by periodically changing their external surface coat, which is endocytosed, along with any attached host antibodies, via a structure called the flagellar pocket. TbBILBO1 is a structural protein of the Flagellar Pocket Collar (FPC) that is essential for FPC biogenesis and parasite survival. Due to the importance of TbBILBO1 for the parasite, protein partners were investigated.In my thesis, I describe, firstly, the characterisation of a novel and essential cytoskeletal protein, FPC6, of the FPC/Hook complex of T. brucei; FPC6 is a partner of TbBILBO1. RNAi Knock-down of FPC6 protein leads to rapid cell death in the blood-stream form of the parasite accompanied with a block in endocytosis. Secondly, I describe the purification and intracellular expression of a nanobody (Nb48), raised against TbBILBO1. The purified Nb is able to identify TbBILBO1 in fixed trypanosomes and denatured protein. Surface Plasmon Resonance analysis confirmed a high affinity of Nb48 to TbBILBO1. Expression of Nb48 as an intrabody in T. brucei, reveals that it binds precisely to its target, TbBILBO1 and leads to rapid cell death. Further exploration of the potential uses of this trypanocidal nanobody is warranted.

In the presence of a solid-liquid or liquid-air interface, bacteria can choose between a planktonic and a sessile lifestyle. Depending on environmental conditions, cells swimming in close proximity to the interface can irreversibly attach to the surface and grow into three-dimensional aggregates where the majority of cells is sessile and embedded in an extracellular polymer matrix (biofilm). We used microfluidic tools and time lapse microscopy to perform experiments with the polarly flagellated soil bacterium Pseudomonas putida (P. putida), a bacterial species that is able to form biofilms. We analyzed individual trajectories of swimming cells, both in the bulk fluid and in close proximity to a glass-liquid interface. Additionally, surface related growth during the early phase of biofilm formation was investigated. In the bulk fluid, P.putida shows a typical bacterial swimming pattern of alternating periods of persistent displacement along a line (runs) and fast reorientation events (turns) and cells swim with an average speed around 24 micrometer per second. We found that the distribution of turning angles is bimodal with a dominating peak around 180 degrees. In approximately six out of ten turning events, the cell reverses its swimming direction. In addition, our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values of mean runtime and rotational diffusion, we presented a model to describe the spreading of a population of cells by a run-reverse random walker with alternating speeds. We successfully recover the mean square displacement and, by an extended version of the model, also the negative dip in the directional autocorrelation function as observed in the experiments. The analytical solution of the model demonstrates that alternating speeds enhance a cells ability to explore its environment as compared to a bacterium moving at a constant intermediate speed. As compared to the bulk fluid, for cells swimming near a solid boundary we observed an increase in swimming speed at distances below d= 5 micrometer and an increase in average angular velocity at distances below d= 4 micrometer. While the average speed was maximal with an increase around 15% at a distance of d= 3 micrometer, the angular velocity was highest in closest proximity to the boundary at d=1 micrometer with an increase around 90% as compared to the bulk fluid. To investigate the swimming behavior in a confinement between two solid boundaries, we developed an experimental setup to acquire three-dimensional trajectories using a piezo driven objective mount coupled to a high speed camera. Results on speed and angular velocity were consistent with motility statistics in the presence of a single boundary. Additionally, an analysis of the probability density revealed that a majority of cells accumulated near the upper and lower boundaries of the microchannel. The increase in angular velocity is consistent with previous studies, where bacteria near a solid boundary were shown to swim on circular trajectories, an effect which can be attributed to a wall induced torque. The increase in speed at a distance of several times the size of the cell body, however, cannot be explained by existing theories which either consider the drag increase on cell body and flagellum near a boundary (resistive force theory) or model the swimming microorganism by a multipole expansion to account for the flow field interaction between cell and boundary. An accumulation of swimming bacteria near solid boundaries has been observed in similar experiments. Our results confirm that collisions with the surface play an important role and hydrodynamic interactions alone cannot explain the steady-state accumulation of cells near the channel walls. Furthermore, we monitored the number growth of cells in the microchannel under medium rich conditions. We observed that, after a lag time, initially isolated cells at the surface started to grow by division into colonies of increasing size, while coexisting with a comparable smaller number of swimming cells. After 5:50 hours, we observed a sudden jump in the number of swimming cells, which was accompanied by a breakup of bigger clusters on the surface. After approximately 30 minutes where planktonic cells dominated in the microchannel, individual swimming cells reattached to the surface. We interpret this process as an emigration and recolonization event. A number of complementary experiments were performed to investigate the influence of collective effects or a depletion of the growth medium on the transition. Similar to earlier observations on another bacterium from the same family we found that the release of cells to the swimming phase is most likely the result of an individual adaption process, where syntheses of proteins for flagellar motility are upregulated after a number of division cycles at the surface. / Bakterien sind einzellige Mikroorganismen, die sich in flüssigem Medium mit Hilfe von rotierenden Flagellen, länglichen Fasern aus Proteinen, schwimmend fortbewegen. In Gegenwart einer Grenzfläche und unter günstigen Umweltbedingungen siedeln sich Bakterien an der Oberfläche an und gehen in eine sesshafte Wachstumsphase über. Die Wachstumsphase an der Oberfläche ist gekennzeichnet durch das Absondern von klebrigen, nährstoffreichen extrazellulären Substanzen, welche die Verbindung der Bakterien untereinander und mit der Oberfläche verstärken. Die entstehenden Aggregate aus extrazellulärer Matrix und Bakterien werden als Biofilm bezeichnet. In der vorliegenden Arbeit untersuchten wir ein Bodenbakterium, Pseudomonas putida (P. putida), welches in wässriger Umgebung an festen Oberflächen Biofilme ausbildet. Wir benutzten photolithographisch hergestellte Mikrokanäle und Hochgeschwindigkeits-Videomikroskopie um die Bewegung schwimmender Zellen in verschiedenen Abständen zu einer Glasoberfläche aufzunehmen. Zusätzlich wurden Daten über das parallel stattfindende Wachstum der sesshaften Zellen an der Oberfläche aufgezeichnet. Die Analyse von Trajektorien frei schwimmender Zellen zeigte, dass sich Liniensegmente, entlang derer sich die Zellen in eine konstante Richtung bewegen, mit scharfen Kehrtwendungen mit einem Winkel von 180 Grad abwechseln. Dabei änderte sich die Schwimmgeschwindigket von einem zum nächsten Segment im Mittel um einen Faktor von 2. Unsere experimentellen Daten waren die Grundlage für ein mathematisches Modell zur Beschreibung der Zellbewegung mit alternierender Geschwindigkeit. Die analytische Lösung des Modells zeigt elegant, dass eine Population von Bakterien, welche zwischen zwei Geschwindigkeiten wechseln, signifikant schneller expandiert als eine Referenzpopulation mit Bakterien konstanter Schwimmgeschwindkeit. Im Vergleich zu frei schwimmenden Bakterien beobachteten wir in der Nähe der Oberfläche eine um 15% erhöhte Schwimmgeschwindigkeit der Zellen und eine um 90 % erhöhte Winkel-geschwindigkeit. Außerdem wurde eine signifikant höhere Zelldichte in der Nähe der Grenzfläche gemessen. Während sich der Anstieg in der Winkelgeschwindigkeit durch ein Drehmoment erklären lässt, welches in Oberflächennähe auf den rotierenden Zellkörper und die rotierenden Flagellen wirkt, kann die Beschleunigung und Akkumulation der Zellen bei dem beobachteten Abstand nicht durch existierende Theorien erklärt werden. Unsere Ergebnisse lassen vermuten, dass neben hydrodynamischen Effekten auch Kollisionen mit der Oberfläche eine wichtige Rolle spielen und sich die Rotationsgeschwindigkeit der Flagellenmotoren in der Nähe einer festen Oberfläche grundsätzlich verändert. Unsere Experimente zum Zellwachstum an Oberflächen zeigten, dass sich etwa sechs Stunden nach Beginn des Experiments größere Kolonien an der Kanaloberfläche auflösen und Zellen für ca. 30 Minuten zurück in die schwimmende Phase wechseln. Ergebnisse von mehreren Vergleichsexperimenten deuten darauf hin, dass dieser Übergang nach einer festen Anzahl von Zellteilungen an der Oberfläche erfolgt und nicht durch den Verbrauch des Wachstumsmediums bedingt wird.

Trypanosoma brucei, un parasite flagellé unicellulaire, est responsable de la trypanosomiase humaine africaine aussi connue comme la maladie du sommeil.Les microtubules (MTs) sous-pelliculaires, le quartet de MTs (MTQ), le flagelle (F) et le collier de la poche flagellaire (CPF) sont les principaux composants du cytosquelette dutrypanosome. À ce jour, une seule protéine du CPF, BILBO1, a été identifiée et caractérisée.Dans cette étude, nous montrons in vivo que BILBO1 forme des polymères capables deconstruire un échafaudage qui permet l’ancrage de protéines partenaires. Ainsi, un crible en double hybride chez la levure a identifié plusieurs protéines partenaires de BILBO1,notamment une nouvelle protéine appelée FPC4. Nous démontrons que FPC4 est une protéine spécifique des kinétoplastides, localisée au CPF mais aussi au hook-complex, une structure proche du CPF. L’interaction FPC4 – BILBO1 est démontrée in vitro et in vivo, etles domaines d'interaction identifiés. En outre, nous démontrons in vivo et in vitro que FPC4est une protéine associée aux microtubules. Nos données suggèrent fortement que FPC4est impliquée dans le processus de séparation des CPFs au cours du cycle cellulaire. Nos résultats mettent en évidence un lien étroit entre le MtQ et le CPF et l'implication probable duhook-complex. Enfin, nous mettons en évidence une structure analogue au hook-complex chez les Leishmanies. L’interaction BILBO1 – FPC4 représente une nouvelle cible thérapeutique et sera caractérisée plus avant. / Trypanosoma brucei, a unicellular flagellated parasite, is responsible for the human African trypanosomiasis also known as sleeping sickness. Sub-pellicular microtubules (MT), the MT quartet (MtQ), the flagellum (F) and the Flagellar Pocket Collar (FPC) are the main components of the T. brucei cytoskeleton. To date, only a single FPC protein, BILBO1, has been identified and characterized. In this study we demonstrate in vivo that BILBO1 forms polymers able to build a scaffold structure that anchors partner proteins. As such, a yeast-2-hybrid screen identified several BILBO1 interacting protein partners. We demonstrate that FPC4 is a kinetoplastid-specific protein, which is localized at the FPC and at the hook complex. Its specific interaction with BILBO1 has been demonstrated in vitro and in vivo, and the interacting domains identified. Furthermore, we demonstrate that FPC4 is a microtubule binding protein. Our data strongly suggest that FPC4 is involved in the separation of the old and the newly formed FPC during the cell cycle. Altogether, our results demonstrate a tight connection and interplay between the MtQ and the FPC and the likely involvement of an adjacent third structure, the hook complex. Finally, we highlight a structure similar to the hook-complex in Leishmania. The BILBO1 – FPC4 interaction represents a new therapeutic target and will be characterized further.