Tesis sobre el tema "Flagellar motility"

Three behavioural mutants of A. tumefaciens C58C1 (mot-l, mot-12 and fla-15) generated by transposon (Tn5) mutagenesis were studied. Analysis was initially at the molecular level, as a cosmid, pDUB1900, from a representative genomic library of C58C1 had been isolated that complemented the mutants. A region of 8624 nucleotides to which the Tn5 insertion sites of the three mutants had been mapped was sequenced completely in both directions. The comparison of this sequence with sequence databases and other computer analyses revealed six flagellar gene homologues (flgI,flgH,fliP,flaA,flaB,flaC), three open reading frames (ORFA, B and C) with no significant sequence identity to any open reading frames in the databases and the partial sequence of the flagellar gene homologue flgG. Computer analysis also showed that theflgH,flgI andfliP homologues, and ORFs A, B and C, could form the downstream region of a larger operon involved in chemotactic and motility functions. However putative transcription signals were also found within the operon. A new mutant (MANl) was created in the last gene (fliP) of the putative operon to investigate the function of possible transcription signals in the open reading frame immediately upstream of it (ORFC). The mot-12 mutant phenotype of fully synthesised but paralysed flagella is brought about by the insertion of Tn5 in ORFC. ORFC contains a possible promoter for fliP. The Tn5 insertion in ORFC should have polar effects upon the expression of fliP, unless the putative promoter can cause expression of fliP. The MANl mutant had a flagella-less phenotype. FliP in other bacteria is required early in the synthesis of flagella and the null phenotype is/7a-. Thus for flagella to be present in mot-12 suggests fliP must have a promoter. The ORFC sequence is highly conserved in R. meliloti and the overall regulation of these flagellar gene homologues may be as an operon with other regulatory signals. Evidence from other operons (including motility operons) with multiple transcription signals is discussed. The flaABC homologues were multiple copies of the gene encoding the flagellin protein of the flagellum. The mot-l phenotype of severely truncated filaments was caused by a Tn5 insertion in flaA. Analysis of the sequence showed flaABC to each have transcription signals that could lead to separate transcription. Transcription analysis by Northern blotting showed flaA to be transcribed monocistronically. Flagella were isolated from A. twnefaciens and the flagellins separated by SDS-PAGE. The migrated distances (relative to those of markers) was not as predicted from the nucleotide sequence. This anomaly could be caused by unequivalent binding of SDS or post-translational modification of FlaA. The A. tumefaciens flagellar genes were most similar to those of R. meliloti. However A. tumefaciens flagella do not exhibit the characteristic cross-hatching of the complex flagella of R. meliloti. This study also showed A. tumefaciens flagella not to be dependent on divalent cations for subunit associations unlike R. meliloti. These properties of A. tumefaciens flagella were similar to those of R. leguminosarum.The open reading frames found were isolated, radiolabelled and used as probesagainst Southern blots containing chromosomal DNA from a variety of soil bacteria, and cosmids known to contain motility genes in R. meliloti. Hybridisation revealed homologous DNA sequences in a number of these bacteria. All the A. tumefaciens open reading frames hybridised to homologous DNA in R. meliloti and are found in the same order in both species. This suggests that there are similarities at the molecular level in motility and chemotaxis functions between R. meliloti and A. tumefaciens as well as in the patterns of chemotaxis and motility observed previously.

The aim of our investigation was to gain insight on the regulation of flagellar movement, at the axonemal level. In our laboratory a panel of monoclonal antibodies (MoAbs) has been produced against the axoneme of the biflagellated algae, Chlamydomonas reinhardtii, a well-characterized model for the study of flagellar movement. Of these MoAbs, L2H12 has been selected, because it has a potent inhibitory effect on the motility of de membranated-reactivated flagella of Chlamydomonas cells. Using video micrography, we demonstrated that low concentrations of L2H12 cause a progressive decrease in the wave amplitude and beat frequency of the flagella. Results of Western blotting of the axonemal proteins indicates that L2H12 recognizes a 105 kDa protein. Analysis of Chlamydomonas radial spoke mutants deficient in one or more radial spoke proteins (RSPs) suggests that this protein is RSP2. Immunoprecipitation of this protein was performed to further characterize it.

Salmonella enterica is considered zoonotic pathogen with capability to colonies on range of plants and animals allowing transmission between them. Whole genome sequence analysis of S. enterica generates a phylogenetic tree comprising of three clades: A1, A2 and B. These 3 clades encompass the known 2,600 serovars used to type S. enterica during clinical outbreaks of salmonellosis. S. enterica exploits the bacterial flagellum to be motile in liquid environments and over surfaces. The genetic regulation of flagellar assembly is an elegant and harmonious system driving assembly of the flagellum from the base upwards. We surveyed the response and changes to flagellar regulation in a cohort of S. enterica serovars. Our analysis encompassed examining phenotypic motility, flagellar gene expression and flagellar abundance depending on nutrient composition. We demonstrated that the timing of flagellar gene expression is consistent across the species but the magnitude of flagellar gene expression varies significantly. The S. enterica flagellar system is bistable, producing a heterogeneous population of motile cells. Our data suggested that population heterogeneity plays a role in the adaptation of S. enterica serovars with respect to motility. The great similarity of the flagellum systems between S.enterica and E.coli gave us a reason to study why flagellar regulation in S.enterica differed from E. coli. Indeed, we replaced the master flagellar regulators, flhDC from E.coli into the S. enterica. We found a significant variation in FlhD4C2 activity through mixing flhD and flhC between both organisms. In conclusion, the diversity and changes we observe in just a small subset of S. enterica serovars and by introducing flhDC homologues has made us reconsider a number of assumptions we make about the regulation of the flagellar system based on model-domesticated strains of S. enterica.

Three different models of motile systems are studied: a vibrating legged robot, a snake-like locomotor, and two kinds of agellar microswimmers. The vibrating robot crawls by modulating the friction with the substrate. This also leads to the ability to switch direction of motion by varying the vibration frequency. A detailed account of this phenomenon is given through a fully analytical treatment of the model. The analysis delivers formulas for the average velocity of the robot and for the frequency at which the direction switch takes place. A quantitative description of the mechanism for the friction modulation underlying the motility of the robot is also provided. Snake-like locomotion is studied through a system consisting of a planar, internally actuated, elastic rod. The rod is constrained to slide longitudinally without slipping laterally. This setting is inspired by undulatory locomotion of snakes, where frictional resistance is typically larger in the lateral direction than in the longitudinal one. The presence of constraints leads to non-standard boundary conditions, that lead to the possibility to close and solve uniquely the equations of motion. Explicit formulas are derived, which highlight the connection between observed trajectories, internal actuation, and forces exchanged with the environment. The two swimmer models (one actuated externally and the other internally) provide an example of propulsion at low Reynolds number resulting from the periodical beating of a passive elastic filament. Motions produced by generic periodic actuations are studied within the regime of small compliance of the filament. The analysis shows that variations in the velocity of beating can generate different swimming trajectories. Motion control through modulations of the actuation velocity is discussed

Thesis (Ph. D.)--University of California, San Diego, 2005.
Title from PDF title page (viewed October 21, 2005) Vita. Includes bibliographical references. Available online via ProQuest Digital Dissertations.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007.
Title from dissertation home page (viewed Sept. 29, 2008). Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0818. Adviser: Clay Fuqua.

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

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.

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

The fitness of any evolutionary unit can be understood in terms of its two basic components: fecundity and viability. The trade-offs between these fitness components drive the evolution of a variety of life-history traits in extant multicellular lineages. Here, I show evidence that the evolution of germ-soma separation and the emergence of individuality at a higher level during the unicellular-multicellular transition are also consequences of these trade-offs. The transition from unicellular to larger multicellular organisms has benefits, costs, and requirements. I argue that germ-soma separation evolved as a means to counteract the increasing costs and requirements of larger multicellular colonies. Volvocalean green algae are uniquely suited for studying this transition since they range from unicells to undifferentiated colonies, to multicellular individuals with complete germ-soma separation. In these flagellated organisms, the increase in cell specialization observed as colony size increases can be explained in terms of increased requirements for self-propulsion and to avoid sinking. The collective flagellar beating also serves to enhance molecular transport of nutrients and wastes. Standard hydrodynamic measurements and concepts are used to analyze motility (self-propulsion) and its consequences for different degrees of cell specialization in the Volvocales as colony size increases. This approach is used to calculate the physical hydrodynamic limits on motility to the spheroid colony design. To test the importance of collective flagellar beating on nutrient uptake, the effect of advective dynamics on the productivity of large colonies is quantified. I conclude first, that when colony size exceeds a threshold, a specialized and sterile soma must evolve, and the somatic to reproductive cell ratio must increase as colony size increases to keep colonies buoyant and motile. Second, larger colonies have higher motility capabilities with increased germ-soma specialization due to an enhancement of colony design. Third, advection has a significant effect on the productivity of large colonies. And fourth, there are clear trade-offs between investing in reproduction, increasing colony size (i.e. colony radius), and motility. This work shows that the evolution of cell specialization is the expected outcome of reducing the cost of reproduction in order to realize the benefits associated with increasing size.

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.

Chromera velia was discovered a decade ago associated with stony corals in the Great Barrier Reef and Sydney Harbour, Australia. This unicellular alveolate possesses housekeeping genes and ultrastructural features typical of apicomplexans, and has active photosynthetic pathways related to dinoflagellates. This unique connection suggests that C. velia can be used to understand the evolution of apicomplexan parasites from their algal ancestors. C. velia exists in either an immotile coccoid state or an active motile flagellated form. The life cycle is not well understood and little is known about what triggers the transformation between states, and how this translates to ecological conditions. With its structural similarity to both apicomplexans and dinoflagellates, the flagellated form of C. velia represents a relatively unexplored part of the life cycle that is critical for understanding the ecological significance of this novel organism. The presence of a flagellated state may enhance competitive fitness of the organism under certain environmental conditions. This thesis investigated the flagellated form of C. velia and explored environmental factors that induce or suppress flagellation. Optimised conditions for flagellation were then used to test the flagellated form as a screening platform to test anti-apicomplexan drugs. In addition, the ecology and biodiversity of C. velia was explored by screening freshly procured coral samples for new chromerid cultures. To explore the flagellated state of C. velia cells were cultured in a range of light, temperature and physical and chemical stress conditions. Light wavelength had a significant effect on flagellation, with different spectra causing the induction or suppression of flagellation. The highest levels of flagellation were seen when C. velia was cultured in blue light or the dark. C. velia exhibited a temporal cycle of motility over 24 hours similar to that of the coral symbiont Symbiodinium, which exhibits motility rhythms in a light-dark environmental cycle. However, in C. velia this pattern continued when the cells were grown in constant dark, suggesting that the cycle is not completely light dependent. High cell density completely suppressed flagellation, and no flagellates were seen in cultures of 106 cells/mL. Non-optimal temperatures and a range of other stressors either killed cells or suppressed flagellation, but did not induce it. A negative correlation was found between light conditions inducing cell multiplication and flagellation, and this has been explored in a proposed new amendment to the life cycle model. Culturing techniques adapted by Moore (2006) were used to screen coral samples for novel chromerids to gain a better understanding of the biodiversity and ecological niche of C. velia. A new strain of C. velia was isolated from L. purpurea present in the Heron Island lagoon, and this additional strain was used in developing a multilocus sequence typing (MLST) system with eight genetic loci that was able to differentiate the three strains of C. velia that are now available. The three strains had considerable diversity at the genetic level, and attempts were made to distinguish them phenotypically by studying growth, flagellation and B vitamin synthesis. The three strains did not require the addition of B vitamins for growth, and possessed a typical algal B7 synthesis pathway, no synthesis or requirement for B12, and an unusual B1 synthesis pathway involving a mosaic of genes with closest homologs of bacterial and fungal origin. New insights on flagellation in C. velia were used to design a flagellation assay to screen compounds for apicomplexan drug development. The Malaria Box, a set of 400 compounds with anti-malarial activity, was provided by the Medicines for Malaria Venture and screened using this assay, finding a number of positive hits. These positive hits were used by structural chemist collaborators at SPECS to synthesise an additional 80 structurally similar compounds that were also screened. A high proportion of compounds found to inhibit flagellation in each assay belong to the diaminopyrimidine class of compounds that are known to affect the folate biosynthesis pathway. A bioinformatic study of C. velia folate synthesis revealed a pathway similar to that of Plasmodium, demonstrating that C. velia has biochemical pathways similar to the parasite and screening may pick up compounds that can be used as starting points for therapeutics. C. velia is closely related, phylogenetically speaking to apicomplexans and the flagellated state is the most structurally similar and also the active form, capable of sensing changes in the environment and moving the cell in response to these changes. To date most studies have only looked at the structure of this form and little has been done to explore its role in the life cycle of C. velia. This study has provided new insights into the ecology and biodiversity of C. velia by identifying triggers for flagellation, producing an amended life cycle model, identifying a new strain of C. velia and designing a set of molecular markers for future population studies. The findings were used to design a flagellation assay able to identify compounds that may be useful starting points for anti-apicomplexan therapeutics.

Trypanosoma brucei est un parasite africain unicellulaire, responsable chez l’homme de la maladie du sommeil et chez les bovins de la Nagana. Il passe par différents stades lors de son cycle de vie, les deux principaux étant la forme sanguicole qui prolifère dans le sang des mammifères infectés, et la forme procyclique qui colonise le tube digestif du vecteur, la mouche glossine.

Les trypanosomes sont extracellulaires, ils possèdent un flagelle qui leur permet de se mouvoir dans les différents milieux qu’ils infestent. La structure de celui-ci contient des éléments conservés au cours de l’évolution. Il constitue donc un excellent modèle de base pour en étudier l’architecture. D’autre part, le flagelle du parasite contient des structures propres à certains kinétoplastides, offrant ainsi une cible thérapeutique aux traitements anti-trypanosomiaux.

Le flagelle est véritablement un organite plurifonctionnel nécessaire à la survie du parasite au sein des divers environnements qu’il rencontre lors de son cycle de développement. Outre son rôle moteur, il permet à la cellule d’échapper au système immunitaire de son hôte mammifère et de s’attacher à l’épithélium des glandes salivaires de l’insecte. Il est également requis pour le bon positionnement des organites, la morphogenèse et la division cellulaire. Enfin, il serait impliqué dans l’activité sensorielle du trypanosome. A ce jour, on ne connait quasiment rien des potentielles voies de « sensing ». Elles doivent pourtant exister, permettant l’appréhension de l’environnement, l’interaction avec les hôtes et la réception de signaux induisant la différenciation.

Cet intérêt pour les voies de signalisation du parasite a abouti à l’étude des composantes de la voie TOR. TOR-Target of Rapamycin est un contrôleur central de la croissance cellulaire qu’il régule en fonction de différents stimuli externes. Il a été démontré depuis que chez T.brucei aussi, TOR régulerait la croissance temporelle et spatiale de la cellule.

La kinase TOR est inhibée par sa liaison avec le complexe rapamycine-FKBP12. Nous avons identifié cette peptidyl-prolyl cis-trans isomérase chez le parasite :TbFKBP12. Elle y serait localisée au niveau du cytosquelette/flagelle. Contrairement à ce qui est observé chez la levure S.cerevisiae, l’isomérase est essentielle chez le trypanosome. Son invalidation par RNAi bloque la cytocinèse des parasites sanguicoles et provoque l’apparition d’axes de clivage internes à la cellule. Chez les formes procycliques par contre, la disparition de la protéine entraîne un défaut sévère de motilité du flagelle qui se traduit par une immobilisation partielle du parasite.

TbFKBP12 est donc impliquée dans l’homéostasie du flagelle chez le trypanosome africain, organite nécessaire à la motilité et à la division cellulaire.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

V. macrocarpon (canneberge) est traditionnellement associé à la prévention des IU, les mécanismes restant mal élucidés. L'effet d'une préparation commerciale de canneberge sur l'adhésion d'E.coli UTI89 aux cellules urothéliales T24, a montré l'importance de pré-incuber les bactéries avec le composé pour obtenir une inhibition dose-dépendante et réversible de l'adhésion. L'étude du transcriptome (E.coli Gene expression microarray, AgilentTechnologies) montre un effet puissant portant sur de nombreux gènes liés aux adhésines (sauf les fimbriae P), au chémotactisme et au flagelle. L'étude en microscopie électronique confirme un effet sur la taille et les structures de surface (adhésines, flagelles), l'étude de la mobilité en microscopie à champ large montre de moindres capacités de déplacement (distances, linéarité). Chez la souris C57BL/6, la pré-incubation n'a pas d'effet significatif sur la colonisation des vessies, ni sur l'adhésion ex vivo aux cellules T24, des bactéries récupérées dans les vessies. Un modèle intégratif d'étude de V.macrocarpon, basé sur des tests simples in vitro (adhésion, swarming, microscopie à champ large) est défini
V.macrocarpon (cranberry) is traditionally associated with the prevention of urinary tract infections although the mechanisms of action remaining poorly elucidated. Preincubation of E.coli UTI89 strain with commercial extracts of V.macrocarpon inhibited adhesion to T24 human urothelial cell line in a dose-dependent and reversible manner. Transcriptomic assay (E.coli Gene expression microarray, Agilent Technologies) highlighted a strong impact on most genes related to adhesion, but P fimbriae, chemotactism and flagella. Electron microscopy study confirmed V.macrocarpon-induced alterations on UTI89 size and surface structures (fimbriae, flagella). In keeping, broad field microscopy (ImarisTrack) evidenced alterations in E.coli motility (track displacement length, duration, speed & straightness). In C57BL/6 mice, pre-incubation of UTI89 with V.macrocarpon extracts failed to impact bladder colonization after intravesical instillations and adhesion to T24 cells of bacteria recovered 3days after instillation. A simple, in vitro model based on adhesion and swarming assays and broad field microscopy is described to evaluate cranberry activity

This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

Centrioles and basal bodies with their characteristic 9+2 structure are found in all major eukaryotic lineages. The correlation between the occurrence of centrioles and the presence of cilia/flagella, but not centrosome-like structures, suggests that the ciliogenesis function of centrioles is ancestral. Here, it is demonstrated that the centriole domain of centrosomes emerged within the Metazoa from an ancestral state of possessing a centriole with basal body function but no functional association with a centrosome. Centrosome structures involving a centriole are metazoan innovations. When an axoneme is still present but no longer fully functional, such as the sensory cilia of Caenorhabditis elegans or, as depicted here, the flagellum of the intracellular amastigote stage of the Leishmania mexicana parasite, the basal body structure is less constrained and can depart from the canonical structure. A general view has emerged that classifies axonemes into canonical motile 9+2 and noncanonical, sensory 9+0 structures. This study reveals this view to be overly simplistic, and additional axonemal architectures associated with potential sensory structures should be incorporated into prevailing models. Here, a striking similarity between the axoneme structure of Leishmania amastigotes and vertebrate primary cilia is revealed. This striking conservation of ciliary structure, despite the evolutionary distance between Leishmania and mammalian cells, suggests a sensory function for the amastigote flagellum. Adding weight to a sensory hypothesis, close examination of Leishmania positioning inside the parasitophorous vacuole revealed frequent contact between the flagellum tip and the vacuole membrane. A sensory function could also explain the retention of a flagellum in Trypanosoma cruzi amastigotes, an intracellular stage that, as shown in this study, emerged independently to the Leishmania amastigote. Basal body appendages, such as pro-basal bodies and microtubule rootlets, also vary widely in their structure. Choanoflagellates, a sister group to the Metazoa, posses an extensive microtubule rootlet system that provides support for their characteristic collar tentacles. This atypical structure is reflected in the underlying molecular components of the choanoflagellate basal body. The importance of choanoflagellates as the closest known relative of metazoans was first revealed by their similarity to choanocytes, the feeding cells of sponges. Although phylogenetic analyses leave little doubt that choanoflagellates are a sister group of animals, comparisons of molecular and structural components of appendages associated with the collar tentacles highlight significant differences and questions the extent to which the collar structures of choanoflagellates and choanocytes can be assumed to be homologous. Finally, the confinement of a centriole-based centrosome to the Metazoa provides little support for the flagellar synthesis constraint as an explanation for the origin of multicellularity. There is, indeed, an apparent constraint; no flagellated or ciliated metazoan cell ever divides. This constraint, however, did not arise until after the incorporation of centrioles into the centrosome in the metazoan lineage and the co-option of centrioles as a structural and functional component of the centrosome. The flagellar synthesis constraint is therefore not an explanation for the origin of multicellularity but a consequence of it.

Le rôle des bactéries est primordial partout dans la nature. Pensons seulement aux impacts qu’elles ont sur la santé humaine. Pour être en mesure de jouer leur rôle dans l’environnement, plusieurs bactéries ont besoin de se déplacer vers des sites spécifiques. Le moyen de locomotion le plus commun est le moteur flagellaire. Pour se propulser, les bactéries flagellaires possèdent un (ou des) moteur rotatif ancré dans leur membrane. Ce moteur transmet sa rotation à un long filament en forme d’hélice se trouvant à l’extérieur de la bactérie via un joint universel se nommant le crochet. Ce moteur fut l’un des premiers moteurs rotatifs biologiques à être découvert. De plus, un grand nombre d’études ont montré l’importance du moteur flagellaire durant les infections bactériennes. Ainsi, il a fait l’objet d’études intensives depuis plusieurs décennies. La vaste majorité de ces études ont toutefois été effectuées dans des milieux simples qui ne représentent qu’une infime partie des milieux biologiques naturels. En effet, les bactéries se déplacent souvent dans des milieux anisotropes, où les propriétés physiques dépendent de la direction. Par exemple, le mucus se trouvant un peu partout dans le corps humain, le liquide synovial qui lubrifie nos articulations, la peau et les biofilms sont tous des milieux qui peuvent être anisotropes et où les bactéries prolifèrent. Cette thèse par article présente les résultats obtenus durant notre étude de la motilité des bactéries flagellaires en milieux anisotropes. Puisque les milieux biologiques naturels sont difficiles à manipuler en laboratoire, un milieu synthétique a d’abord été choisi afin de mimer les propriétés de ces milieux. Deux types de milieux anisotropes ont été testés, les cristaux liquides (LCs) 5CB et DSCG. Seul le LC DSCG a été retenu puisque les bactéries ne peuvent pas pénétrer le LC 5CB. Pour créer le LC DSCG, des molécules sont dissoutes dans l’eau. À faible concentration, le milieu est isotrope, et à haute concentration le milieu devient anisotrope (un LC). Dans un premier temps, la vitesse et l’orientation du corps des bactéries ont été observées en faisant passer le LC DSCG de la phase isotrope à la phase anisotrope. Ces mesures ont d’abord confirmé que, dans un milieu anisotrope, les bactéries se déplacent en ligne droite et renverse leur mouvement plutôt que d’effectuer une marche aléatoire comme dans des milieux isotropes. L’observation du comportement bactérien a également démontré la présence d’une zone de prétransition dans les solutions isotropes de DSCG. À ces concentrations de DSCG, les molécules commencent à s’organiser sous forme de bâtonnets. Cette organisation explique pourquoi les bactéries deviennent collantes (via la force de déplétion), et pourquoi la viscosité augmente dans la zone de prétransition. Pour comprendre comment les bactéries peuvent renverser leur mouvement dans des milieux anisotropes, les filaments ont également été étudiés. Ces observations ont démontré que, durant le change de direction de la bactérie, le crochet n’est plus capable de jouer son rôle de joint universel et se bloque momentanément, permettant ainsi de changer l’orientation du filament. Cette réorientation du filament permet non seulement le renversement du mouvement de la bactérie dans le LC, mais également la réorientation du filament dans d’autres milieux comme dans des milieux poreux. Ce constat agrémenté de résultats provenant de la littérature nous permet de croire que le crochet bloqué est un phénomène universel se produisant dans tous les milieux. Pour terminer, la microscopie à champ sombre par guidage de lumière ainsi qu’une technique de microrhéologie seront exposées. Ces techniques ont été utilisées durant la caractérisation de la zone de prétransition. Tout au long de ce travail, il sera également souligné en quoi notre approche multidisciplinaire a été bénéfique.
Bacteria play an essential role in nature. We can simply think of their impact on human health to convince ourselves. To be able to play their role in the environment, bacteria often need to reach specific locations. The most common bacterial locomotion system is the flagellar motor. To propel themselves, the flagellated bacteria possess one (or few) rotary motor anchored in their membrane. This motor transfers its rotation to a long helical filament located outside the bacterium thanks to a universal joint called the hook. This motor was the first biological rotary motor discovered. Furthermore, several studies have shown the importance of the flagellar motor during bacterial infections. Thus, it has been the subject of intensive studies for several decades. Most of these studies, however, have been conducted in simple media that represent only a small fraction of natural biological environment. Indeed, bacteria often move in anisotropic media, where the physical properties depend on the direction. For example, mucus found throughout the human body, synovial fluid that lubricates our joints, skin and biofilms are all media that can be anisotropic and where bacteria proliferate. This thesis by article presents our study of the motility of flagellar bacteria in anisotropic media. Since natural biological media are difficult to manipulate in the laboratory, a synthetic medium was first chosen to mimic the properties of natural anisotropic media. Two types of anisotropic media were tested, the liquid crystals (LCs) 5CB and DSCG. Only the LC DSCG has been used since bacteria cannot penetrate the LC 5CB. To create the DSCG LC, molecules of disodium cromoglycate (DSCG) are dissolved in a water-based solvent. At low concentration, the medium is isotropic, and at high concentration the medium becomes anisotropic (a LC). First, the speed and the orientation of the body of the bacteria were recorded while changing the concentration of the DSCG LC to bring the solution from the isotropic phase to the anisotropic phase. These measurements first confirmed that, in an anisotropic environment, the bacteria move in a straight line and reverse their movement rather than performing a random walk as in isotropic media. Observation of bacterial behavior also demonstrated the presence of a pretransition zone in isotropic solutions of DSCG. At these concentrations of DSCG, the molecules begin to organize into rods. This organization explains why bacteria become sticky (via the depletion force), and why the viscosity increases in the pretransition zone. To understand how bacteria can reverse their motion in anisotropic media, the filaments have also been studied. These observations have shown that during the change of direction of the bacteria, the hook is no longer a universal joint and momentarily locks, thus changing the orientation of the filament. This reorientation of the filament does not only reverse the movement of the bacteria in the LC, but it also triggers the reorientation of the filament in other media as in porous media. This observation, supplemented by results from literature, suggests that the blocked hook is a universal phenomenon occurring in all environments. Finally, light-guided dark field microscopy and a microrheological technique will be exposed. These techniques were used during the characterization of the pretransition zone. Throughout this work, it will also be highlighted how our multidisciplinary approach has been beneficial.

Les cils et flagelles sont constitués d’un cylindre de neuf doublets de microtubules périphériques appelé axonème. Ils contiennent au moins 500 protéines et leur construction s’effectue essentiellement par addition de nouvelles sous-unités à l’extrémité distale. Les composants du flagelle y sont acheminés par le transport intraflagellaire (IFT), le déplacement de « trains » formés de deux complexes de protéines, entre la membrane flagellaire et les doublets de microtubules par des moteurs moléculaires de type kinésine et dynéine. Mon projet de thèse s’articule autour du rôle et du fonctionnement de l’IFT en utilisant comme modèle d’étude le protiste Trypanosoma brucei. Les objectifs de ma thèse étaient (i) de déterminer comment les trains IFT sont assemblés en établissant le lien entre leur composition moléculaire et leur structure et (ii) d’établir le trajet emprunté par les trains IFT au sein de l’organite. En combinant des approches de microscopie photonique et de microscopie électronique après ciblage par ARNi de gènes encodant des protéines constituants les trains IFT, nous avons mis en évidence leur contribution à la construction des trains IFT et la régulation de leur longueur. Nous proposons un nouveau modèle pour expliquer la formation des trains et leur entrée dans le compartiment flagellaire. Par microscopie électronique tridimensionnelle (FIB-SEM), nous avons démontré qu’après leur assemblage, les trains IFT sont localisés au niveau de seulement 4 doublets de microtubules sur les 9 disponibles. Ces résultats ont été obtenus à la fois in vitro et ex vivo sur des parasites se développant chez la mouche tsé-tsé. La comparaison des résultats avec la littérature met en exergue la flexibilité du transport en fonction de l’anatomie des cils et flagelles
Cilia and flagella are essential organelles composed of 9 doublet microtubules. They contain at least 500 proteins and their construction is mainly done by adding new subunits at the distal end. They are transported byIntraflagellar transport (IFT), the movement of trains composedof two protein complexes between the flagellar membrane and the microtubule doublets by driven by molecular kinesin and dynein motors. My thesis project is based on the role and functioning of IFT using the protistTrypanosoma brucei as a model organism. The goal of my thesis project was (i) to determine how IFT trains are assembled by establishing the link between their molecular composition and their structure and (ii) to establish the route taken by IFT trains within the flagella. By combining light microscopy and electron microscopy approaches after RNAi targeting of genes coding for IFT train components, we have demonstrated their contribution to the construction of IFT trains. We propose a new model to explain train formation and their entry in the flagellum. By three-dimensional electron microscopy (FIB-SEM), we have also shown where IFT trains are located. Trains are specifically found on 4 microtubule doublets out of the 9 available. These results have been obtained bothin vitro and ex vivousing parasites developing in the tsetse fly.Comparison of the results with the literature highlights the flexibility of transport depending on the anatomy of cilia and flagella

Les bactéries du genre xanthomonas sont responsables de nombreuses maladies des plantes, telles que la nervation noire des brassicacées causée par x. campestris pv. campestris (xcc). Lors des phases précoces du processus infectieux, ces bactéries doivent identifier des sites favorables à leur pénétration dans les tissus et les atteindre afin de s'internaliser dans les tissus végétaux et s’y multiplier. Le chimiotactisme est le mécanisme par lequel les bactéries détectent des signaux et se dirigent vers des attractants ou s’éloignent de signaux répulsifs. L’objectif de ce travail est de comprendre les rôles du chimiotactisme et de la mobilité flagellaire dans la fitness des xanthomonas. Nous avons montré que la mobilité flagellaire n’est pas une caractéristique partagée par tous les xanthomonas mais qu’environ 5% des souches perdent cette capacité sans altération majeure de leur fitness in planta. Un senseur du chimiotactisme, dénommé hsb1, probablement acquis par transfert horizontal, présente un groupe d’allèles spécifique à x. campestris. Une mutation de hsb1 dans la souche xcc atcc 33913 entraine une diminution de l’internalisation de cette souche dans les tissus de plantes hôtes combinée à une augmentation de l’internalisation dans les tissus des plantes non-hôtes. Hsb1 perçoit un signal émis par les blessures des feuilles de chou. Un glucosinolate, la sinigrine, et un acide aminé, la l-phénylalanine, sont détectés in vitro par ce senseur, mais ne sont pas métabolisés. Des travaux complémentaires seront nécessaires pour identifier le signal détecté par ce senseur et envisager la conception de méthodes de lutte basées sur la confusion d’informations
Xanthomonads are responsible for plant diseases such as black rot of Brassicaceae caused by X. campestris pv. campestris (Xcc). During the early stages of the infection, pathogenic bacteria such as Xcc must detect favorable sites and ingress into host plant tissues to colonize and multiply in the apoplast or the xylem vessels. Chemotaxis is the mechanism used by bacteria to detect attractants and repellents and adapt in consequence its direction. The aim of this work is to understand the roles of chemotaxis and flagellar motility in the fitness of xanthomonads. We showed that flagellar motility is not a general feature of xanthomonads. About 5 % of tested strains lost this ability without major impact on their fitness in planta. A chemotaxis sensor, named Hsb1, probably acquired by horizontal transfer shows a group of alleles that are specific of X. campestris. In Xcc ATCC 33913, a mutation in hsb1 resulted in a decreased penetration of this strain in the host plant tissues combined with an increase penetration in the non-host plant tissues. Hsb1 sense a signal from wounds of cabbage leaves. In vitro, a glucosinolate, the sinigrin, and an amino acid, the L-phenylalanine are detected by Hsb1 but are not metabolized. Further work is needed to identify the signal detected by the sensor and to design control methods based on confusion

Helicobacter pylori is a well-known pathogen able to colonize the human stomach of more than half of the world population. Luckely, only the 10% of the affected people manifest severe incidences, like chronic gastritis, peptic ulcers and, among those, only in 1% of cases MALT (Mucosal associated lymphoid tissue) lymphoma and gastric adenocarcinomes are recognized [1]. The project described in this thesis focus mainly on the structural determination and functional characterization of the proteins involved in the assembly of the flagellar architecture. The presence of a flagellate phenotype is paramount for the bacterial colonization processes. Its role is strictly related to the chance of avoiding the acidic milieu in the gas- tric environment and its periodical mechanical clearances, allowing H. pylori to swim through the mucus layer and adhere to gastric epithelial cells [2]. Once the epithelium is reached, the regular colonization process takes place, starting with the adhesion to the cells via a set of adhesines, which cover both the bacterial membrane and flagella [3]. Its propulsion in the motion is guaranteed by a tuft of 5-7 polar flagella, long from 2 to 5 μm, and characterized by a S shape [4]. The flagellum is composed by about thirty proteins that can increase up to hundred considering the components involved in chemotatic and regulatory mechanism [5]. The flagellum is a complex rotary nano-machine, which can be divided in two main components: i) the hook-basal bady portion, where the engine is located; ii) the filament, representing the machinery propeller [6]. During the thesis, the proteins from the hook region of the flagellum and the adhesine recovering the filament were analyzed. The strategy adopted included the amplification of the selected genes from the purified Helicobacter pylori chromosomal DNA (in particular the strains G27 and P12), the cloning in one or two expression vectors, followed by the expression of the protein in E. coli cells and the specific purification from the bacterial lysis supernatant. Unfortunately, the soluble proteins from the hook do not share a significance sequence similarity with any other proteins already known in literature, so their behaviour in solution was not easily predictable. The flagellar proteins analyzed along the work of thesis were: FlgE, FlgE2, FlgK and HpaA (as a flagellar sheath protein). Although all of them were successfully cloned, expressed and purified, only the protein FlgE2 gave sufficiently results to lead to some preliminary structural studies. In chapter 3 the analysis of the flagellar proteins is reported. FlgK is a hook-associated protein involved in the junction between the filament and the hook [7] and FlgE is directly involved in the assembly of the hook [8]. The proteins were characterized in solution and although several crystallization screens were performed, none of them produced crystals suitable for X-ray diffraction analysis. The most relevant limit was in the production of a large amount of protein, that was eluted as an aggregated form during size-exclution chromatography. The protein FlgE2 is involved in the assembly of the hook as well. It seems to be peculiar for Helicobacter and Campilobater species, but it has not been characterized further. During the thesis, the protein was successfully purified as a monomer and a tetramer. In both the cases, good quality crystals for the X-ray diffraction studies were produced. Several attempts were performed in solving the structure by molecular replacement, using the orthologous from Salmonella typhimurium as template, but none of them was completely successful. However a preliminary model has been designed and is discussed in the chapter. Since the protein does not share a high percentage of similaty with its orthologous proteins, seleniomethionine-labeled protein was produced in order to solve the phase problem and improve the phases obtained by the molecular replacement. The last part of the job is still ongoing. Besides, the functional role of the protein was explored, checking the interaction between the hook structural components and its cognate chaperon, FlgD. The crystal structure of the protein FlgD has been recently solved [9] and demonstrated the presence of a binding site located at the C-terminus. To confirm the binding, a microscale thermophoresis analysis was performed. To achieve the attachment on the epithelial cell, a set of bacterial adhesines are needed and the role of HpaA protein is presented in chapter 4. H. pylori adhesin A is a surface-located lipoprotein that was initially described as a sialic acid binding adhesin, expressed on the surface of gastrointestinal cells [10]. It is supposed to act as a flagellar sheath protein that facilitates the attachment to the cell through flagellar filaments [11]. Owing to its exposed position on the filament, the protein is supposed to be a good candidate as a vaccine target [12]. Due to its high tendency to aggregate, the protein was expressed and purified in a fused form with a super-folder GFP [13]. It produced good quality crystals for X-ray diffraction analysis purpose. However, after the analysis, the crystals resulted to be composed only by sfGFP. The protein HP1457 is related to a different project and it is reported in chapter 5. The protein belongs to a class of proteins that has been recognized, only recently, to act as a outer-membrane proteins that contributes to the stimulation of the peptidoglycan synthase PBP1B [14]. It is enclosed in a translational unit characterized by the presence of genes coding for high immunogenic, secreted proteins, like HP1454 [15], HP1455 (lipoprotein with unknown function) and the protein HP1456, recognized to be a Lpp20 [16]. The protein was fully characterized in solution and several crystallization trials were performed. Unfortunately, none of those led to obtain protein crystals.
Helicobacter pylori é un batterio Gram-negativo presente in piú del 50%della popolazione mondiale. Tuttavia, i ceppi piú aggressivi del batterio colpiscono solo il 10% delle persone affette, causando gastriti croniche e ulcera peptica. Tra queste, solo l’1% manifesta gravi patologie come MALT linfoma ed adenocarcinoma gastrico [1]. Il progetto descritto in questo lavoro di tesi é finalizzato alla determinazione strutturale ed alla caratterizzazione di proteine coinvolte nell’architettura del flagello di H. pylori. Difatti, la presenza del flagello é fondamentale per la colonizzazione da parte del batterio dell’intero ambiente gastrico. Il suo ruole é direttamente collegato all’abilitá del batterio di evitare la permanenza nell’ambiente gastrico, permettengli di attraversare lo strato di muco che aderire all’epitelio gastrico [2]. Grazie ad un set di adesine localizzate sia sulla membrana batterica, sia sui flagelli, H. pylori é in grado di aderire alle cellule epiteliali, cominciando il regolare processo di colonizzazione [3]. Inoltre, la presenza di un seti di 5-7 flagelli polarizzati (lunghi tra 2 e 5 μm) e la sua forma ad S garantiscono al batterio la propulsione e la velocitá necessaria per superare la barriera di muco e raggiungere l’epitelio [4]. In generale, la struttura del flagello é costituita da circa trenta diverse proteine, ma il numero puó crescere fino a cento considerando anche le proteine coinvolte nei meccanismi di chemotassia e di regolazione della crescita del flagello [5]. L’architettura generale del flagello é quella di una una complessa nanomacchina, caratterizzata dalla presenza di due principali componenti: i) la porzione dell’uncino e della parte basale, dove é collocato il motore della stuttura; ii) il filamento, che rappresento il vero propulsore della macchina [6]. Nel corso del progetto di dottorato, sono state analizzate le proteine che costituiscono la regione dell’uncino e ricoprono il filamento. La metodica utilizzata prevede l’amplificazione iniziale dei geni target utilizzando il DNA genomico dei ceppi G27 e P12 come templato. I geni amplificati sono stati inseriti in uno o due vettori di espressione, e le proteine ricombinanti sono state prodotte in colture di E. coli e purificate, tramite diversi metodi cro- matografici, dalla parte solubile della sospensione di lisi batterica. Le proteine dell’uncino di H. pylori non presentano un’elevata similaritá di sequenza con le altre proteine giá riportate in letteratura, pertanto la loro reattivitá in soluzione non é facilmente prevedibili a priori. FlgE, FlgE2, FlgK and HpaA (come proteina di rivestimento del flagello) sono le proteine del flagello analizzate durante questo lavoro di dottorato. Sebbene, tutte siano state clonate, espresse e purificate, solo FlgE2 ha fornito dei risultati sufficienti a condurre degli studi strutturali preliminari. I vari studi condotti sulle proteine del flagello sono riportati nel capitolo 3. FlgK é defina una hook-associated protein, che funge da giuntura tra il filamento e l’uncino [7], mentre FlgE é direttamente coinvolta nella polimerizzazione dell’uncino [8]. Entrambe le proteine sono state ampiamente caratterizzate in soluzione ma, sebbene siano stati preparati numerosi screen di cristallizzazione, nessuna delle due ha portato ad ottenere cristalli analizzabili tramite diffrazione a raggi-X. Il limite maggiore osservato nella produzione delle due proteine é l’eluizione di una grande quantitá di forma aggregata, durante il processo di purificazione via cromatografia ad esclusione dimensionale. Anche la proteina FlgE2 é coinvolta nel processo di polimerizzazione dell’uncino. Tuttavia questa sembra essere peculiare solo per le specie batteriche di Helicobacter e Campilobacter e, pertanto, non si hanno numerosi informazioni a riguardo. Durante il lavoro di tesi, la proteina é stata purificata sia in forma monomerica che in forma tetramerica e, in entrambi i casi, sono stati ottenuti cristalli analizzabili tramite diffrazione da raggi-X. Tuttavia, molti tentavi sono stati svolti per risolvera la struttura tramite molecular replacement, utilizzando come templato la struttura della proteina ortologa di Salmonella typhimurium, ma nessuno di questo ha avuto interamente successo. Tuttavia, é stato possibile descrivere un modello preliminario, che verrá successivamente discusso all’interno del capitolo. Poiché HpFlgE2 non presenta un’elevata similaritá con StFlgE2, é stata prodotta una variante in cui sono state sostituite le metionine con seleniometionine; in questo modo si é provato a migliorare calcolando le fasi relative agli atomi pesanti (Se) e successivamente espandendole tramite i dati raccolti per la proteina nativa. Quest’ultima parte del lavora é ancora in via di definizione. Inoltre, date le scarse conoscenze riportate in letteratura riguardanti la proteina, si é cercato di definirne la reale funzione a partire dall’interazione con lo specifico chaperone FlgD. La struttura cristallografica della proteina FlgD é stata risolta recentement [9], rivelando la presenza di un motivo lineare collocato nella parte C-terminale, rappresentativo di una zona di legame con la proteina coniugata. Il legame é stato confermato tramite termoforesi in microscala. Per potersi legare alle cellule epitaliali, H. pylori ha bisogno di un set di proteine (ade-sine) che ne permettano l’adesione. La proteina HpaA (Helicobater pylori adhesine A),analizzata nel capitolo 4, é una lipoproteina, localizzata sulla superfice del flagello ed inizialmente é stata ipotizzata affine all’acido sialico, esposto sulla superficie delle cellule gastriche [10]. Si suppone che la proteina possa operare come proteina di rivestimento delflagello e che faciliti il legame sulla superficie delle cellule attraverso l’adesione dei flagelli[11]. Inoltre, data la sua posizione esposta, HpaA é stata considerato un ipotetico target per lo svilluppo di vaccini [12]. Infine, la proteina da sola presenta un’elevata tendenza a creare degli aggreagati in soluzione, pertanto é stata espressa e purificata in fusione con una super-folder GFP [13]. La proteina é stata successivamente cristallizata ed é stata analizzata tramite diffrazioni da raggi-X. Sfortunatamente, l’analisi dei dati di diffrazione ha portato alla conclusione che i cristalli erano formati esclusivamente da sfGFP. Infine, nel capitolo 5 viene analizzata la proteina HP1457. Questa appartiene ad una classe di proteina recentemente riconusciute operare come outer-membrane proteine, coinvolte nella stimolazione della peptidoglicano-sintasi, PBP1B [14]. Inoltre, la proteina HP1457 appartiene ad un operone caratterizzato dalla presenza di geni che codificano per proteine di secrezione altamente immunogeniche. Tra queste: HP1454 [15], HP1455 (una lipoproteina di funzione ancora sconosciuta), HP1456 (identificata come la lipoproteina Lpp20) [16]. HP1457 é stata ampiamente caratterizzata in soluzione e sono stati effettuati numerosi tentativi di cristallizzazione su diversi costrutti, ma nessuno di questi ha portato ad ottenere cristalli.

La chronologie des principaux évènements flagellaires survenant dans l'établissement du mouvement des spermatozoïdes de mammifères en relation à la fois avec des phénomènes physiologiques de maturation et de capacitation et les modifications du milieu environnant a été établie grace à des techniques microphotographiques et cinématographiques. L'analyse du mouvement flagellaire a montré l'existence d'un modèle de motilité commun à la maturation et à la capacitation. Un tel modèle apparait aussi au cours de l'induction de la motilité des spermatozoïdes testiculaires et de la réactivation des spermatozoïdes matures demembranes. Dans tous les cas, les spermatozoïdes présentent une courbure non propagée dans la première partie du flagelle avec des déplacemetns erratiques et circulaires. Le rôle joué par cette courbure ainsi que son origine possible sont discutés en relation avec l'ultrastructure et la physiologie du flagelle
No summary available

L'analyse de la mobilite des cellules ciliees et flagellees trouve de nombreuses applications en biologie et en medecine. Nous presentons des methodes de quantification de la forme et du mouvement des spermatozoides humains et de dunaliella bioculata (une algue marine) par analyse d'image microcinematographique et microvideographique. La reconnaissance des formes cellulaires est realisee par une approche syntaxique. Les mouvements de formation, de propagation et de relachement des ondes ciliaires et flagellaires sont modelises par des splines biparametriques. Les trajectoires cellulaires sont decrites par une modelisation fractale. Ces methodes ont ete appliquees a la classification automatique des anomalies morphologiques des spermatozoides humains, a la comparaison des battements ciliaires et flagellaires et a l'etude de l'effet d'un produit toxique (le lindane) sur le battement ciliaire

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Salmonella Enteritidis (SE) causa o paratifo aviário em aves e frequentemente está relacionada aos surtos de infecção alimentar em seres humanos. A contribuição do flagelo versus motilidade na interação patógenohospedeiro requer estudos mais aprofundados. Para melhor entendimento da contribuição individual desses fatores de virulência em aves, pintinhos de um dia de vida foram desafiados oralmente com estirpe selvagem de SE, uma mutante não-móvel mas flagelada (SE ΔmotB) e outra mutante aflagelada (SE ΔfliC). Excreção fecal e colonização de fígado, baço e conteúdo cecal pelas estirpes de SE foram avaliadas. Além disso, também foi realizada a avaliação das alterações macroscópicas e microscópicas. Nos estágios iniciais da infecção, ambos mutantes mostraram menor capacidade de colonizar o ceco, além de menor recuperação no baço por SE ΔfliC comparando a estirpe selvagem SE. Após 7 dpi não havia diferenças na contagem das três estirpes em conteúdo cecal, fígado e baço. Análises histopatológicas demonstraram que estirpes flageladas (SE ΔmotB e SE) induziram reatividade linfóide em inglúvio, ceco, íleo e fígado. No entanto, nos estágios iniciais da infecção a estirpe SE ΔfliC não estimulou a reatividade linfóide em lâmina própria de ceco e íleo mas induziu discretos focos necróticos em fígado. Portanto, neste estudo a presença de estrutura flagelar e motilidade parece exercer um papel nos estágios iniciais da colonização intestinal e infecção sistêmica por SE nas aves.
Salmonella Enteritidis (SE) causes fowl paratyphoid in poultry often related to outbreaks of food-borne diseases in humans. The contribution of flagella and motility in host pathogen interaction require further investigation. To better understand the individual contribution of these virulence factors in poultry, one day old chickens were challenged orally with wildtype strain of SE, a nonmotile but fully flagellated (SE ΔmotB) and aflagellated mutant (SE ΔfliC). Faecal excretion and colonization of liver, spleen and cecal contents by the SE strains were assessed. Additionally, the assessment of gross and microscopic alterations was also performed. At the early stages of infection both mutants showed lower capacity to colonize the ceca, besides the lower recovering in spleen of SE ΔfliC comparing to the wild type of SE. After 7 dpi there were no differences among the counts of the three strains in ceca, liver and spleen. Histopathological analyses demonstrated that flagellated strains (wild type SE and SE ΔmotB) induced lymphoid reactivity in crop, ceca, ileum and liver. On the other hand, in the early stages of infection, SE ΔfliC strain did not stimulate lymphoid reactivity in lamina propria of ceca and ileum but induced discrete necrotic foci in liver. Thus in the present study the flagellar structure and motility seemed to play a role at the early stages of the intestinal colonization and systemic infection by SE in the chicken.
FAPESP: 2014/02014-1

La motilité constitue un atout pour les bactéries tant dans leurs déplacements vers les environnements les plus favorables que dans la pathogénicitéou l'adhésion et la formation des bio films. En contrepartie, la motilité demande de grandes dépenses énergétiques et les filaments des flagelles portent des caractères antigéniques marques. Il n'est donc pas étonnant qu'un système très élaboré contrôle la synthèse des flagelles suivant les conditions environnementales. Nous avons aborde les bases moléculaires de ces processus chez E. Coli. Les résultats obtenus montrent que le complexe camp-cap et la protéine h-ns, mais pas la protéine para logue stpa, contrôlent la synthèse des flagelles au niveau de l'opéron maître flhdc. Camp-cap active directement la transcription, tandis que l'effet positif d'h-ns dépend de la présence de la région régulatrice complète incluant l'extrémité 5 de l'arnm. L'analyse des profils d'expression dans un mutant hns montre la position hiérarchique élevée d'h-ns dans le contrôle de la physiologie bactérienne, en particulier en réponse aux facteurs environnementaux comme le pH acide. La caractérisation de revertants spontanés motiles dans un contexte hns a révèlé un contrôle coordonne de la motilité et de la résistance au pH acide. Par ailleurs, nos résultats montrent un remarquable parallélisme entre la régulation de l'opéron flhdc par h-ns et par les conditions environnementales, impliquant les effets locaux d'h-ns sur la topologie de l'ADN. La présence de régions régulatrices similaires dans les opérons homologues à flhdc,

Plusieurs bactéries possèdent des flagelles qui leur permettent de se déplacer dans leur milieu. Ce sont des moteurs rotatifs, imbriqués dans la membrane, qui font tourner des filaments hélicoïdaux à plus de 100 Hz et qui propulsent les bactéries dans leur environnement. La source d'énergie des moteurs flagellaire est le gradient électrochimique de protons de part et d'autre de la membrane dont l'énergie potentielle est convertie en mouvement de rotation. Plusieurs facteurs influencent la rotation des filaments, notamment la concentration de certaines protéines dans le cytoplasme des bactéries. Afin d'étudier plus facilement les caractéristiques du moteur flagellaire, un système in vitro a été développé pour contrôler les conditions de rotation des moteurs flagellaires d'Escherichia coli. Pour ce faire, les bactéries ont été coincées individuellement à l'extrémité d'une micropipette de verre. Une partie de la membrane de la bactérie située à l'intérieur de la micropipette a été perforée en utilisant l'ablation laser femtoseconde. La perméabilisation de la membrane de la bactérie a permis le contrôle externe de la source d'énergie du moteur et le remplacement du contenu cytoplasmique par le liquide à l'intérieur de la micropipette. Avec le contrôle des conditions de rotation du moteur, il a été possible d'observer la relation linéaire entre la vitesse de rotation des moteurs et la différence de potentiel électrique appliquée. La rotation des filaments des bactéries a également été soutenue pendant plus de 30 minutes grâce à un gradient de pH. La diffusion de protéines fluorescentes à l'intérieur des bactéries a permis de confirmer que notre technique pourrait être utiliser pour étudier l'effet de certaines protéines, notamment CheY-P, sur la rotation des moteurs. Enfin, notre technique a également permis des observations préliminaires de la dynamique d'entrée et de sortie des unités génératrices du couple dans les moteurs. Ce nouvel outil pour l'étude du moteur flagellaire devrait permettre d'approfondir notre compréhension du mécanisme de la génération du couple dans le moteur en fournissant des données permettant de mettre des contraintes aux modèles théoriques.

Serratia sp. ATCC 39006 (S39006) is known for producing carbapenem and prodiginine antibiotics; 1-carbapen-2-em-3-carboxylic acid (car) and prodigiosin. It displays different motility mechanisms, such as swimming and swarming aided by flagellar rotation and biosurfactant production. In addition, S39006 produces gas vesicles to float in aqueous environments and enable colonization of air-liquid interfaces. Gas vesicles are thought to be constructed solely from proteins expressed from a gene cluster composed of two contiguous operons, gvpA1-gvpY and gvrA-gvrC. Prior to this study, three cognate regulators, GvrA, GvrB, and GvrC, encoded by the right hand operon were known to be essential for gas vesicle synthesis. Post-transcriptional regulators such as RsmA-rsmB were also known to be involved in the inverse regulation of gas vesicles and flagella based motility. Furthermore, gas vesicle formation, antibiotic production, and motility in S39006 were affected by cell population densities and de-repressed at high cellular densities through a quorum sensing (QS) system. The aim of this research study was to identify novel regulatory inputs to gas vesicle production. Mutants were generated by random transposon mutagenesis followed by extensive screening, then sequencing and bioinformatic identification of the corresponding mutant genes. After screening, 31 mutants and seven novel regulatory genes impacting on cell buoyancy were identified. Phenotypic and genetic analysis revealed that the mutations were pleiotropic and involved in cell morphology, ion transport and central metabolism. Two new pleiotropic regulators were characterized in detail. Mutations in an ion transporter gene (trkH) and a putative transcriptional regulator gene (floR) showed opposite phenotypic impacts on flotation, flagella-based motility and prodigiosin, whereas production of the carbapenem antibiotic was affected in the transcription regulator mutant. Gene expression assays with reporter fusions, phenotypic assays in single and double mutants, and proteomics suggested that these regulatory genes couple different physiological inputs to QS and RsmA-dependent and RsmA-independent pathways.

During colonization of the cystic fibrosis airway Pseudomonas aeruginosa converts from non-mucoid to a mucoid phenotype, characterized by the production of the exopolysaccharide alginate. Alginate production has been shown to enhance survival by promoting biofilm formation, evading complement killing, and resisting phagocytosis. The mechanism by which alginate protects P. aeruginosa from phagocytosis is unclear. To investigate the role of alginate in the inhibition of phagocytosis, a human monocytic cell line (THP-1) and a murine alveolar macrophage cell line (MH-S) were used to determine the effects of alginate on macrophage binding, signaling, and phagocytosis. Phagocytosis assays using the mucoid cystic fibrosis clinical isolate FRD1, and its non-mucoid isogenic algD mutant FRD1131, revealed that alginate inhibits opsonic and non-opsonic phagocytosis. The inhibitory effect of alginate production is intrinsic to the bacteria as exogenous alginate was unable to protect non-mucoid FRD1131 from phagocytosis. Decreased binding of FRD1 compared to FRD1131 was also demonstrated by using the actin polymerization inhibitor cytochalasin D to inhibit phagocytosis. Furthermore, studies using blocking antibodies to CD11b and CD14 found that both of these receptors were important for the phagocytosis of FRD, and it is likely that these receptors are blocked by alginate. Alginate production by P. aeruginosa may reduce lipid raft formation, however, it was not found to affect acid sphingomyelinase activity, which is important for ceramide formation within the lipid raft. Decreased binding led to decreased signaling in macrophages demonstrated by reduction in level and alteration in kinetics of phosphorylation of AKT and ERK1/2 kinases. Signaling pathway inhibitors revealed that PI3K, but not MEK, activation was critical for phagocytosis of P. aeruginosa. Despite altered intracellular signaling in murine macrophages, both mucoid and non-mucoid P. aeruginosa induced similar levels of IL-8 and MIP-2 from human and murine macrophages, respectively. By understanding the pathways involved in mediating efficient phagocytosis of clinical isolates, it may be possible to develop a treatment to promote clearance by the resident alveolar macrophages. These experiments may serve as a model to evaluate the effectiveness of such treatments. This approach also provides valuable insight into previously unknown mechanisms of phagocytosis of P. aeruginosa.

La tubuline est une protéine majeure des cils et des flagelles qui subit des modifications post-traductionnelles (acétylation, tyrosination/detyrosination, glutamylation, glycylation) donnant naissance à une diversité d'isoformes de tubuline. L'étude de l'expression de ces isoformes de tubuline dans les flagelles de spermatozoïdes de mammifères a été entreprise à l'aide de techniques biochimiques : électrophorèses et immunotransferts. La distribution de ces isoformes de tubuline dans les microtubules des axonèmes a été précisée par immunocytochimie quantitative en microscopie électronique en utilisant des anticorps dirigés contre les modifications post traductionnelles. Les résultats ont révélé que contrairement à d'autres isoformes de tubuline, la tubuline glutamylee présente un marquage longitudinal proximo-distal décroissant avec une prédominance du marquage sur les doublets 1-5-6, correspondant au plan du battement flagellaire. Les études similaires effectuées sur la tubuline glycylee ont montré que la tubuline monoglycylee présente une distribution proximo-distale croissante avec une prédominance du marquage sur les doublets 3-8, correspondant à la perpendiculaire du plan du battement flagellaire. En accord avec l'effet inhibiteur des anticorps sur la motilité démontre par d'autres auteurs, ces marquages spécifiques interdoublets suggèrent un rôle des tubulines glutamylee et glycylee dans la régulation du battement flagellaire. Cette régulation implique des interactions entre les isoformes de tubuline et des protéines associées à l'axonème, en particulier, celles qui forment les structures périaxonémales caractéristiques des flagelles de spermatozoïde des mammifères. Afin de tester la validité de cette hypothèse, une analyse quantitative de la distribution des mêmes isoformes de tubuline a été entreprise, pour comparaison, sur des modèles d'axonèmes nus : cils de cellules pulmonaires de rongeur, cils de paramécie, flagelles de chlamydomonas et flagelles de spermatozoïdes d'oursin. En accord avec notre hypothèse, les axonèmes nus ne présentent aucune spécificité de distribution des tubulines glutamylee et glycylee respectivement dans les doublets 1-5-6 et 3-8. En revanche, les résultats ont révélé que la composition et l'organisation des isoformes de tubuline sont spécifiques dans ces différents modèles de cils et de flagelles suggérant autant de modèles d'organisation fonctionnelle et de régulation du battement de l'axonème.

Little is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages.

flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming but not swimming motility. Based on a chance observation of a ‘green’ colony phenotype of flhE mutants on pH indicator plates containing glucose, I have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica, but is seen in the absence of glucose and unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, but gets worse after the switch and upon filament assembly, leading to cell lysis. I show that FlhE is a periplasmic protein, which co-purifies with flagellar basal bodies. Also, co-localization of fluorescent fusion proteins suggests a plausible interaction between FlhE and FlhA, implicated in channeling protons for PMF-driven secretion. These results imply that FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system. I have obtained crystals of the FlhE protein. X-ray data show that the FlhE crystal belongs to space group P212121 and is diffracted to 2.02 Å. Completion of this study will contribute to a better understanding of the proposed role of FlhE.
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This work explores the effects of body shape and configuration of flagella on motility of Helicobacter pylori, a helical-shaped bacterium that inhabits the viscoelastic gastric mucosa and causes gastritis, ulcers and gastric cancer. Although it is well known that different shapes produce different hydrodynamic drag thus altering the speed and that helical shapes generate additional thrust this has not been quantitatively established for flagellated bacteria. Using fast time-resolution and high-magnification two-dimensional phase-contrast microscopy to simultaneously image and track individual H. pylori and its rod-shaped isogenic mutant in broth and mucin solutions, the shape as well as rotational and translational speed was determined. In collaboration with Professor Henry Fu and Mehdi Jabbarzadeh the experimental data was used to validate the method of regularized Stokeslets by directly comparing the observed speeds to numerical calculations. The results show that due to relatively slow body rotation rates, the helical shape makes at most a 15% contribution to speeds. In order to explore the effects of arrangement of flagella on motility three different Helicobacter spp. were examined: H. suis (bipolar, multiple flagella), H. cetorum (bipolar, single flagellum) and H. pylori (unipolar, multiple flagella) swimming in broth and mucin. Results show that regardless of media, the flagella bundles of bipolar bacteria can assume one of two configurations interchangeably: extended away from the body or wrapped around it. H. suis predominantly swims with the lagging flagella extended behind the body and the leading flagella wrapped around it, but cases where both bundles are extended or both are wrapped have also been observed. In addition the effects of varying pH on motility of H. suis in broth and mucin were investigated. In broth the rotational speed is not significantly affected by varying pH and the peak of the speed distribution shifts to lower values as the pH decreases. However in mucin the rotational speed decreases by a factor of 20 from pH5 to 4 and the motion is completely hindered below pH4. This indicates that H. suis is unable to move below pH4, in agreement with previous findings on H. pylori, due to gelation of mucin below pH4.

Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines.
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