Estudio del mecanismo de translocación y permeabilización de la toxina adenilato ciclasa de Bordetella pertussis

220 p.De entre todos los factores de virulencia secretados por la bacteria causante de la tos ferina, Bordetella pertussis, la toxina adenilato ciclasa (o ACT) es esencial para la colonización del tracto respiratorio y en el establecimiento de la enfermedad. La ACT forma parte de las toxinas RTX (del inglés Repeats in ToXin), siendo ésta una proteína bifuncional con un dominio formador de poros que se ocupa de la permeabilización de la membrana y un dominio catalítico, el cual se transloca directamente a través de la membrana, catalizando de forma no regulada el ATP intracelular de las células diana en cAMP a niveles suprafisiológicos. La combinación de ambas actividades provoca la citotoxicidad general producida por la toxina.En esta tesis doctoral se han estudiado ambos procesos en células y en membranas modelo. Por una parte se ha descrito una nueva actividad para la toxina, una actividad fosfolipasa A, la cual es indispensable para la translocación directa a través de la membrana del dominio catalítico. Por otra parte, se ha estudiado el proceso de formación de poros en membranas modelo y en células eucariotas, describiendo procesos de inserción, oligomerización, estabilidad y permeabilización.CSIC BIOFISIKA INSTITUTU

Estudio del mecanismo de translocación y permeabilización de la toxina adenilato ciclasa de Bordetella pertussis

220 p.De entre todos los factores de virulencia secretados por la bacteria causante de la tos ferina, Bordetella pertussis, la toxina adenilato ciclasa (o ACT) es esencial para la colonización del tracto respiratorio y en el establecimiento de la enfermedad. La ACT forma parte de las toxinas RTX (del inglés Repeats in ToXin), siendo ésta una proteína bifuncional con un dominio formador de poros que se ocupa de la permeabilización de la membrana y un dominio catalítico, el cual se transloca directamente a través de la membrana, catalizando de forma no regulada el ATP intracelular de las células diana en cAMP a niveles suprafisiológicos. La combinación de ambas actividades provoca la citotoxicidad general producida por la toxina.En esta tesis doctoral se han estudiado ambos procesos en células y en membranas modelo. Por una parte se ha descrito una nueva actividad para la toxina, una actividad fosfolipasa A, la cual es indispensable para la translocación directa a través de la membrana del dominio catalítico. Por otra parte, se ha estudiado el proceso de formación de poros en membranas modelo y en células eucariotas, describiendo procesos de inserción, oligomerización, estabilidad y permeabilización.CSIC BIOFISIKA INSTITUTU

Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin

Adenylate cyclase toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between toxin's two main activities, production of supraphysiological cAMP levels by its N-terminal adenylate cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.This study was supported by grants from the Basque Government (Grupos Consolidados IT849) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-P (AEI/FEDER, UE) to H.O

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins

Permeabilization of the plasma membrane represents an important threat for any cell, since it compromises its viability by disrupting cell homeostasis. Numerous pathogenic bacteria produce pore-forming toxins that break plasma membrane integrity and cause cell death by colloid-osmotic lysis. Eukaryotic cells, in turn, have developed different ways to cope with the effects of such membrane piercing. Here, we provide a short overview of the general mechanisms currently proposed for plasma membrane repair, focusing more specifically on the cellular responses to membrane permeabilization by pore-forming toxins and presenting new data on the effects and cellular responses to the permeabilization by an RTX (repeats in toxin) toxin, the adenylate cyclase toxin-hemolysin secreted by the whooping cough bacterium Bordetella pertussis, which we have studied in the laboratory.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-PAEI/FEDER, UE)

Fast Marching-based globally stable motion learning

In this paper, a novel motion learning method is introduced: Fast Marching Learning (FML). While other learning methods are focused on optimising probabilistic functions or fitting dynamical systems, the proposed method consists on the modification of the Fast Marching Square (FM2) path planning algorithm. Concretely, FM2 consists of expanding a wave through the environment with a velocity directly proportional to the distance to the closest obstacle. FML modifies these velocities in order to generalise the taught motions and reproduce them. The result is a deterministic, asymptotically globally stable learning method free of spurious attractors and unpredictable behaviours. Along the paper, detailed analysis of the method, its properties and parameters are carried out. Comparison against a state-of-the-art method and experiments with real data is also included.This work is supported by the Spanish Ministry of Science and Innovation under the projects DPI2010-17772 and CSD2009-00067.Publicad

3D robot formations path planning with fast marching square

This work presents a path planning algorithm for 3D robot formations based on the standard Fast Marching Square (FM2) path planning method. This method is enlarged in order to apply it to robot formations motion planning. The algorithm is based on a leader-followers scheme, which means that the reference pose for the follower robots is defined by geometric equations that place the goal pose of each follower as a function of the leader’s pose. Besides, the Frenet-Serret frame is used to control the orientation of the formation. The algorithm presented allows the formation to adapt its shape so that the obstacles are avoided. Additionally, an approach to model mobile obstacles in a 3D environment is described. This model modifies the information used by the FM2 algorithm in favour of the robots to be able to avoid obstacles. The shape deformation scheme allows to easily change the behaviour of the formation. Finally, simulations are performed in different scenarios and a quantitative analysis of the results has been carried out. The tests show that the proposed shape deformation method, in combination with the FM2 path planner, is robust enough to manage autonomous movements through an indoor 3D environment.Acknowledgments This work is funded by the project num ber DPI2010-17772, by the Spanish Ministry of Science and Innovation, and also by RoboCity2030-II-CM project (S2009/DPI-1559), funded by Programas de Actividades I+D en la Comunidad de Madrid and co-funded by Structural Funds of the EU.Publicad

Fast methods for Eikonal equations: An experimental survey

Fast methods are very popular algorithms to compute time-of-arrival maps (distance maps measured in time units) solving the Eikonal equation. Since fast marching was proposed in 1995, it has been applied to many different applications, such as robotics, medical computer vision, fluid simulation, and so on. From then on, many alternatives to the original method have been proposed with two main objectives: reducing its computational time and improving its accuracy. In this paper, we collect the main single-threaded approaches, which improve the computational time of the standard fast marching method and study them within a common mathematical framework. Then, they are evaluated using isotropic environments, which are representative of their possible applications. The studied methods are the fast marching method with the binary heap, the fast marching method with Fibonacci heap, the simplified fast marching method, the untidy fast marching method, the fast iterative method, the group marching method, the fast sweeping method, the locking sweeping method, and the double dynamic queue method.This work is funded by the projects: "RoboCity2030-DIH-CM Madrid Robotics Digital Innovation Hub (Robtica aplicada a la mejora de la calidad de vida de los ciudadanos. Fase IV; S2018/NMT-4331), funded by Programas de Actividades I+D en la Comunidad de Madrid and cofunded by Structural Funds of the EU,'' and "Investigacion para la mejora competitiva del ciclo de perforacion y voladura en mineriai y obras subterraneas, mediante la concepcion de nuevas tecnicas de ingenieriai, explosivos, prototipos y herramientas avanzadas (TUNEL).'

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program KK-2015/0000089). A.E. was recipient of a fellowship from the University of the Basque Country (UPV/EH) and D.G.-B. was recipient of a fellowship from the Bizkaia Biophysics Foundation

Cholesterol stimulates the lytic activity of Adenylate Cyclase Toxin on lipid membranes by promoting toxin oligomerization and formation of pores with a greater effective size

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore-forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore-forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein-protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins.Rocío Alonso is gratefully acknowledged for excellent technical assistance. This study was supported by grants from the Spanish Ministerio de Economia y Competitividad BFU2017-82758-P (H.O.) and of Basque Government (Grupos Consolidados IT1264-19). D.G.B was recipients of a fellowship from the Bizkaia Biophysics Foundation, and JA was recipient of a fellowship from the Basque Government

Hacking ético para profundizar en la implementación de sistemas informáticos seguros

Memoria ID-052 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2020-2021