Дисертації з теми "Intracellular vacuoles"

The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases like gastritis, duodenal and gastric ulcers, and gastric cancer. It has evolved a range of mechanisms to aid in colonization and persistence, manipulating the host immune response to avoid clearance. A major factor in this is the secreted vacuolating cytotoxin VacA which has a variety of effects on host cells. VacA is endocytosed and forms anion-selective channels in the endosome membrane, causing the compartment to swell. The resulting VacA-containing vacuoles (VCVs) can take up most of the cellular cytoplasm. Even though vacuolation is VacA's most prominent and namesake effect, the purpose of the vacuoles is still unknown. VacA exerts influence on the host immune response in various ways, both pro- and anti- inflammatorily. Most importantly, it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell activation and proliferation and thereby suppressing the host immune response. Furthermore, VacA is transported to mitochondria, where it activates the mitochondrial apoptosis pathway. Within the cell, VacA has only been shown to localize to endocytic compartments/VCVs and mitochondria. Considering its diverse effects, however, the existence of other cellular sites of action seems plausible. In this study, the VCV proteome was comprehensively analyzed for the first time in order to investigate VCV function. To this end, three different strategies for VCV purification from T-cells were devised and tested. Eventually, VCVs were successfully isolated via immunomagnetic separation, using a VacA-specific primary antibody and a secondary antibody coupled to magnetic beads. The purified vacuoles were then measured by mass spectrometry, revealing not only proteins of the endocytic system, but also proteins usually localized in other cellular compartments. This apparent recruitment of proteins involved in all kinds of cellular pathways indicates a central function of VCVs in VacA intoxication effects. In a global evaluation, the VCV proteome exhibited an enrichment of proteins implicated in immune response, cell death, and cellular signaling; all of these are processes that VacA is known to influence. One of the individual proteins contained in the sample was STIM1, a calcium sensor normally residing in the endoplasmic reticulum (ER) that is important in store- operated calcium entry (SOCE). This corroborates the findings of a concurrent report, in which VacA severely influenced SOCE and colocalized with STIM1. A direct interaction of STIM1 with VacA was examined in a pull-down assay, but could be neither shown nor excluded. Immunofluorescence experiments conducted in HeLa cells confirmed the presence of VacA in the ER and also found it to traffic to the Golgi apparatus, identifying these two cellular compartments as novel VacA target structures. The exact route of VacA transport remains unclear, but the involvement of both the ER and the Golgi suggests the possibility of retrograde trafficking, analogous to other bacterial toxins like shiga and cholera toxins. In summary, the elucidation of the VCV proteome and the discovery of the ER and the Golgi apparatus as VacA target structures have generated intriguing starting points for future studies. The detection of many proteins implicated in VacA intoxication effects in the VCV proteome leads to the proposal of VCVs as signaling hubs that may coordinate the complex meshwork of VacA effects. Further investigation of individual proteins is expected to help greatly in illuminating this matter.

Listeria monocytogenes est une bactérie pathogène intracellulaire facultative responsable d’une pathologie grave, la listériose. Si de très nombreux travaux ont permis de caractériser les mécanismes de virulence de cette bactérie, il existe peu de données sur les mécanismes conduisant au portage asymptomatique de L. monocytogenes dans les hôtes mammifères. L’un de ces mécanismes pourrait être une phase de persistance intracellulaire. Lors d’infections prolongées de cellules épithéliales humaines en culture, comme des hépatocytes et des cellules de trophoblastes, L. monocytogenes change de mode de vie intracellulaire. Après la phase active de dissémination de cellule en cellule, les bactéries arrêtent de polymériser l’actine et se retrouvent piégées dans des vacuoles à simple membrane marquées par la protéine endosomale LAMP1. L’objectif de ma thèse était de caractériser ces « Listeria-Containing Vacuoles » (LisCVs). Nous avons montré que les LisCVs sont des compartiments acides, partiellement-dégradatifs, marquées par la protéase lysosomale cathépsine D. Leur formation coïncide avec la disparition du facteur de polymérisation d’actine ActA de la surface bactérienne et la capture des bactéries cytosoliques dépourvues d’actine par des membranes cellulaires. Dans ces compartiments, les bactéries entrent en croissance ralentie ; une sous-population résiste aux stress et peut survivre au-delà de trois jours d’infection. L’utilisation de la gentamicine lors du protocole d’infection n’est pas responsable de la formation des LisCVs. Cependant, cet antibiotique permet la sélection des bactéries vacuolaires, en inhibant spécifiquement la croissance des bactéries cytosoliques. La formation des LisCVs n’est pas spécifique des souches de laboratoire. Toutefois l’efficacité du phénomène pourrait diverger selon les séquençotypes des souches de L. monocytogenes. Les bactéries vacuolaires ont la capacité de sortir des vacuoles et de retourner vers un état motile et réplicatif, après le passage des cellules infectées. Lorsque l’expression du gène actA reste inactive, comme dans les mutants ∆actA, des formes de Listeria vacuolaires persistent dans les cellules hôtes dans un état viable mais non cultivable (VBNC). Ces formes VBNC peuvent être transmises au cours des divisions des cellules hôtes. L’ensemble de ces résultats révèle une nouvelle phase de persistance dans le processus infectieux intracellulaire de L. monocytogenes lors des infections prolongées de certaines cellules épithéliales. Cette propriété pourrait contribuer au portage asymptomatique de ce pathogène dans les tissus épithéliaux, allonger la période d'incubation de la listériose, et rendre les bactéries tolérantes à l’antibiothérapie
Listeria monocytogenes is a facultative intracellular pathogenic bacterium responsible for a serious disease, listeriosis. Although much work has been done to characterize the virulence mechanisms of this bacterium, there is little data on the mechanisms leading to the asymptomatic carriage of L. monocytogenes in mammalian hosts. One of these mechanisms could be a phase of intracellular persistence. During prolonged infections of human epithelial cells in culture, such as hepatocytes and trophoblast cells, L. monocytogenes changes its intracellular lifestyle. After the active phase of cell-to-cell spread, the bacteria stop polymerizing actin and become trapped in single-membrane vacuoles labeled with the endosomal protein LAMP1.The aim of my thesis was to characterize these "Listeria-Containing Vacuoles" (LisCVs). We have shown that LisCVs are acidic, partially degradative compartments, labeled by the lysosomal protease cathepsin D. Their formation coincides with the disappearance of actin polymerization factor ActA from the bacterial surface and the capture of actin-free cytosolic bacteria by cell membranes. In these compartments, bacterial growth is slowed; a subpopulation is resistant to stress and can survive beyond three days of infection. The use of gentamicin during the infection protocol is not responsible for the formation of LisCVs. However, this antibiotic allows selection of vacuolar bacteria, by specifically inhibiting the growth of cytosolic bacteria. The formation of LisCVs is not specific to laboratory strains. However, the efficacy of the phenomenon could diverge according to the sequence types of L. monocytogenes strains. Vacuolar bacteria have the ability to exit the vacuoles and return to a motile and replicative state during the subculture of infected cells. When expression of the actA gene remains inactive, as in ΔactA mutants, vacuolar Listeria forms persist in host cells in a viable but non-culturable (VBNC) state. These VBNC forms can be transmitted during host cell divisions. All these results reveal a new phase of persistence in the intracellular infectious process of L. monocytogenes during prolonged infections of a subset of epithelial cells. This property could contribute to asymptomatic carriage of this pathogen in epithelial tissues, extend the incubation period of listeriosis, and make bacteria tolerant to antibiotic therapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Estreptococos do grupo B (EGB) comumente colonizam adultos saudáveis, sem sintomas, mas sob certas circunstâncias possui a capacidade de invadir tecidos do hospedeiro, evadir da detecção imunológica e causar doenças invasivas graves. Por conseguinte, os EGB continuam sendo uma das principais causas de mortalidade neonatal, pneumonia, sepse e meningite. Contudo, a patogênese desta infecção ainda está pouco elucidada. O sorotipo V é freqüentemente associado à doença invasiva em mulheres adultas não gestantes e o segundo mais prevalente em mulheres grávidas. O principal objetivo deste trabalho foi estudar a aderência, invasão e persistência intracelular de amostras pertencentes ao sorotipo V (88641-vagina/portador e 90186-sangue/paciente) usando as células epiteliais respiratórias A549. As amostras de EGB demonstraram capacidade de aderir e invadir as células epiteliais A549, mas somente a amostra 90186-sangue apresentou maior invasão quando comparada com a de vagina (P <0.001). Ambas as amostras demonstraram persistência intracelular sem replicação no interior das células A549. Apenas o isolado 90186-sangue sobreviveu dentro das células epiteliais até 24h de incubação (P <0,05). A fusão dos lisossomas das células epiteliais com vacúolos contendo bactérias foi observada em células A549 tratadas com Lyso Tracker Grenn DND-26 para todas as amostras testadas. Nossos dados indicam pela primeira vez que as amostras viáveis do sorotipo V permanecem dentro de vacúolos ácidos epiteliais. Curiosamente, a amostra 90186- sangue induziu vacuolização celular e a amostra 88641-vagina promoveu a morte celular após 7h de incubação. Finalmente, nossos resultados aumentam o nosso conhecimento sobre eventos celulares da fagocitose e da patogênese das doenças invasivas promovidas pelos EGB.
Group B Streptococcus (GBS) commonly colonizes healthy asymptomatic adults, yetunder certain circumstances displays the ability to invade host tissues, evade the immune system and cause serious invasive disease. Consequently, GBS remains the major cause of neonatal pneumonia, sepsis and meningitis. However, the pathogenesis of this infection is poorly understood. The serotype V is frequently associated with invasive diseases in non-pregnant adults and the second most prevalent in pregnant women. The aim of this work was to study the adherence; invasion and persistence intracellular of the GBS serotype V (88641-vagina/carriers and 90186-blood/patient) in epithelial cells A549. All GBS strains showed ability to adhere and invade the epithelial A549 cells, but GBS 90186-blood was more invasive than the vagina isolate (P<0,001). Both strains persisted intracellular, but without replicating into the A549 cells. Only 90186-blood strain survived within epithelial cells even after a 24h incubation (P<0,05). Fusion of epithelial lysosomes with bacteria containing phagocytic vacuoles was observed in A549 cells treated with Lysotracker Grenn DND-26 for all strains tested. Our data indicate for the first time that viable strains of serotype V remain within acidic epithelial vacuoles. Interestingly, the 90186-blood strain induced cellular vacuolization and 88641-vagina strain caused cell death after 7h incubation. Lastly, our results increase our knowledge about cellular events of phagocytosis and pathogeneses of GBS diseases.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.

The cytoplasm of eukaryotic cells contains several membrane-delimited compartments of specific molecular compositions and functions. Among those, the vacuole of fungal cells is often described as an organelle equivalent to the lysosomes of animal cells and the vacuoles of plant cells. These compartments indeed share two similar features: they contain a wide variety of hydrolases and are the most acidic compartments of the cell, which accounts for their key role in the intracellular degradation of macromolecules. In humans, dysfunctions of the lysosomes often give rise to lysosomal related diseases, such as lysosomal storage disorders. These are a class of metabolic disorders caused by the accumulation of non-degraded macromolecules or impaired export of hydrolytic degradation products. Cystinosis is an autosomal recessive disorder (1/200 000 incidence) generally associated with renal dysfunctions. It is caused by the accumulation and crystallization of cystine, the disulfide of cysteine, into the lumen of lysosomes. Cystinosin, the causative gene product of cystinosis, is present at the lysosomal membrane and catalyses the export of cystine from this compartment. The human cystinosin is a member of the Lysosomal Cystine Transporter (LCT) family. LCT proteins are conserved in all eukaryotic species and are defined by the presence of highly conserved PQ-loop motifs.

During this thesis work, we have studied three LCT proteins of the yeast Saccharomyces cerevisiae, named Ypq1, Ypq2 and Ypq3 (Yeast PQ-loop proteins 1, 2 and 3). We first showed that these proteins localize to the vacuolar membrane. We next studied the roles of these proteins, the regulation of their genes and the mechanisms and signals implicated in their delivery to the vacuolar membrane. We also contributed to the functional characterization of a mammalian homologue of yeast Ypq proteins, named rPqlc2.

In the first part of this work, we report that the Ypq proteins are most probably implicated in the export of basic amino acids from the vacuole to the cytosol. More precisely, Ypq2 and Ypq3 behave like vacuolar arginine and lysine exporters, respectively. Interestingly, the mammalian rPqlc2 protein expressed in yeast reaches the vacuolar membrane and functions as an orthologue of the Ypq proteins. Our results also reveal that the expression of the YPQ3 gene is regulated by the Lys14 transcription factor, responsible for the transcriptional activation of the LYS genes encoding enzymes implicated in the biosynthesis of lysine. We have also noted that, in general, the expression of the expression of the YPQ genes is regulated according to the quality of the nitrogen source available in the extracellular medium, eg. YPQ3 is sensitive to the nitrogen catabolite repression regulatory mechanism.

In the last part of this thesis work, we investigated the intracellular trafficking of the Ypq proteins and show that these predominantly reach the vacuolar membrane via the ALP (alkaline phosphatase) pathway due to the presence of a dileucine-based sorting signal in their sequences. Interestingly, a similar mechanism seems responsible for targeting to the yeast vacuole of the mammalian rPqlc2 protein.

Une caractéristique des cellules eucaryotes est leur organisation en compartiment internes délimité par une membrane lipidique, appelé organelles. Ces compartiments intracellulaires présentent une composition lipidique et protéique particulaire conforme à leur identité et fonction. Les lysosomes de cellules de mammifères et la vacuole fongique jouent un rôle clé dans la digestion intracellulaire de macromolécules et de ce fait leurs lumières sont enrichis d’enzymes hydrolytiques nécessaires à cette action. Des disfonctionnements du lysosome peuvent être la conséquence de pathologie chez l’homme, regroupé sous le nom de maladie lysosomale, lié à un à une accumulation de macromolécules non digéré ou un default d’export des produits d’hydrolysé depuis la lumière du lysosome. La cystinose est une maladie autosomale récessive avec une faible fréquence d’incidence (1/200 000) qui regroupe trois formes cliniques :deux formes rénales graves et une forme extra-rénale. Cette maladie est due à une accumulation et cristallisation de cystine dans la lumière du lysosome qui est corrélé à des mutations ponctuelles dans le gène CTNS qui code pour l’exporteur de cystine, la cystinosine. Cette protéine est un membre de la famille LCT (Lysosomal Cystine Transporter) qui possède des représentants chez les cellules animales, végétales et fongiques. Les protéines de la famille possèdent une taille et une topologie prédite similaire (7 segments transmembranaires) et on retrouve aussi au sein de ces protéines deux exemplaires de motifs PQ. Lors de ce travail de thèse nous nous sommes intéressés à trois membres de la famille LCT chez Saccharomyces cerevisiae que nous avons nommé Ypq1, Ypq2 et Ypq3 pour Yeast PQ-loop proteins. Ces protéines n’ayant pas fait l’objet de nombreuses études, nous nous sommes orientés vers une analyse fonctionnelle et transcriptionnelle. De plus, nous avons également étudié les mécanismes et signaux impliqué dans leur adressage vers la vacuole. Finalement, nous avons également inclus dans notre étude un homologue mammalien de ces protéines, rPqlc2.

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Doctorat en Sciences
info:eu-repo/semantics/nonPublished

The mechanism of protein sorting to the vacuole in yeast was studied both in vitro and in vivo. A series of experiments were performed to reconstitute transport of carboxypeptidase Y (CPY) from Golgi vesicles to vacuoles. In order to investigate this process, microsomes were purified from sec, pep4-3 mutant strains that accumulate inactive proCPY in the Golgi when incubated at the nonpermissive temperature. These were mixed with purified vacuoles isolated from a mutant lacking CPY activity, but containing active proteinases A and B. Transported proCPY is maturated by these proteinases to active form. Experiments indicate that maturation of CPY is due to the correct transport of proCPY from microsomes to vacuoles because:- Firstly, the reaction is temperature sensitive, requires ATP and is stimulated by the addition of soluble factors (S100). Secondly, the addition of proteinase A and B inhibitors to the reaction mixtures has a negligible effect on the maturation process. Thirdly, disrupting the membranes by the addition of Triton X-100 before addition of the proteinase inhibitors, inhibited the maturation of proCPY. Fourthly, the majority of CPY activity was observed in the sedimented fraction of the reaction mixtures rather than supernatant fractions. Lastly, analysis with western blot shows a clear band of mature CPY only in the sedimented fraction of the reaction mixtures with ATP. This in vitro system will be invaluable in investigating the molecular events of vacuolar biogenesis. For in vivo sorting of proteins to the vacuole, a series of experiments were performed that involved the genetic fusion of the CPY promoter and prepro-sequence of CPY to the bacterial Gus (β-glucuronidase) reporter gene. The Gus gene was expressed in yeast with high efficiency and the results of sub-cellular fractionation indicated that the Gus product was distributed in all cell components. Using a centromeric vector gave similar results but with a lower efficiency of Gus expression. Removal of 90bp from Gus, including Gus initiation codon does not completely inhibit Gus expression either in bacteria or in yeast. Fusion of the shortened Gus with the CPY prepro-fragment and expression in yeast led to the correct sorting of the CPY-Gus hybrid protein to the vacuole. This CPY-Gus fusion is potentially useful in the genetic analysis of mutations defective in vacuolar protein sorting.

Le système endomembranaire ou système de sécrétion est constitué de compartiments spécifiques et distincts et de vésicules qui assurent le transport entre ces différents compartiments. Les protéines sécrétées, solubles ou membranaires, sont introduites de façon co-traductionnelle dans le réticulum endoplasmique (RE). Du RE, ces protéines sont transportées tout au long du système endomembranaire de sécrétion jusqu'à leur compartiment cible: l'appareil de Golgi, la vacuole le tonoplaste, la membrane plasmique ou le compartiment extracellulaire. Des travaux récents ont permis d'identifier des signaux spécifiques d'adressage et de rétention qui par leur interaction avec des récepteurs, permettent le transport de certaines protéines vers leur localisation finale. Nos travaux font appel aux approches de la biologie cellulaire et moléculaire pour l'étude du transport et de l'adressage des protéines dans le système endomembranaire de sécrétion chez les plantes. Nous avons modifié par mutagénèse dirigée les signaux d'adressage d'une protéine recombinante modèle, la sporamine. De plus nous avons développé un système d'expression stable de ces protéines recombinantes dans des cellules de tabac (nicotiana tabacum CV BY2). Nous avons ainsi obtenu des cellules transgéniques exprimant fortement une même protéine modèle transportée vers la vacuole (sporamine), vers le milieu extracellulaire (dpro-sporamine) ou retenue dans la fraction microsomale (SpoHDEL ou dproHDEL). L'étude de la localisation intracellulaire de SpoHDEL a permis de montrer l'accumulation de cette protéine dans un compartiment présentant une activité IDPase

Les aquaporines sont des canaux hydriques qui contrôlent la perméabilité à l'eau des membranes cellulaires, au cours du développement ou en réponse à des stress. La dynamique de l'expression des aquaporines de plantes et leur rôle physiologique ont été examinés dans deux organes modèles, le pollen et la racine d'Arabidopsis. Le pollen mature contient une cellule végétative et deux cellules de sperme. Des analyses transcriptomiques ont récemment identifié AtTIP1;3 et AtTIP5;1 comme deux aquaporines spécifiques du pollen. Dans ce travail, des protéines reportrices fluorescentes ont permis d'établir que AtTIP1;3 et AtTIP5;1 s'expriment spécifiquement dans la membrane vacuolaire de, respectivement, la cellule végétative et les cellules de sperme. Ces études révèlent aussi la grande plasticité dynamique des vacuoles, de la maturation du pollen jusqu'à la fécondation. Des approches de génétique inverse suggèrent un rôle des deux aquaporines dans la reproduction de la plante. La seconde partie de ce travail concerne les effets concomitants des stress oxydants, inhibant la conductivité hydraulique des racines et provoquant une accumulation intracellulaire des aquaporines initialement sur les membranes plasmiques. Le dernier processus a été disséqué par des approches de biochimie, pharmacologie et microscopie. La co-expression avec des marqueurs des endomembranes a révélé que l'isoforme AtPIP2;1 subit une accumulation dans les endosomes tardifs en réponse à l'H2O2. Ce processus peut être bloqué par l'auxine synthétique 1-NAA, mais non par l'inhibiteur d'endocytose tyrphostine A23. La grande stabilité des aquaporines internalisées suggère que l'H2O2 déclenche un mécanisme de séquestration réversible de celles-ci. Au-delà de données originales sur la régulation cellulaire des aquaporines, ce travail apporte un éclairage nouveau sur la dynamique des membranes intracellulaires des plantes, au cours du développement ou en réponse à des stress
Aquaporins are membrane water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The dynamic expression of aquaporins in planta, as well as their role in plant water relations, were investigated in two representative model organs, the pollen and roots of Arabidopsis. Mature pollen consists of a vegetative cell and two sperm cells. Transcriptomics recently identified AtTIP1;3 and AtTIP5;1 as two pollen exclusive aquaporins. Here, we investigated their in vivo temporal and spatial expression pattern. Fluorescently-tagged chimeras revealed that AtTIP1;3 and AtTIP5;1 have a distinct and specific localisation in the vacuolar membrane of the vegetative and sperm cells, respectively. The two aquaporins also revealed the dynamic plasticity of vacuoles from pollen maturation to embryo fecundation. Loss of function approaches suggest an implication of both proteins in plant reproduction. The second part of this work focused on the oxidative stress-induced internalisation of root plasma membrane aquaporins and its concomitant drop in root hydraulic conductivity. The former process was described in great detail by combined biochemical, pharmacological and microscopic approaches. Co-expression analyses of the AtPIP2;1 isoform with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in late endosomal compartments. This process could be antagonized by the auxin analog 1-NAA, but not by the endocytosis blocker tyrphostin A23. Life-time analyses established the high stability of the internalised protein suggesting that H2O2 triggers a mechanism for intracellular and reversible sequestration of plasma membrane aquaporins. Besides information on cell regulation of aquaporins, the overall work gives novel and complementary insights into the dynamic remodelling of plant internal membranes during development and stress responses

Les Chlamydia sont des bactéries intracellulaires strictes dont trois espèces sont pathogènes pour l'Homme. Elles sont responsables, selon les souches, d'infections oculaires et génitales et d'infections respiratoires. Après avoir induit leur propre entrée, les bactéries se développent dans un compartiment délimité par une membrane, appelé inclusion. Au cours du cycle infectieux, les bactéries transloquent, par leur appareil de sécrétion de type trois (STT), des protéines dont certaines s'ancrent dans la membrane de l'inclusion où elles sont en contact avec le cytosol de la cellule hôte, les protéines Inc. Mon travail de thèse a porté sur les protéines bactériennes sécrétées, au cours du cycle infectieux, dans la cellule hôte. Une approche globale nous a permis d'identifier de 24 nouvelles protéines de Chlamydia sécrétées par l'appareil de STT. Ce travail a ouvert la voie à l'étude fonctionnelle de ces protéines bactériennes qui sont en contact avec l'hôte. De manière plus spécifique, nous avons étudié la fonction d'une protéine de l'inclusion, IncA, au cours du cycle infectieux. Nous avons montré que IncA présente des similitudes structurales et fonctionnelles avec les protéines eucaryotes, les SNAREs, facteurs essentiels de la fusion des membranes cellulaires. IncA interagit dans un modèle cellulaire, et in vitro, avec certaines SNAREs. De plus, dans des expériences de fusion de liposomes in vitro, la fusion membranaire induite par un complexe SNARE des endosomes tardifs est inhibée par IncA. Ce travail a permis de proposer que IncA puisse mimer les SNAREs et participer au détournement du trafic intracellulaire induit par Chlamydia
Chlamydiae are obligate intracellular pathogens of humans and animals. Depending on the species, they are responsible for ocular and genital infections, and respiratory diseases. After inducing their own entry, the bacteria develop in a membrane-bound compartment, called the inclusion. During the infectious cycle, they translocate a subset of proteins via a type three secretion (TTS) apparatus into the host cytosol. Among these, the Inc proteins remain anchored in the inclusion membrane where they face the host cytosol. My work has focused on bacterial proteins secreted into the host cell. By a global approach, we have identified 24 new proteins secreted by the TTS apparatus of Chlamydia. This work has opened the functional studies of these bacterial proteins that are in contact with the host cell cytosol. More specifically, we have studied the function of an inclusion protein, IncA. We have shown that IncA, from different chlamydial species, share structural and functional homologies with the SNARE family of eukaryotic proteins, which are essential factors for cellular membrane fusion events. We have shown that IncA interact with SNAREs in both a cellular and an in vitro model. Moreover, in liposome fusion assays, IncA inhibit membrane fusion induced by a cognate SNARE complex specific from the late endosomal compartment. We propose that IncA, by mimicking SNAREs proteins, participate in the control of the interactions between the inclusion membrane and intracellular compartments of the host cell

Chez la levure Saccharomyces cerevisiae Las17p est l’homologue de WASp (Wiskott-Aldrich syndrome protein des cellules mammifères), responsable de l’activation de la polymérisation d’actine dépendante d’Arp2/3 qui forme des tâches corticales. Les résultats préliminaires du laboratoire avaient démontré que la peroxine Pex13p est capable d’interagir via son domaine Src homology 3 (SH3) avec Las17p, in vitro dans un essai GST pull-down. Cette étude est alors basée sur la recherche d’un lien éventuel entre la machinerie de polymérisation d’actine et les peroxysomes. La peroxine 13 (Pex13p) comme Pex14p et Pex5p fait partie intégrante du complexe d’amarrage à la membrane peroxysomale et est essentielle pour l’import dans les peroxysomes. Mes résultats favorisent l’hypothèse que le cytosquelette d’actine pourrait affecter les peroxysomes non pas directement sur l'import mais à travers un effet sur leur biogénèse, non encore précisé ayant un impact sur leur taille et leur nombre. Le gène hMTM1 sous une forme mutée est responsable de la myopathie myotubulaire liée au chromosome X (XLMTM). Il code pour une phosphatase à phosphoinositides, membre de la famille des myotubularines comprenant un unique homologue levure, Ymr1p. L’objet de la deuxième partie de ma thèse était de mieux comprendre la fonction intracellulaire et éventuellement la préférence de substrat de cette enzyme en utilisant le modèle levure S. Cerevisiae. Suivant les résultats obtenus, l’expression hétérologue de la myotubularine humaine chez la levure S. Cerevisiae produit une enzyme active, provoquant l’élargissement des vacuoles. Le phénotype observé est dû à l’activité phosphatase et donc à la diminution du taux de PtdIns(3,5)P2. Les résultats suggèrent un rôle de hMTM1 dans le trafic membranaire et la maturation des endosomes précoces et tardifs et dans l’homéostasie vacuolaire
In yeast Saccharomyces cerevisiae Las17p is the yeast homologue of WASP (Wiskott-Aldrich syndrome protein) in mammalian cells and a principal activator of actin polymerization Arp2/3 dependant that forms actin patches. Preliminary results from a GST pull-down assay in the laboratory had shown that Pex13p was able to interact, via its domain Src homology domain 3 (SH3), with Las17p. This study is based on the search for an eventual link between components of the actin polymerization machinery and peroxisomes. The peroxine Pex13p, together with Pex14p and Pex5p, is a part of the import complex at the peroxysomal membrane. My results favour the hypothesis that the actin cytoskeleton might regulate not the import into peroxisomes directly, but through an effect on peroxysomal biogenesis having an impact on peroxysomal size and number. Mutated forms of the hMTM1 gene are responsible for the X-linked myotubular myopathy (XLMTM) and it encodes a phosphoinositides phosphatase, a member of the myotubularine family, having an unique homologue in yeast S. Cerevisiae, Ymr1p. According to the results, heterologous expression of hMTM1 in yeast S. Cerevisiae leads to the production of an active enzyme, causing an enlarged vacuole phenotype. This phenotype is due to the phosphatase activity and therefore to the decrease in the level of PtdIns(3,5)P2. The results suggest a role for hMTM1 in membrane trafficking, in early and late endosome maturation and in maintaining vacuolar homeostasy

Resultats obtenus chez melilotus alba et acer pseudoplatanus. Methodologie permettant l'obtention de vacuoles isolees et d'une fraction cytosolique purifiee. Identification, caracterisation et localisation dans la cellule, des systemes proteolytiques responsables de la degradation des proteines. Specificite des proteases vis-a-vis de differents substrats proteiques. Definition d'un modele d'etude permettant une approche in vivo du phenomene afin d'apprecier le niveau d'intervention de la vacuole dans les processus proteolytiques

Dans la cellule végétale, les protéines solubles vacuolaires et extracellulaires entrent dans le système endomembranaire de sécrétion au niveau du réticulum endoplasmique. Ces protéines sont ensuite transportées via des vésicules de transport jusqu'à leur compartiment de stockage, la vacuole et le compartiment extracellulaire, en passant par l'appareil de Golgi. Dès leur insertion dans la lumière du RE, ces protéines vont subir des modifications co-traductionnelles, en particulier la N-glycosylation et le repliement qui leur conféreront stabilité et activité biologique. Des suspensions cellulaires de tabac BY-2 exprimant simultanément plusieurs protéines-marqueur vacuolaires et extracellulaires nous ont permis d'étudier les implications des modifications co-traductionnelles dans le transport de protéines et de glycoprotéines à travers le système endomenbranaire de sécrétion. Nous avons montré qu'un defaut de repliement d'une glycoprotéine vacuolaire peut d'une part entraîner une modification de sa N-glycosylation et d'autre part sa réorientation vers la surface cellulaire. Les mécanismes de tri et la maturation des glycoprotéines vacuolaires ont été également étudiés en présence de drogues perturbant le fonctionnement de l'appareil de Golgi. Enfin, le transport protéique dans le système endomembranaire de sécrétion a été étudié dans des conditions où la N-glycosylation est inhibée par la tunicamycine. Ainsi, nous avons montré qu'en absence de N-glycosylation, le transport à la vacuole des protéines et des glycoprotéines a lieu mais se trouve fortement ralenti. En revanche, la sécrétion des glycoprotéines extracellulaires est totalement inhibée alors que, dans les mêmes conditions, les protéines sont transportées vers le compartiment extracellulaire. Nous avons pu préciser que les glycoprotéines extracellulaires non-glycosylées en presence de tunicamycine sont dégradées dans le système endomembranaire de sécrétion par un mécanisme pré-golgien et indépendant des protéasomes.

Salmonellen spp. sind invasive, intrazelluläre Pathogene, die in einem membranumhüllten Kompartiment innerhalb der infizierten Wirtszelle überleben. Wie auch andere intrazelluläre Pathogene repliziert Salmonella in dieser intrazellulären Nische, obwohl es anscheinend von sowohl extra- als auch intrazellulären Nährstoffquellen isoliert ist. Wir zeigen hier, dass intrazelluläre Salmonella den Proteinabbau des Wirts ausnutzen, um Aminosäuren in Form von Peptiden zu erhalten. Dieser spezielle, auch als Chaperon-vermittelte Autophagie bekannte, Abbauweg spielt eine Rolle im Transport zytosolischer Proteine zum Abbau im Lysosom. Ein Salmonellenmutant, der nur in Anwesenheit von Peptiden im Medium als Aminosäurenquelle wächst, wies intrazellulär eine Wachstumsrate auf, die der des Wildtyps ähnlich war. Dies deutet darauf hin, dass Peptide intrazellulär für Salmonella zugänglich sind. Wir fanden heraus, dass die Salmonella-enthaltende Vakuole (SCV, Salmonella containing vacuole) die Wirtproteine LAMP-2A und Hsc73, Kernkomponenten von CMA, anzieht, jedoch nicht lysosomale Proteine wie LAMP-2B und LIMP-2. Im Gegensatz zum Salmonellawildtyp zeigte der peptidabhängige Mutantentstamm stark verringertes Wachstum, wenn die Wirtszellen mit CMA-Inhibitoren behandelt wurde. Diese Ergebnisse zeigen einen neuen Mechanismus auf, durch den ein intrazelluläres Pathogen vom membranumhüllten Kompartiment aus Zugriff auf Cytosol der Wirtzelle zur Beschaffung von Nährstoffen hat. Wir schlagen vor, dass diese Ergebnisse eine Erklärung für die Rückfälle von persistenten Salmonellainfektionen liefern können. Des Weitern schlagen wir diesen Mechanismus als moegliches Ziel antibakterieller Therapeutika zur Bekämpfung intrazellulärer Pathogene vor.
Salmonella spp. are invasive, intracellular pathogens which survive and proliferate within a membrane-bound compartment inside infected host cells. Like other intracellular pathogens, Salmonella replicates within this intracellular niche, despite its apparent isolation from both extra- and intracellular sources of nutrients. Here, we show that intracellular Salmonella acquire amino acids in the form of peptides by co-opting the host protein degradation pathway known as chaperone-mediated autophagy (CMA) involved in the transport of cytosolic proteins to the lysosome for degradation. A mutant of Salmonella strictly dependent upon peptides in growth media as a source of amino acids, showed intracellular growth similar to the wild-type strain in host cells, indicating intracellular access to peptides. We found that the Salmonella-containing vacuole (SCV) acquires the host cell proteins LAMP-2A and Hsc73, key components of CMA, but excludes lysosomal proteins such as LAMP-2B and LIMP-2. In contrast to wild-type Salmonella, the peptide-dependent mutant strain showed a severe reduction in growth when host cells were treated with inhibitors of CMA.. These results reveal a novel means whereby an intracellular pathogen can access the host cell cytosol to acquire nutrients from within its membrane-bound compartment. We suggest these results may provide an explanation for relapse infections resulting from persistent Salmonella infections, and suggest a possible means of targeting antibacterials against intracellular pathogens.

Tesis entregada a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al grado de Doctor en Ciencias con Mención Microbiología.
Salmonella Typhimurium is an enteric pathogen able to infect different animal hosts, including humans. In immunocompetent humans, S. Typhimurium mainly causes gastroenteritis, a disease characterized by an inflammatory diarrhoea with massive neutrophil infiltration in the ileum and colon. The infective cycle of Salmonella starts with the ingestion of bacteria that reach the small intestine and invade epithelial cells by its apical face. After crossing the epithelial barrier, bacteria are captured by phagocytic cells of the immune system present in the sub-epithelium, such as macrophages, neutrophils and dendritic cells, being contained within a membrane bound compartment. Here, Salmonella subverts the endocytic route, avoiding the fusion of this compartment with the lysosomes and generating the Salmonella-containing vacuole (SCV). In this compartment, Salmonella is able to survive and replicate. The ability to modify this intracellular niche explains the ability of this pathogen to survive intracellularly. To carry out this process, Salmonella employs two Type Three Secretion Systems (T3SS) and an arsenal of secreted effector proteins in order to take control over the eukaryotic cell. An important aspect of Salmonella’s life cycle that has not been studied in detail is its survival in the environment, where bacteria are exposed to predation by protozoa, and specially by amoebae. These organisms are specialized phagocytes that feed on bacteria and fungi. To address this interaction, we and other groups use amoeba models to characterise the molecular processes involved in the survival of intracellular pathogens within environmental protozoa. Among these model organisms, the social amoeba Dictyostelium discoideum is amenable for molecular analyses in laboratory settings and several tools have been developed in this organism for the study of different aspects of its interaction with bacterial pathogens. Recently, our group described that S. Typhimurium is able to survive intracellularly in the social amoeba Dictyostelium discoideum, and that mutants in genes required for virulence in other infection models present survival defects in this host. Despite of this, the mechanisms that allow the intracellular survival of this pathogen in this kind of organism have not been studied in detail. This Thesis proposed the characterization at the cellular level of this interaction, with a focus on two secreted effector proteins of S. Typhimurium that are directly related to SCV generation and modification in other cell models: SopB and SifA. Our results show that these effectors are needed for intracellular survival of S. Typhimurium in D. discoideum. Furthermore, by means of a combination of microscopy and proteomic analyses we were able to characterise the protein composition of the vacuolar compartment containing Salmonella in this host. Our results show that known markers linked to this compartment in other cell types and the autophagy machinery play a role in the biogenesis of this intracellular niche in D. discoideum.
Salmonella Typhimurium es un patogeno enterico que tiene la capacidad de infectar diversos hospederos animals, incluyendo el ser humano. En individuos inmunocompetentes, S. Typhimurium provoca gastroenteriris, una enfermedad diarreica inflamatoria caracterizada por la masiva infiltracion de neutrofilos en el ileon y el colon. El ciclo infectivo de Salmonella comienza con la ingestion de las bacterias que al llegar al intestino delgado invaden las celulas epiteliales por la cara apical. Luego de cruzar la barrera epitelial, las bacterias son capturadas por las celulas fagociticas del sistema inmune que residen en el sub-epitelio, como macrofagos, neutrofilos y celulas dendriticas, quedando contenida en un compartimento membranoso. En esta etapa, Salmonella interviene la ruta endocitica, evitando la fusion de este compartimento con el lisosoma y generando la vacuola contenedora de Salmonella (Salmonella-containing vacuole: SCV). En este compartimento, Salmonella es capaz de sobrevivir y replicarse. La habilidad de modificar este nicho intracelular explica la habilidad de este patogeno de sobrevivir intracelularmente. Para esto, Salmonella utiliza dos Sistemas de Secrecion Tipo Tres (Type Three Secretion Systems: T3SS) y un arsenal de proteinas efectoras secretadas para tomar control sobre la celula eucarionte. Por otra parte, un importante aspecto del ciclo de vida de Salmonella que no ha sido estudiado en detalle es su supervivencia en el ambiente, donde las bacterias se encuentran expuestas a la depredacion por protozoos y en particular, amebas. Estos organismos son fagocitos profesionales que se alimentan de bacteria y hongos. Recientemente, nuestro grupo describio que S. Typhimurium es capaz de sobrevivir intracelularmente en la ameba social Dictyostelium discoideum y que mutantes en genes requeridos para la virulencia en numerosos modelos de infeccion tambien presentan defectos de supervivencia en este hospedero. A pesar de esto, los mecanismos que le permiten a este patogeno en este tipo de organismo no han sido estudiado en detalle. Para entender esta interaccion, nosotros y otros grupos usamos modelos de ameba a fin de caracterizar los procesos moleculares involucrados en la supervivencia de patogenos intracelulares en el interior de protozoos ambientales. Dentro de estos organismos modelo, la ameba social Dictyostelium discoideum es sencilla para el analisis molecular en condiciones de laboratorio. Por otra parte, numerosas herramientas se han desarrollado en este organismo para el estudio de diversos aspectos de su interaccion con patogenos bacterianos. Esta Tesis propuso caracterizar a nivel cellular esta interaccion, enfocandonos en dos proteinas efectoras secretadas de S. Typhimurium que estan directamente relacionadas a la formacion y modificacion de la SCV en otros modelos celulares: SopB y SifA. Nuestros resultados muestran que estos efectores son necesarios para que S. Typhimurium sobreviva intracelularmente en D. discoideum. Adicionalmente, mediante una combinacion de tecnicas de microscopia y analisis proteomicos pudimos caracterizar la composicion proteica de este compartimento vacuolar que contiene a Salmonella en este hospedero. Nuestros resultados muestran que marcadores asociados a la SCV en otras lineas celulares se encuentran en elcompartimento que se genera en D. discoideum y que la maquinaria de autofagia juega un rol importante en la biogenesis de este nicho intracelular en D. discoideum.
FONDECYT grants 1140754 y 1171844, CONICYT Doctoral Fellowship 21140615.
Enero 2020.

Les lymphocytes T CD8 jouent un rôle central dans l'immunité protectrice contre les pathogènes intracellulaires tels que le parasite Toxoplasma gondii (T. gondii). T. gondii réside à l'intérieur d'une cellule hôte et dans une vacuole parasitophore. L'interface entre l'hôte et le parasite comprend une membrane limitant la vacuole ainsi qu'un réseau intravacuolaire (IVN) composé de tubules membranaires fortement incurvés et dont la fonction reste incertaine. Beaucoup d'effecteurs parasitaires, incluant des sources potentielles d'antigènes pour les lymphocytes T CD8, sont sécrétés par T. gondii dans la vacuole et adressés à différents endroits dans la vacuole ou au-delà, dans la cellule hôte. A l'heure actuelle, les mécanismes contrôlant l'adressage des antigènes parasitaires dans la cellule hôte demeurent mal compris et nous ne savons pas comment le transport intracellulaire des protéines de T. gondii influence leur disponibilité par la voie de présentation CMH I et leur capacité à induire l'immunité T CD8. En utilisant une approche multidisciplinaire combinant la génétique inverse de T. gondii, la microscopie, la présentation antigénique in vitro et l'expérimentation animale, mes travaux de thèse ont montré que l'insertion d'un antigène immunodominant à la membrane limitante de la vacuole constitue une des clés de l'immunogénicité. J'ai également montré que l'association de cet antigène à l'IVN limite sa présentation par les molécules du CMH I et réduit les réponses T CD8 spécifiques de cet antigène chez la souris. L'IVN pourrait jouer un rôle immuno-modulateur dans lequel il limiterait l'accès de protéines parasitaires sécrétées au cytosol de la cellule hôte et à la voie CMH I
CD8 T cells play a key role in protective immunity against intracellular pathogens such as Toxoplasma gondii (T. gondii) parasite. T. gondii resides inside host cell in parasitophorous vacuole. The host-T. gondii interface comprises a vacuole limiting membrane and a highly curved membraneous IntraVacuolar Network (IVN) of uncertain function. Many parasite effectors, including potential epitopes for CD8 T cells, are secreted by T. gondii to and across the boundary of their parasitophorous vacuole. Currently, the mechanisms controlling the targeting of parasite antigens to host cell are misunderstood et we don't kwnow how the intracellular transport of T. gondii proteins impacts on their access to MCH I pathway and their ability to induce CD8 T cell immunity. Using a multidisciplinary approach which combined reverse genetics in T. gondii, microscopy, antigen presentation measurements and in vivo experiments, I showed that insertion of a T. gondii dominant antigen at the vacuole limiting membrane is key for immunogenicity, yet that association of this antigen to high curvature IVN limits its presentation and curtails specific CD8 responses in mice. The IVN may play a role in immune modulation by limiting the access of parasite proteins to host cytosol and thus to MHC I pathway

Brucella est un pathogène bactérien intracellulaire responsable de la brucellose animale et humaine. Des études in vitro ont démontré que Brucella est capable de contourner la voie d’endocytose pour assurer sa survie intracellulaire à la fois dans les cellules phagocytaires et nonphagocytaires. Les brucelles assurent leur réplication par la biogenèse d’une vacuole de réplication issue du réticulum endoplasmique (ER) appelée « Brucella-containing vacuole » (BCV). L’identification de protéines spécifiques localisées sur la membrane de la BCV est essentielle pour comprendre comment Brucella est capable de maintenir ces interactions avec le ER et de continuer à se répliquer au sein de ce compartiment. Nous avons caractérisé pour la première fois la composition protéique de sa niche de réplication par des approches protéomiques. Pour cela, nous avons développé une méthode de fractionnement subcellulaire afin d’isoler les BCV. L’analyse de la composition protéique de la membrane de la BCV par électrophorèse sur gel en 2 dimensions et par spectrométrie de masse a permis l’identification de plusieurs protéines eucaryotes et procaryotes. Nous avons obtenu des protéines du ER et des protéines ribosomales qui valident le modèle : la niche de réplication de Brucella est une vacuole qui dérive du ER. De plus, nous avons identifié plusieurs protéines eucaryotes et bactériennes pouvant contribuer à la virulence de Brucella dans les cellules hôtes. Nous avons focalisé notre étude sur 2 protéines eucaryotes : la glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et la petite protéine G Rab 2, qui sont recrutées au niveau de la vacuole de Brucella. Ce complexe a été décrit précédemment pour être localisé sur les vésicules et les tubules de transport (VTC) et pour réguler le trafic des membranes des VTC entre le ER et l’appareil de Golgi. La destruction de l’appareil de Golgi par des traitements prolongés à la bréfeldine A réduit la réplication de B. Abortus. De plus, l’inhibition de l’expression de GAPDH par l’ARN interférence et le blocage de Rab 2 dans sa forme GDP inhibent fortement la réplication de B. Abortus. Ces résultats révèlent un rôle essentiel pour GAPDH et Rab 2 dans la virulence de B. Abortus à l’intérieur des cellules
Brucella is a facultative intracellular pathogenic bacteria which causes severe infections and abortion in animals and a long-lasting febrile disease in humans. Essential to Brucella virulence is its ability to survive and replicate inside host cells. In vitro studies showed that Brucella initially avoids or escapes the endocytic pathway to ensure its intracellular survival both in phagocytic and nonphagocytic cells. Bacteria secure their replication via the biogenesis of an endoplasmic reticulumderived replicative organelle named “Brucella-containing vacuole” (BCV). Identification of specific proteins on the BCV membrane is essential to understand how Brucella sustain interactions with the endoplasmic reticulum (ER) and keeps replicating within its compartment. We characterize for the first time the protein composition of its replicative niche by proteomic approches. We developed a subcellular fractionation method to isolate BCVs. The analysis of BCV membrane proteins by 2 dimensional gel electrophoresis and mass spectrometry allowed the identification of several eukaryotic and prokaryotic proteins. We obtained ER proteins and ribosomal proteins that validate the model: the replicative niche of Brucella is an ER-derived vacuole. Furthermore, we have identified several interesting host proteins as well as some bacterial proteins that may contribute to Brucella virulence within host cells. We focused our study on two eukaryotic proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2, which are recruited to the vacuolar membrane of Brucella. This complex was previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between ER and Golgi. Disruption of the Golgi apparatus with prolonged brefeldin A treatment reduced B. Abortus replication. Furthermore, inhibition of GAPDH expression by small interfering RNA and blockage of Rab 2 GTPase in a GDP-locked form strongly inhibited B. Abortus replication. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. Abortus virulence within host cells

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase.
This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.