Heart failure contributes to nearly 60,000 deaths per year in the USA and is often caused by hypertension and preceded by the development of left ventricular hypertrophy (LVH). LVH is usually accompanied by intensive interstitial and perivascular fibrosis which may contribute to arrhythmogenic sudden cardiac death. Emerging evidence indicates that LV dysfunction in patients and animal models of cardiac hypertrophy is closely associated with perivascular inflammation. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of pressure-overload-induced LVH: transverse aortic constriction (TAC). Early perivascular inflammation was indicated by accumulation of macrophages and T lymphocytes 24 hours post-TAC and which peaked at day 7. Coronary luminal platelet deposition was observed along with macrophages and lymphocytes at day 3. Also, LV protein levels of VEGF and MCP-1 were significantly increased. Consistent with lymphocyte accumulation, cardiac expression of IL-10 mRNA was elevated. Furthermore, circulating platelet-leukocyte aggregates tended to be higher after TAC, compared to sham controls. Platelets have been shown to modulate perivascular inflammation and may facilitate leukocyte recruitment at sites of inflamed endothelium. Therefore, we investigated the impact of thrombocytopenia in the response to TAC. Immunodepletion of platelets decreased early perivascular accumulation of T lymphocytes and IL-10 mRNA expression, and altered subsequent coronary artery remodeling. The contribution of lymphocytes was examined in Rag1-/- mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation which will contribute to long-term LV remodeling. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. To determine the contribution of granule release from inflammatory cells in the development of LVH, we used Unc13dJinx (Jinx) mice, which contain a single point mutation in Unc13d gene resulting in defects in Munc13-4. Munc13-4 is a limiting factor in vesicular priming and fusion during granule secretion. Therefore, Jinx mice have defects in degranulation of platelets, NK cells, cytotoxic T lymphocytes, neutrophils, mast and other cells. With the use of bone marrow transplantation, Jinx chimeric mice were created to determine whether the ability of hematopoietic cells to secrete granule contents affects the development of LVH. Wild-type mice (WT) that were transplanted with WT bone marrow (WT>WT) and WT mice that received Jinx bone marrow (Jinx>WT) developed LVH and a classic fetal reprogramming response early after TAC (7 days), but at later times (5 weeks), Jinx>WT mice failed to sustain the cardiac hypertrophic response observed in WT>WT mice. No difference in cardiac fibrosis was observed at early or late times. Repetitive injection of WT platelets or platelet releasate restored cardiac hypertrophy in Jinx>WT mice. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted, likely from platelets. In conclusion, our studies demonstrate that platelets and lymphocytes are involved in early perivascular inflammation post-TAC, which may contribute to later remodeling in the setting of LVH. Factors released from hematopoietic cells, including platelets, in a Munc13-4-dependent manner are required to promote cardiac hypertrophy. These findings focus attention on modulating perivascular inflammation and targeting granule cargo release to prevent the development and consequences of LVH.

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.

The duodenal mucosa is regularly exposed to acid, digestive enzymes and ingested noxious agents. It is thus critical to maintain a protective barrier to prevent the development of mucosal injury and inflammation, which are often observed in situations when barrier function is impaired. The rate of mucosal bicarbonate secretion, the regulation of epithelial paracellular permeability and motility are each key components of duodenal barrier function. The hormone melatonin is present in high levels in the gastrointestinal tract and it has been hypothesized that melatonin exerts protective properties. This thesis aims to investigate the impact of exogenous melatonin on the regulation of duodenal barrier function and motility in anesthetized rats in vivo. In addition, duodenal tissue was examined histologically and the expression levels of tight junction proteins and melatonin receptors were assessed with qRT-PCR. It was found that melatonin stimulated mucosal bicarbonate secretion and decreased basal paracellular permeability. Exposing the duodenal mucosa to the well-characterized barrier breaker ethanol increased mucosal bicarbonate secretion, paracellular permeability and motility. Omission of luminal Clˉ abolished, while pretreatment with a nicotinic receptor antagonist reduced, the ethanol-induced bicarbonate secretion suggesting that the secretory response to ethanol is meditated via Clˉ/HCO3ˉexchangers and enteric neural pathways. Melatonin reduced the ethanol-induced increases in paracellular permeability and motility either when injected intravenously or when administered in drinking water for two weeks. The actions of melatonin were abolished by the melatonin receptor antagonist luzindole and by nicotinic acetylcholine receptor inhibition. Two weeks oral administration of melatonin up-regulated the expression levels of melatonin receptors, down-regulated the expression of ZO-3 while the expression of ZO-1, ZO-2, claudin 2-4, occludin and myosin light chain kinase were unaffected. Superficial epithelial changes in a few villi were seen in response to ethanol exposure, an effect that was histologically unchanged by melatonin pretreatment. In conclusion, the results suggest that melatonin plays an important role in the neurohumoral regulation of gastrointestinal mucosal barrier function and motility via receptor- and enteric neural-dependent pathways in vivo in rats. Melatonin might be a candidate for treatment of barrier dysfunction in humans.

In seawater, drinking is a fundamental part of the osmoregulatory strategy for teleost fish, and presents a unique challenge. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid from imbibed seawater is crucial to compensating for water losses to the surrounding hyperosmotic environment. Alongside solute-linked water transport (driven by NaCl cotransport), intestinal bicarbonate (HCO3-) secretion also benefits fluid absorption directly (via apical Cl-/HCO3- exchange), and indirectly through the formation of calcium carbonate (CaCO3) thus removing the osmotic influence of Ca2+ within the gut fluid. For the European flounder (Platichthys flesus), elevated luminal Ca2+ has proven to be a specific, potent stimulator of HCO3- secretion both in vitro and in vivo where these actions are presumably modulated by an extracellular Ca2+-sensing receptor (CaR). The focus of this work was to learn more about how intestinal HCO3- secretion is regulated, the role of Ca2+, and more specifically the CaR. To achieve this, in vitro ‘gut sac’ experiments investigated how luminal Ca2+ influenced HCO3- secretion, and associated ion and fluid transport. Contrary to expectation, increasing Ca2+ from 5 to 20 mM did not stimulate HCO3- secretion. In an attempt to elucidate the role of CaCO3 precipitation in fluid absorption, and further explore the physiological implications of HCO3- secretion, the intestine was perfused in vivo with salines containing varying concentrations of Ca2+ (10, 40 and 90 mM). The production and secretion of HCO3-, in addition to CaCO3 formation increased accordingly with Ca2+, and was associated with a dramatic 25 % rise in the fraction of fluid absorbed by the gut. Additional in vitro experiments, utilising the Ussing chamber, helped establish some of the characteristics of intestinal HCO3- secretion by the euryhaline killifish (Fundulus heteroclitus), but was unresponsive to elevated mucosal Ca2+. Further attempts to potentiate the activity of the CaR, and application of the receptor agonists gadolinium (Gd3+) and neomycin, failed to produce responses consistent with the effect of Ca2+ observed previously, either in vitro or in vivo. With no evidence supporting a direct role for an extracellular, intestinal CaR in HCO3- secretion it was argued that secretion would be principally regulated by two factors, the ability of the epithelia to generate high levels of intracellular HCO3- and the rate of CaCO3 formation.

Endocardial endothelial cells (EECs) can exert a significant influence on cardiac function by releasing various factors such as nitric oxide (NO), prostanoids, endothelin-1 (ET-1) and angiotensin II (Ang II). Recently, results obtained in our laboratory demonstrated the presence of NPY and its receptors, Y[subscript 1] and Y[subscript 2], as well as ET-1 and its receptors, ET[subscript A] and ET[subscript B], at the level of endocardial endothelial cells (EECs). We have also shown that NPY induces a sustained rise in the intracellular calcium level of these cells, and that only right ventricular EECs have the capacity of secreting NPY. Moreover, the evidence in the literature has become plentiful about complex interactions existing between ET-1 and other cardioactive mediators, such as NO and Ang II. Based on the above-mentioned data, the objective of this study was to investigate if a dialogue equally exists between the systems of NPY and ET-1 at the level of human right (hREECs) and left (hLEECs) ventricular EECs. Using the technique of indirect immunofluorescence coupled to 3-D confocal microscopy, as well as ELISA, our results show that increasing concentrations of NPY (10[superscript -15], 10[superscript -10] and 10[superscript -5]M) induce the release of ET-1 from REECs and LEECs in a time- and dose-dependent fashion. However, right ventricular EECs seem to have a higher ET-1 secretory capacity as compared to their left counterparts. Upon the use of selective antagonists for the NPY receptors, Y[subscript 1], Y[subscript 2] and Y[subscript 5], and the ET-1 receptors, ET[subscript A] and ET[subscript B], our results demonstrated that in REECs the NPY-induced release of ET-1 seems to be primarily due to Y[subscript 2] receptor activation, with the subsequent activation of the ET[subscript A] and ET[subscript B] receptors by the released ET-1. On the other hand, in LEECs, the NPY-evoked secretion of ET-1 seems to be mainly the result of Y[subscript 5] receptor activation by NPY. Unlike REECs, the ET-1 released by NPY in this type of cells does not seem to be contributing further to its own release by activation of its ET[subscript A] and ET[subscript B] receptors. Therefore, our results suggest that NPY is a regulator of ET-I secretion at the level of human EECs, and that this secretory process of ET-1 is different between the right and left ventricular cells. Moreover, these results serve to highlight and endorse the important sensory and tuning roles that right and left ventricular EECs possess, respectively. The ability of EECs to contribute to the local as well as systemic release of factors, such as NPY and ET-1, can affect not only the excitation-secretion coupling of EECs and the excitation-contraction coupling of cardiomyocytes, but also the physiological and pathophysiological state of the underlying, heart muscle.

This thesis presents an investigation into the role of the recently identified two pore channels (TPCs) in β-cell stimulus-secretion coupling. TPCs are the receptors for calcium mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP) located in the membrane of acidic intracellular calcium stores. It is proposed that they are responsible for the ATP-sensitive potassium channel (Katp channel) independent pathway of stimulus-secretion coupling; and that this pathway is not subordinate to the KAT? channel dependent pathway; but an alternative explanation of stimulus-secretion coupling in its own right. The first section of this thesis presents a characterisation of sub-membrane cal- cium signals observed in primary mouse β-cells in response to glucose and the membrane-permeable acetoxymethyl ester form of NAADP (NAADP-AM) using the non-ratiometric fluorescent calcium indicator fluo-4 and total internal reflection (TIRF) microscopy. These are compared to global cytosolic calcium changes observed with epifluorescence microscopy. Factors affecting the shape and time course of re- sponses are investigated, and pharmacological tools used to provide evidence for the role of intracellular calcium release from acidic stores mediated by NAADP. Having characterised the calcium responses of β-cells using TIRF; the second part of the thesis examines the effects of knocking out TPC2 (single KO), or both TPC1 and TPC2 (DKO) on these responses; after an initial assessment of pancreatic islet and β-cell morphology using electron microscopy. Gender differences in β-cell responses to glucose and NAADP are assessed in both wild type and knockout animals. Finally, the third section presents the discovery of elementary calcium release events in pancreatic β-cells. The current project visualises what are likely the triggering events for the global calcium signals examined in sections one and two. They take the form of localised calcium release in response to NAADP-AM and glucose; akin to sparks and puffs observed by stimulation with cADPR and IP3. Optical quantal analysis demonstrates the quantal nature of the events and estimates the size of the unitary calcium release unit (CRU) for NAADP. .

Introduction : La Fibrose Kystique (FK) est la maladie autosomique récessive la plus fréquente chez les Caucasiens et est due à une mutation du gène Cystic Fibrosis Transmembrane Regulator (CFTR), codant pour un canal chlore. La principale mutation est la délétion de l'acide aminé phénylalanine en position 508. En raison de l’augmentation de l'espérance de vie, de nouvelles complications telles que le diabète associé à la FK (DAFK) ont vu le jour. Le DAFK semble principalement dû à un défaut de sécrétion d'insuline. Des études ont montré que les femmes et les personnes homozygotes ΔF508 ont un risque plus élevé de développer le DAFK. Objectifs : Comparer la sécrétion d'insuline entre les hommes et les femmes FK selon leur génotype CFTR. Notre hypothèse était que les femmes FK présentaient une sécrétion d'insuline moins élevée que des hommes. Méthodes : Deux cents sujets adultes sans diabète connu ont été recrutés dans la clinique de FK du CHUM et inclus dans cette étude. Cent seize ont été revus après un suivi de 24 ± 10 mois. Leur génotype CFTR a été extrait à partir des dossiers médicaux. Tous les sujets ont subi une hyperglycémie provoquée par voie orale de 2-h (HGPO) afin de déterminer leur tolérance au glucose : normale (NGT), intolérance (IGT) ou DAFK. Des échantillons de sang ont été prélevés aux temps 0, 30, 60, 90, et 120 min de l’HGPO. À partir de ces derniers, la sécrétion d'insuline et la sensibilité à l’insuline des sujets ont été évaluées en utilisant les indices de Stumvoll et les aires sous la courbe de l’insuline durant l’HGPO. Résultats : Pour une excursion glycémique comparable, il y avait des différences significatives dans les concentrations d'insuline entre les hommes et les femmes et selon le génotype CFTR. Ainsi, les femmes et les sujets hétérozygotes avaient des concentrations d’insuline plus élevées que les hommes et les sujets homozygotes. Cela restait significatif quelle que soit leur tolérance au glucose. Le calcul du disposition index représentant la sécrétion d'insuline ajustée pour le degré de sensibilité à l’insuline a suggéré une sécrétion d'insuline plus élevée chez les femmes que les hommes. Le suivi prospectif nous a permis de déterminer que cette sécrétion plus élevée d’insuline était associée à une évolution plus favorable pour la tolérance au glucose. Fait intéressant, cette constatation n'était vraie que pour les femmes. Conclusion : Dans une vaste cohorte prospective observationnelle de patients FK sans diabète connu, nous avons démontré qu’en dépit d’un âge et d’une fonction pulmonaire semblables, les femmes présentaient une sécrétion d'insuline supérieure à celle des hommes et que cela pourrait avoir un effet protecteur, à court terme, chez celles-ci pour le développement du DAFK. / Introduction: Cystic Fibrosis (CF) is the most common autosomal recessive disease among Caucasians and is caused by a mutation in the gene encoding for a chloride channel, the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. The main change is the deletion of the phenylalanine amino acid at position 508. Due to increasing life expectancy, new complications such as CF related diabetes (CFRD) have emerged. CFRD seems mainly due to a defect in insulin secretion. Studies have shown that women and people with homozygous ΔF508 have a higher risk of developing CFRD. Objectives: To compare insulin secretion between men and women according to their CFTR genotype. Our hypothesis was that, in CF, women had a lower insulin secretion than men. Methods: Two hundred adult subjects without known diabetes were recruited from the CF clinic at the CHUM and included in this study. One hundred and sixteen were reviewed after a follow-up of 24 ± 10 months. Their CFTR genotype was extracted from medical records. All subjects underwent a 2-h oral glucose tolerance test (OGTT) to determine their glucose tolerance: normal (NGT), intolerance (IGT) or CFRD. Blood samples were collected at 0, 30, 60, 90, and 120 min of the OGTT. Insulin secretion and insulin sensitivity were evaluated using the Stumvoll indices and the area under the curve (AUC) of insulin during the OGTT. Results: For a similar glycemic excursion, there were significant gender differences in insulin concentrations and according to the CFTR genotype. Thus, women and heterozygous subjects had insulin concentrations higher than men and homozygous. This remained significant regardless of their glucose tolerance. The calculation of the disposition index, representing insulin secretion adjusted for the degree of insulin sensitivity, suggested a higher insulin secretion in women than in men. Prospective follow-up showed that higher insulin secretion was associated with more favorable evolution of glucose tolerance. Interestingly, this finding was only applicable for women. Conclusion: In a large prospective observational cohort of CF patients without known diabetes, we demonstrated that, despite similar age and pulmonary function, women had a higher insulin secretion than men and that this could have a protective effect for the development of CFRD.

Legionella pneumophila est une bactérie gram/négative de l'environnement qui infecte et se multiplie au sein des protozoaires aquatiques, comme les amibes. Elle peut également infecter les macrophages pulmonaires humains, causant une forme sévère de pneumonie appelée légionellose (ou maladie du légionnaire). L'importance du Système de Sécrétion de Type IV (SST4) Icm/Dot est clairement démontrée, de même que celle du Système de Sécrétion de Type II (SST2) Lsp. Des études bioinformatiques ont suggéré l'existence d'un Système de Sécrétion de Type I (SST1), mais aucune étude n'a à ce jour démontré sa fonctionnalité, ni même un éventuel rôle dans la virulence. Ce travail consiste à étudier la fonctionnalité et le rôle dans la virulence d'un SST1 potentiel de L. pneumophila. La fonctionnalité de pompes d'efflux potentielles, possédant une structure très proche d'un SST1, est également investiguée. Des mutants de L. pneumophila invalidés pour les gènes codant pour ce SST1 potentiel (lssB, lssD et tolC) ont été construits : ils possèdent une virulence fortement atténuée vis-à-vis de plusieurs types de cellules hôtes. L'entrée dans la cellule est affectée chez ces mutants, bien que le reste du cycle intracellulaire ne soit pas altéré. La fonctionnalité de ce SST1 a été démontrée par la sécrétion de protéines hybrides entre un rapporteur et la protéine RtxA, qui fait partie de la famille des protéines RTX (Repeat/in ToXins), classiquement sécrétées par des SST1. Enfin, la fonctionnalité de plusieurs pompes d'efflux potentielles a été démontrée : des composés toxiques expulsés par ces pompes ont été identifiés, ainsi qu'une pompe majeure (HelA/HelB/HelC). D'autres analyses sont en cours pour caractériser plus précisément l'importance de ces pompes d'efflux / Legionella pneumophila is a gram/negative pathogen that infects and survives within protozoans, such as amoebas. It can also infect human lung macrophages, causing a disease called Legionnaire’s disease. The Type IV Secretion System Icm/Dot is known to be involved in the virulence of L. pneumophila, as well as the Type II Secretion System Lsp. Bioinformatics studies suggested the presence of a Type I Secretion System (T1SS), but its functionality and importance have not been demonstrated to date. This work aims to study the functionality and the implication in the virulence of the putative T1SS of L. pneumophila. Investigation efforts also concerned putative efflux pumps, which share high similarity with T1SS. Mutant strains of L. pneumophila were constructed by deletion of genes encoding the T1SS (lssB, lssD and tolC): they are defective for the entry into the host cells. The creation of the Legionella replicative vacuole is not altered though. Then the functionality of the LssB/LssD/TolC T1SS was demonstrated in a heterologous host: the reconstructed T1SS allows the secretion of hybrid proteins created by fusing a reporter with parts of the RTX protein RtxA. Finally, the functionality of several putative efflux pumps was also demonstrated: several substrates were identified for those efflux pumps, as well as a major pump (HelA/HalB/HelC). Investigations are currently made to decipher the importance of such efflux systems

Chlamydia trachomatis est une bactérie intracellulaire obligatoire. Ce pathogène de l’Homme est la première cause infectieuse de cécité ainsi que de maladies sexuellement transmissible d’origine bacterienne.Utilisant une souche de C. trachomatis L2 exprimant une protéine fluorescente, nous avons développé des méthodes de microscopie et de cytométrie en flux permettant de suivre les différentes étapes du développement de la bactérie. Ces méthodes faciliteront les futures études de l’infection par Chlamydia.Chlamydia interagit avec différents processus cellulaires, et plus particulièrement via la sécrétion d’effecteurs par le système de sécrétion de type 3 (ST3). Nous avons identifié une famille de protéines possédant un signal de ST3 qui partagent un domaine, le DUF582, présent uniquement chez les Chlamydia pathogènes.Nous avons montré que les 5 protéines DUF582 de C. trachomatis sont exprimées à partir du milieu du cycle infectieux. Nous avons démontré que la protéine Hrs interagit avec le DUF582 et que la protéine DUF582 CT619 interagit avec Tsg101. Hrs et Tsg101 sont d’importants composants de la machinerie ESCRT impliquée dans de nombreux processus de fission membranaire.Utilisant l’interférence ARN, nous avons montré que Hrs et Tsg101 ne sont requis ni pour l’entrée, ni pour le développement de la bactérie. Ceci suggère que les protéines DUF582 bloquent des processus dépendant de Hrs/Tsg101. A l’inverse, la bactérie pourrait utiliser la machinerie ESCRT mais l’existence de mécanismes redondants expliquerait l’absence de phénotype dans les expériences d’interférence. Nous discutons trois hypothèses concernant le rôle des protéines DUF582 dans l’infection. / Chlamydia trachomatis is an obligate intracellular human pathogen. It is the first infectious cause of blindness and the most common cause of sexually transmitted diseases of bacterial origin. Using a strain of C. trachomatis serovar L2 expressing a fluorescent protein we developed microscopy and flow cytometry based methods to quantify several steps of its developmental cycle. These methods will facilitate future studies aimed at testing anti-bacterial compounds or various culture conditions. Chlamydiae interfere with many cellular processes, in particular via the secretion of bacterial proteins through a type 3 secretion (T3S) system. We identified a family of proteins that possess T3S signals. They share a domain designated as DUF582, which is only found in pathogenic chlamydiae. We showed that the five DUF582 proteins of C. trachomatis are expressed from the mid phase of infection. We demonstrated that the protein Hrs is a common interactor for the DUF582. In addition the N-terminal part of the DUF582 protein CT619 interacts with Tsg101. Hrs and Tsg101 are both important components of the ESCRT machinery, which is an ancient machinery required for several processes involving membrane fission.Using RNA interference we showed that Hrs and Tsg101 are dispensable for bacterial entry and growth. This last result suggest that DUF582 proteins actually prevent Hrs and/or Tsg101 driven processes. Alternatively, the bacteria might highjack the ESCRT machinery but redundant mechanisms would explain the absence of phenotype on bacterial development observed in the silencing experiments. We discuss three hypotheses as to the possible role of the DUF582 proteins in infection.