Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm.

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Chlamydia-like organisms: ecology, pathogenesis and virulence factors

Members of the Chlamydiales order are strict intracellular bacteria with the fascinating ability to infect a huge variety of animais, insects, and protists. They are closely related to their hosts, and to survive during a long évolution process they evolved an arsenal of tools to always keep an unstable equilibrium, avoiding to be too much deleterious for hosts, while being able to survive. Each chlamydial species will exhibit a specific set of virulence factors that will allow it to overcome the specific defence of the host. In this thesis, we have tackled chlamydial biology according to three axes: ecology and biodiversity, pathogenesis, and virulence factors. First we were able to identify enough chlamydial DNA within ticks in Switzerland, to make whole genome sequencing allowing to describe a family for which no genome were available for now. This new ecological niche of Chlamydiales bacteria was further investigated by showing the ability of Chlamydia-related bacteria to grow within tick cells. Additionally, we determined the pathogenic potential of Chlamydia-related bacteria in respiratory and génital tract infections and by developing animal models that will be useful to investigate the rôle of virulence factors. Finally, we described some key players of struggles between hosts and chlamydiae. Besides demonstrating that Chlamydia-re\ated bacteria can activate inflammasome pathway, we identified several virulence factors among which one is secreted by the Type III sécrétion system within host cytoplasm. More investigations on closed relationship between chlamydiae and their host are required. It appears that each chlamydial species exhibit specificities. It's will be a challenge to understand these unique and fascinating microorganisms, which are among the most enigmatic of the bacteria. -- Les membres de l'ordre des Chlamydiales sont des bactéries intracellulaires strictes ayant la capacité fascinante d'infecter une grande variété d'animaux, d'insectes et de protistes. Elle sont intimement liées a leurs hôtes, et pour survivre pendant leur évolution, elles ont développés un arsenal de solutions qui leur permet de maintenir un équilibre instable, en évitant d'être trop délétère pour leurs hôtes, tout en survivant. Chaque espèce de chlamydia aura développé un ensemble de facteurs de virulence spécifique qui lui permettront de surmonter les défenses spécifiques de leurs hôtes. Dans ce travail de thèse, nous avons abordé la compréhension de la biologie des chlamydias selon 3 axes: leurs écologie et biodiversité, leur pathogénèse et leurs facteurs de virulence. Nous avons tout d'abord pu trouver assez d'ADN de chlamydia dans des tiques en Suisse, pour pouvoir séquencer un génome complet, ce qui nous a permis de décrire une famille de chlamydia pour laquelle aucun génome n'existait jusqu'alors. Nous avons également pu caractériser plus en détails cette nouvelle niche écologique des Chlamydiales en montrant la capacité, des bactéries apparentées au chlamydias, de se répliquer dans des cellules de tiques. De plus, nous avons pu décrire le potentiel pathogène des bactéries apparentées aux chlamydias, dans les infections génitales et respiratoires, en établissant des modèles animaux. Ces modèles animaux pourront être utiles pour étudier le rôle des facteurs de virulence de ces bactéries. Finalement, nous avons décrit certains acteurs majeurs de la lutte entre les chlamydias et leurs hôtes. En plus de démontrer la capacité des bactéries apparentées aux chlamydias d'activer la voie de l'inflammasome, nous avons identifié plusieurs facteurs de virulence parmi lesquels un est sécrété dans le cytoplasme de la cellule hôte par le système de sécrétion de type III. D'avantage d'études sur la relation intime entre les chlamydias et leurs hôtes sont nécessaires car chaque espèce a ces spécificités. La compréhension de ces microorganismes énigmatiques et fascinants reste en défi passionnant

Tunable Double-Network GelMA/Alginate Hydrogels for Platelet Lysate-Derived Protein Delivery

International audienceHydrogels (gels) are attractive tools for tissue engineering and regenerative medicine due to their potential for drug delivery and ECM-like composition. In this study, we use rheology to characterize GelMA/alginate gels loaded with human platelet lysate (PL). We then characterize these gels from a physicochemical perspective and evaluate their ability to transport PL proteins, their pore size, and their rate of degradation. Finally, their biocompatibility is evaluated. We describe how alginate changes the mechanical behavior of the gels from elastic to viscoelastic after ionic (calcium-mediated) crosslinking. In addition, we report the release of ~90% of PL proteins from the gels and relate it to the degradation profile of the gels. Finally, we evaluated the biocompatibility of the gels. Thus, the developed gels represent attractive substrates for both cell studies and as bioactive materials

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila.

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 108 W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium

Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells

<i>Waddlia chondrophila</i> replicate within mouse lungs.

<p><b>A)</b> Bacterial loads in lung homogenates of mice inoculated intranasally with 2 x 10⁸ <i>W</i>. <i>chondrophila</i>. The horizontal line represents the median value. <b>B)</b> Representative electron microscopy picture of a mouse lung 3 days post-inoculation with 2 x 10⁸ <i>W</i>. <i>chondrophila</i> (n = 3). The white arrowhead is showing an inclusion full of <i>W</i>. <i>chondrophila</i>. A: air in lung alveolus, EB: elementary body, RB: Reticulate body, mi: mitochondria. Note that <i>W</i>. <i>chondrophila</i> EB exhibits here a typical crescent shape that has been reported to be induced by fixative used for electron microscopy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150909#pone.0150909.ref041" target="_blank">41</a>]. Scale bar = 1 μm. Magnification 13000x.</p

<i>Waddlia chondrophila</i> induce mice mortality and body weight loss.

<p><b>A)</b> C57BL/6 mice (n = 6 animals per group) were inoculated intranasally with 5 x 10⁷, 1 x 10⁸ and 2 x 10⁸ <i>W</i>. <i>chondrophila</i>. A mock control group was processed in parallel (n = 5 mice). The Kaplan-Meier survival curve is shown. <b>B)</b> Body weight of mice that survived or died after <i>W</i>. <i>chondrophila</i> infection, and mock control treated mice. Results are expressed as the mean percentage (± SEM) of animal weight prior to inoculation.</p

Dense infiltration of specific immune cells in the lungs of infected mice.

<p>Mice were inoculated intranasally with 2 x 10⁸ <i>W</i>. <i>chondrophila</i> or a mock control. Adjacent lung sections were stained with haematoxylin and eosin 3 days post-inoculation. Scale bar = 50 μm. <b>A)</b> Typical dense lympho-plasmacytic (white arrows) and neutrophilic (black arrows) infiltration of the lung alveolar and interstitial spaces of mice infected with <i>W</i>. <i>chondrophila</i>. Infiltration is peri-bronchiolar (black star) colonization of the bronchiole by neutrophils and lymphocytes that is typical of a subacute pneumonia. Magnification 400x. <b>B)</b> Typical focal inflammation in lungs of mock control mice, with presence of a few neutrophils (black arrow) and more lymphocytes (white arrow) in peri-bronchial and peri-bronchiolar area. Magnification 400x.</p