Pseudomonas aeruginosa prise en flagrant délit de casse ! Pseudomonas aeruginosa caught in the act !

International audienceDes armes qui attaquent le génome eucaryote de façon encore mystérieuse Pseudomonas aeruginosa est une bactérie pathogène opportuniste infectant principalement les individus aux défenses immunitaires affaiblies, les patients ayant une rupture de leurs barrières cutanées (chirurgie, brûlures,...) ou subissant un geste invasif (pose d'une sonde, d'un cathéter) [1]. D'autres sujets sont particulièrement à risque, ainsi 80% des personnes atteintes de mucoviscidose sont chroniquement infectées par cette bactérie. Les infections à P. aeruginosa sont le plus souvent contractées en milieu hospitalier et elles demeurent un défi pour le corps médical en raison de la multi-résistance des souches aux antibiotiques [2]. La pathogénicité liée aux infections aiguës ou chroniques à P. aeruginosa est multifactorielle [3]. L'un des systèmes de virulence majeurs associé aux infections aiguës est le système de sécrétion de type 3 (SST3) [4]. Il est constitué d'une aiguille creuse érigée à la surface de la bactérie qui s'insère dans la membrane plasmique de la cellule hôte et permet l'injection de toxines bactériennes (ExoY, ExoT et ExoS ou ExoU) dans le cytoplasme de la cellule infectée. Nous avons recherché les dommages potentiels causés par P. aeruginosa au patrimoine génétique de la cellule hôte. Pour la première fois, nos travaux montrent que le SST3 est associé à des cassures double-brin de l'ADN, lésions très dangereuses pour le génome, et à une réaction de la cellule hôte qui va tenter de réparer les dégâts [5]

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB.

International audienceBACKGROUND: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. RESULTS: The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. CONCLUSIONS: The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA

A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.</p> <p>Results</p> <p>A bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding <it>fdx </it>gene in the <it>Proteobacteria </it>phylum, without occurrence in <it>Archaea </it>and <it>Eukaryota</it>. The transcript was found to be monocistronic in <it>Pseudomonas aeruginosa</it>, and not part of an operon in most bacteria. Only <it>fdx </it>genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in <it>Pseudomonas aeruginosa </it>and in <it>Escherichia coli</it>. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the <it>fdx </it>gene in <it>Pseudomonas aeruginosa </it>abolished growth, unless a plasmid copy of the gene was provided to the deleted strain.</p> <p>Conclusions</p> <p>The gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in <it>Pseudomonas aeruginosa</it>. These data identify a new potential antimicrobial target in this and other pathogenic <it>Proteobacteria</it>.</p

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

International audienceHighly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action

How do small and medium architectural firms deal with architectural complexity? A look into digital practices

peer reviewedDigital design tools are nowadays deeply rooted in most architectural design processes. Either used as a way to communicate a designed artifact, or rather as a medium to test innovative morphologies (lately referred to as non-standard architecture), these design tools profoundly shape the way architects envision complexity all along their day-to-day tasks. The large teams of "star-architects" were able to develop dedicated and specific strategies and digital tools to overcome some of the technological gaps they were faced to when using commercial packages. But how are small and medium firms dealing with the inherent complexities of such software? This contribution analyzes how those firms (and more specifically the Walloon ones) deal with the growing complexity of digital tools, both in terms of use and interdisciplinarity

Ruptures et démesures de l'architecture non standard à l'ère du numérique: la paramétrisation comme outil de réconciliation.

peer reviewedL’intégration de l’outil numérique au processus de conception architectural a ouvert les champs morphologiques et a favorisé l’émergence de l’architecture non standard. Cette contribution identifie les principaux bouleversements pratiques et réflexifs liés à l’émergence de cette architecture parfois démesurée. Il les résume en trois ruptures entre les différentes phases du projet-objet et les acteurs du projet. Nous proposons ensuite de considérer la modélisation paramétrique comme l’un des outils d’une maîtrise renouvelée du processus de génération de la forme, et de la forme elle-même

Practical Trajectories of Parametric Tools in Small and Medium Architectural Firms

peer reviewedInitially used as an extension of hand-drawing tools, digital design tools and moreover parametric ones are nowadays deeply modifying the architectural design process. Big offices with star-architects were able to adopt these tools but most architects working in a small office are still trying to cope with these parametric design tools. Several questions arise in this regard: what digital tools do architects usually use? Do they express interest for new technologies and software such as parametric ones? What is their understanding of the term “parametric architecture”? Why is this kind of tools still not largely adopted? Going through the results of an online survey, this paper first discusses the meaning of parametric design for architects. The contribution then analyzes the Belgian case regrouping mostly small and medium offices. It reflects particularly on the way architects do or do not implement these new digital tools in their workflows, and it sheds light on the fact that parametric tools also have the potential to free the creativity of SME’s