Predicting root defence against herbivores during succession

P>1. Root herbivores and pathogens interfere with basic below-ground plant function, and can thereby affect plant fitness and spatial and temporal patterns in natural plant communities. However, there has been little development of concepts and theories on below-ground plant defence, a deficit that is in contrast to the abundance of theorizing for above-ground plant parts.2. A review of the past 10 years of research on below-ground plant-herbivore interactions has revealed that, similar to above-ground tissues, root defences can be expressed constitutively or induced upon herbivore attack, and can be classified into direct and indirect traits, tolerance, and escape. Indeed, it has been shown that roots tolerate herbivory by outgrowing or re-growing lost tissues, or resist it by producing secondary metabolites that are toxic to herbivores or attract natural enemies of herbivores.3. We propose that, similar to above-ground plant-herbivore theories, the partition of abiotic and biotic factors over ecological succession can serve as the basis for predicting investment in defence strategies below-ground.4. Investigation of herbivore pressure and root responses along primary and secondary successional gradients suggests that: (i) roots are often fast growing, thinner and softer in early compared to later succession. (ii) Insect and nematode herbivore pressure increases until mid-succession and later decreases. (iii) Mycorrhizal abundance increases with succession, and the composition of fungal species changes through succession, often shifting from arbuscular mycorrhizae to ecto-mycorrhizae.5. Based on these findings, and on classical (above-ground) plant defence theory, we suggest the following set of testable hypotheses for below-ground plant defence: (i) During succession, early plants invest most of their resources in growth and less in defences (associated with a general lack of herbivores and pathogens, and with limited availability of resources in the system), therefore relying more on re-growth (tolerance) strategies. (ii) During mid-succession, a buildup of herbivore pressure facilitates replacement by plant species that exhibit greater direct and indirect defence strategies. (iii) Constitutive and inducible levels of defences may trade-off, and early successional plants should rely more on induction of defences after herbivore attack, whereas late successional plants will increasingly rely on constitutively produced levels of physical and chemical defence. (iv) Successional changes in microbial associations have consequences for root defence by improving plant nutrition and defence expression as well as directly competing for root space; however, toxic or impenetrable root defences may also limit association with root symbionts, and so may constrain the expression of root defence.

Immune responses to airborne fungi and non-invasive airway diseases.

Inhalation of fungal particles is a ubiquitous way of exposure to microorganisms during human life; however, this exposure may promote or exacerbate respiratory diseases only in particular exposure conditions and human genetic background. Depending on the fungal species and form, fungal particles can induce symptoms in the lung by acting as irritants, aeroallergens or pathogens causing infection. Some thermophilic species can even act in all these three ways (e.g. Aspergillus, Penicillium), mesophilic species being only involved in allergic and/or non-allergic airway diseases (e.g. Cladosporium, Alternaria, Fusarium). The goal of the present review is to present the current knowledge on the interaction between airborne fungal particles and the host immune system, to illustrate the differences of immune sensing of different fungal species and to emphasise the importance of conducting research on non-conventional mesophilic fungal species. Indeed, the diversity of fungal species we inhale and the complexity of their composition have a direct impact on fungal particle recognition and immune system decision to tolerate or respond to those particles, eventually leading to collateral damages promoting airway pathologies.

Bacterial subfamily of LuxR regulators that respond to plant compounds.

Pseudomonas fluorescens are rhizobacteria known for their biocontrol properties. Several antimicrobial functions are crucial for this process, and the experiments described here investigate the modulation of their expression during the plant-bacterium interaction. The role of a LuxR family regulator in interkingdom signaling has been investigated using genome-scale transcriptome analysis, gene promoter studies in vivo and in vitro, biocontrol assays, and response to plant compounds. PsoR, a LuxR solo or orphan regulator of P. fluorescens, was identified. PsoR is solubilized and activates a lux-box-containing promoter only in the presence of macerated plants, suggesting the presence of a plant molecule(s) that most likely binds to PsoR. Gene expression profiles revealed that genes involved in the inhibition of plant pathogens were affected by PsoR, including a chitinase gene, iron metabolism genes, and biosynthetic genes of antifungal compounds. 2,4-Diacetylphloroglucinol production is PsoR dependent both in vitro and in vivo. psoR mutants were significantly reduced for their ability to protect wheat plants from root rot, and damping-off caused by Pythium ultimum infection. PsoR most likely senses a molecule(s) in the plant and modulates expression of genes that have a role in biocontrol. PsoR and related proteins form a subfamily of LuxR family regulators in plant-associated bacteria.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines.

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented here demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. In vivo immunization studies and tumor protection experiments unambiguously establish the value of saffron saponins as candidate adjuvants. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen. This preclinical study provides an in depth immunological characterization of a new saponin as a vaccine adjuvant, and encourages its further development for use in vaccine formulations.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Rôle des molécules de co-stimulations et de CD93 dans la différenciation en plasmocytes

Le développement des cellules B est constitué d'une première phase qui se déroule dans la moelle en absence d'antigène et d'une deuxième phase qui se déroule dans les organes lymphoïdes secondaires et qui débute uniquement en présence d'antigène. Cette deuxième partie est extrêmement importante et doit être très bien régulée pour lutter efficacement contre les pathogènes, ainsi que pour éviter de nombreuses maladies de type auto-immunes. Ce travail est basé à l'origine sur l'étude de souris mutantes dans lesquelles une protéine des cellules T est modifiée, impliquant une très forte activation des cellules B en absence d'antigène et de manière non spécifique. Ces souris constituent donc un outil de travail très intéressant pour étudier tout d'abord le mécanisme aboutissant à l'activation des cellules B dans ce contexte particulier. De plus comme ces souris contiennent énormément de cellules sécrétant des anticorps, à savoir les plasmocytes, il est facile d'étudier leur phénotype. Cela nous a permis de démontrer qu'un récepteur membranaire, CD93 est exprimé à leur surface. Cette observation a ensuite été confirmée dans des souris normales, de type sauvage. L'utilisation de ce marqueur de surface nous a permis de caractériser plus en détail les étapes du développement des plasmocytes. De plus nous avons tenté de trouver la fonction jouée par cette molécule à la surface de ces cellules, en utilisant des souris dans lesquelles ce récepteur a été supprimé. Si les premières étapes de l'activation des cellules B étaient normales, ces souris n'étaient par contre pas capables de produire des anticorps à long-terme dans le sang. Nous avons pu montrer que la survie des plasmocytes en l'absence de CD93 est moins efficace dans la moelle, probablement du au fait qu'en absence de cette molécule, les plasmocytes ont plus de difficultés à adhérer dans ce que l'on appelle des niches de survie. Nous avons essayé ensuite de déterminer si CD93 peut être utilisé comme cible thérapeutique dans le cadre de maladies auto-immunes ou de lymphomes. Bien que CD93 soit exprimé à la surface des cellules d'intérêt dans les souris souffrant de lupus, il n'a pas été possible de les éliminer avec un anticorps dirigé contre CD93. De plus nous n'avons pas pu mettre en évidence l'expression de CD93 à la surface des plasmocytes humains induits in vitro. SUMMARY : Antigen dependent B cell activation is a key aspect of the adaptive immunity which is involved in the efficient response against pathogens, but also in vaccination and in numerous pathologies. The aim of this project was to investigate two key aspects of the late B cell development, namely the role of costimulatory molecules in the immunological synapse between T and B cells and the characterization of a new plasma cell marker, CD93. This work was initially based on the study of the LatY136F mutant mouse. The latter harbors a point mutation in the LAT adaptor protein which is involved in T cell receptor signaling. As a consequence of this mutation, CD4 T cells in the periphery expand strongly and are polarized in a TH2 manner leading to a normal but exaggerated B cell response. For this reason, these mice provide a useful tool to investigate different aspects of the late B cell development. The first part of the project was focused on the role played by costimulatory molecules in LotY136F CD4 T cell mediated B cell activation. In vitro studies showed that CD80/CD86, IL-4 and LFA-1 were required for LatY136FT cells to activate B cells whereas CD40 and IcosL were not necessary. In vivo we showed that CD80/CD86 was required for initial T cell expansion whereas CD40 and IcosL deficiency led to a less efficient B cell activation. The large amount of plasma cells present in LatY136F mice allows investigating in more details their phenotype and CD93 was found to be expressed on their surface, This observation was confirmed in wild type B cells activated either in vivo or in vitro with T-independent or T-dependent antigens. Moreover we found that CD93 expression can occur either before CD138/Blimp-1 induction or after, showing that two independent pathways can lead to the formation of CD93/CD138 double positive population, which was shown to be the more mature. Indeed, their phenotype correlated with modified transcriptional network, high isotype switched antibody secretion and cell cycle arrest. Analysis of CD93 deficient mice demonstrated that the initial B cell activation after immunization was normal, but also showed that these mice failed to maintain a high antibody secretion level at later time points both after primary and boost immunization. This was shown to be due to a less efficient survival of the long-lived plasma cells in the bone marrow niches, most likely related with a defective adhesion process in absence of CD93. We investigated the possibility to use CD93 as a target to treat plasma cell pathologies, but even if this molecule is expressed on cells of interest in the bone marrow of lupus mice, it was not possible to deplete them using anti-CD93 antibodies. Moreover we were not able to show its expression on the surface of in vitro activated B cells and multiple myeloma cell lines of human origin. In conclusion, our data helped understand both the mechanisms leading to the polyclonal B cell activation occurring in the LatY136F KI mouse and the role played by CD93 on the surface of plasma cells, which could potentially open the way to therapeutic application.

Production of Pseudomonas aeruginosa Intercellular Small Signaling Molecules in Human Burn Wounds

Pseudomonas aeruginosa has developed a complex cell-to-cell communication system that relies on low-molecular weight excreted molecules to control the production of its virulence factors. We previously characterized the transcriptional regulator MvfR, that controls a major network of acute virulence functions in P. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (HAQs)-4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Though HHQ and PQS are produced in infected animals, their ratios differ from those in bacterial cultures. Because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human P. aeruginosa acute wound infection and whether their ratio is similar to that observed in P. aeruginosa-infected mice. We found that a clinically relevant P. aeruginosa isolate produced detectable levels of HAQs with ratios of HHQ and PQS that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. These molecules could be isolated from wound tissue as well as from drainage liquid. These results demonstrate for the first time that HAQs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection.

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection.

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.

Backseat drivers: the hidden influence of microbial viruses on disease.

Because viral replication depends on the vigour of its host, many viruses have evolved incentives of fitness to pay their keep. When the viral host is a human pathogen, these fitness factors can surface as virulence: creating a Russian doll of pathogenesis where pathogens within pathogens complicate the disease process. Microbial viruses can even be independently immunogenic, as we recently reported for leishmania-virus. Thus, the incidence of this 'hyperpathogenism' is becoming an important clinical consideration and by appreciating the microbial-virus as a backseat driver of human disease, we could exploit its presence as a diagnostic biomarker and molecular target for therapeutic intervention. Here we discuss the prevalence of clinically relevant hyperpathogenism as well as the environmental sanctuaries that breed it.

Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Discovery of novel arenavirus receptors and entry factors

Arenaviruses are enveloped negative-strand RNA viruses that contain a bi-segmented genome. They are rodent-borne pathogens endemic to the Americas and Africa, with the exception of lymphocytic choriomeningitis virus (LCMV) that is world-wide distributed. The arenaviruses include numerous important human pathogens including the Old World arenavirus Lassa virus (LASV), the causative agent of a severe viral hemorrhagic fever in humans with several hundred thousand infections per year in Africa and thousands of deaths. Viruses are obligatory intracellular parasites, strictly depending on cellular processes and factors to complete their replication cycle. The binding of a virus to target cells is the first step of every viral infection, and is mainly mediated by viral proteins that can directly engage cellular receptors, providing a key determinant for viral tropism. This early step of infection represents a promising target to block the pathogen before it can take control over the host cell. Old World arenaviruses, such as LASV and LCMV, bind to host cells via attachment to their main receptor, dystroglycan (DG), an ubiquitous receptor for extracellular matrix proteins. The engagement of DG by LASV results in a fast internalization and transfer the virus to late endosomal compartment suggesting that the virus binding to DG causes marked changes in the dynamics of the receptor. These events could result in the clustering of the receptor and subsequent induction of signaling that could be modulated by the virus. Recently, numerous findings also suggest the presence of alternative receptor(s) for LASV in absence of the main DG receptor. In my first project, I was interested to investigate the effects of virus-receptor binding on the tyrosine phosphorylation of the cytoplasmic domain of DG and to test if this post-translational modification was crucial for the internalization of the LASV-receptor complex. We found that engagement of cellular DG by a recombinant LCMV expressing the envelope GP of LASV in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. Virus-induced dissociation of utrophin and consequent virus internalization were affected by the broadly specific tyrosine kinase inhibitor genistein. We speculate that the detachment of virus- bound DG from the actin-based cytoskeleton following DG phosphorylation may facilitate subsequent endocytosis of the virus-receptor complex. In the second project, I was interested to characterize the newly indentified LASV alternative receptor Axl in the context of productive arenavirus infection. In a first step, we demonstrated that Axl supports productive infection by rLCMV-LASVGP in a DG-independent manner. In line with previous studies, cell entry of rLCMV-LASVGP via Axl was less efficient when compared to functional DG. Interestingly, Axl-mediated infection showed rapid kinetics similar to DG-dependent entry. Using a panel of inhibitors, we found that Axl-mediated cell entry of rLCMV-LASVGP involved a clathrin-independent pathway that critically depended on actin and dynamin and was sensitive to EIPA but not to PAK inhibitors, compatible with a macropinocytosis-like mechanism of entry. In a next step, we aimed to investigate the molecular mechanism by which rLCMV-LASVGP recognizes Axl. Phosphatidylserine (PS) is the natural ligand of Axl via the adaptor protein Gas6. We detected the presence of PS in the envelope of Old World arenaviruses, suggesting that PS could mediate Axl-virus binding, in a mechanism of apoptotic mimicry already described for other viruses. Whether envelope PS and/or the GP of LASV plays any role in virus entry via Axl is still an open question. The molecular mechanisms underlying host cell-virus interaction are of particular interest to answer basic scientific questions as well as to apply key findings to translational research. Understanding pathogen induced-signaling and its link to invasion of the host cell is of great importance to develop drugs for therapeutic intervention against highly pathogenic viruses like LASV. - Les Arenavirus sont des virus enveloppés à ARN négatifs organisés sous forme de génome bisegmenté. Ils sont véhiculés par les rongeurs et se retrouvent de manière endémique aux Amériques et en Afrique avec l'exception du virus de la chorioméningite lymphocytaire (LCMV) qui lui est distribué mondialement. De nombreux pathogènes humains font parti de la famille des Arenavirus dont le virus de l'Ancien Monde Lassa (LASV), un agent responsable de fièvres hémorragiques sévères chez les humains. Le virus de Lassa cause plusieurs centaines de milliers d'infections par année en Afrique ainsi que des milliers de morts. De manière générale, les virus sont des parasites intracellulaires obligatoires qui dépendent strictement de processus et facteurs cellulaires pour clore leur cycle de réplication. L'attachement d'un virus à sa cellule cible représente la première étape de chaque infection virale et est principalement dirigée par des protéines virales qui interagissent directement avec leur récepteurs cellulaires respectifs fournissant ainsi un indicateur déterminant pour le tropisme d'un virus. Cette première étape de l'infection représente aussi une cible prometteuse pour bloquer le pathogène avant qu'il ne puisse prendre le contrôle de la cellule. Les Arenavirus de l'Ancien Monde comme LASV et LCMV s'attachent à la cellule hôte en se liant à leur récepteur principal, le dystroglycan (DG), un récepteur ubiquitaire pour les protéines de la matrice extracellulaire. La liaison du DG par LASV résulte en une rapide internalisation transférant le virus aux endosomes tardifs suggérant ainsi que l'attachement du virus au DG peut provoquer des changements marqués dans la dynamique moléculaire du récepteur. Ces événements sont susceptibles d'induire un regroupement du récepteur à la surface cellulaire, ainsi qu'une induction subséquente qui pourrait être, par la suite, modulée par le virus. Récemment, plusieurs découvertes suggèrent aussi la présence d'un récepteur alternatif pour LASV en l'absence du récepteur principal, le DG. Concernant mon premier projet, j'étais intéressée à étudier les effets de la liaison virus- récepteur sur la phosphorylation des acides aminés tyrosines se trouvant dans la partie cytoplasmique du DG, le but étant de tester si cette modification post-translationnelle était cruciale pour Γ internalisation du complexe LASV-DG récepteur. Nous avons découvert que l'engagement du récepteur DG par le virus recombinant LCMV, exprimant la glycoprotéine de LASV, dans des cellules épithéliales humaines induit une phosphorylation de résidu(s) tyrosine se situant dans le domaine cytoplasmique du DG. La liaison de la glycoprotéine de LASV au DG induit par la suite la dissociation de la protéine adaptatrice utrophine du complexe virus-DG récepteur. Nous avons observé que cette dissociation de l'utrophine, induite par le virus, ainsi que son internalisation, sont affectées par l'inhibiteur à large spectre des tyrosines kinases, la génistéine. Nous avons donc supposé que le détachement du virus, lié au récepteur DG, du cytosquelette d'actine suite à la phosphorylation du DG faciliterait l'endocytose subséquente du complexe virus-récepteur. Dans le second projet, j'étais intéressée à caractériser le récepteur alternatif Axl qui a été récemment identifié dans le contexte de l'infection productive des Arenavirus. Dans un premier temps, nous avons démontré que le récepteur alternatif Axl permet l'infection des cellules par le virus LCMV recombinant LASV indépendamment du récepteur DG. Conformément aux études publiées précédemment, nous avons pu observer que l'entrée du virus recombinant LASV via Axl est moins efficace que via le récepteur principal DG. De façon intéressante, nous avons aussi remarqué que l'infection autorisée par Axl manifeste une cinétique virale d'entrée similaire à celle observée avec le récepteur DG. Utilisant un éventail de différents inhibiteurs, nous avons trouvé que l'entrée du virus recombinant rLCMV-LASVGP via Axl implique une voie d'entrée indépendante de la clathrine et dépendant de manière critique de l'actine et de la dynamine. Cette nouvelle voie d'entrée est aussi sensible à l'EIPA contrairement aux inhibiteurs PAK indiquant un mécanisme d'entrée compatible avec un mécanisme de macropinocytose. L'étape suivante du projet a été d'investiguer le mécanisme moléculaire par lequel le virus recombinant rLCMV-LASVGP reconnaît le récepteur alternatif Axl. La phosphatidylsérine (PS) se trouve être un ligand naturel pour Axl via la protéine adaptatrice Gas6. Nous avons détecté la présence de PS dans l'enveloppe des Arenavirus du Vieux Monde suggérant que la PS pourrait médier la liaison du virus à Axl dans un mécanisme de mimétisme apoptotique déjà observé et décrit pour d'autres virus. Cependant, il reste encore à déterminer qui de la PS ou de la glycoprotéine de l'enveloppe virale intervient dans le processus d'entrée de LASV via le récepteur alternatif Axl. Les mécanismes moléculaires à la base de l'interaction entre virus et cellule hôte sont d'intérêts particuliers pour répondre aux questions scientifiques de base ainsi que dans l'application de découvertes clés pour la recherche translationnelle. La compréhension de la signalisation induite par les pathogènes ainsi que son lien à l'invasion de la cellule hôte est d'une importance considérable pour le développement de drogues pour l'intervention thérapeutique contre les virus hautement pathogènes comme LASV.

Adult native septic arthritis: 10 years in review in order to establish local guidelines on empiric antibiotic therapy.

Objective: Antibiotic stewardship includes development of practice guidelines incorporating local microbiology and resistance patterns. In case of septic arthritis (SA), addition of vancomycin to the empiric therapy and broad-spectrum antibiotherapy in some clinical settings are subjects of discussion. Our objective was to review the local epidemiology of native septic arthritis in adults, in order to establish local guidelines for empiric therapy. Methods: Retrospective study based on positive synovial fluid cultures and hospital discharge diagnoses of SA obtained from 1999 to 2008 in patients _16 years. Medical records were reviewed to assess the diagnosis and complete relevant clinical information. Results: During this ten-year period, we identified 233 SA on native joints in 231 patients. 107 episodes (46%) were obtained through positive synovial fluid cultures, and 126 episodes (54%) through the discharge diagnosis. 147 SA (63%) were large joint infections (LJI). 35 SA (15%) occurred in intravenous drug users. Preexisting arthropathy was present in 51% of cases. 42% of patients with small joint infection (SJI) were diabetic, vs. 23% with LJI (p = 0.003). When available, synovial fluid direct examination was positive in 35% of cases. Etiologic agents are reported in the table. Five of the 11 MRSA SA (45%) occurred in known carriers. SJI were more frequently polymicrobial (24% vs. 1%, p<0.001). For LJI, an empiric treatment with amoxicillin/clavulanate (A/C) would have been appropriate in 85% of cases. MRSA (8 cases) and tuberculous (7 cases) arthritis would have been the most frequently untreated pathogens. Addition of vancomycin to A/C in MRSA carriers would rise the adequacy to 87%. In contrast, A/C would cover only 75% of SJI (82% if restricted to non-diabetic patients). MRSA (3 cases) and P. aeruginosa (9 cases, 7 monomicrobial) would be the main untreated pathogens. An anti-pseudomonal penicillin would have been appropriate in 94% of cases of SJI (P = 0.002 vs. A/C, p = 0.19 if diabetic patients not included). Conclusions: Treatment with A/C seems adequate for empiric coverage of LJI in our setting. Broad-spectrum antibiotherapy was significantly superior for SJI in diabetic patients, due to different causative bacteria. In an area of low MRSA incidence, our results do not justify a systematic empiric therapy for MRSA, which should be considered in a known carrier.