Propionibacterium acnes: An Underestimated Pathogen in Implant-Associated Infections.

The role of Propionibacterium acnes in acne and in a wide range of inflammatory diseases is well established. However, P. acnes is also responsible for infections involving implants. Prolonged aerobic and anaerobic agar cultures for 14 days and broth cultures increase the detection rate. In this paper, we review the pathogenic role of P. acnes in implant-associated infections such as prosthetic joints, cardiac devices, breast implants, intraocular lenses, neurosurgical devices, and spine implants. The management of severe infections caused by P. acnes involves a combination of antimicrobial and surgical treatment (often removal of the device). Intravenous penicillin G and ceftriaxone are the first choice for serious infections, with vancomycin and daptomycin as alternatives, and amoxicillin, rifampicin, clindamycin, tetracycline, and levofloxacin for oral treatment. Sonication of explanted prosthetic material improves the diagnosis of implant-associated infections. Molecular methods may further increase the sensitivity of P. acnes detection. Coating of implants with antimicrobial substances could avoid or limit colonization of the surface and thereby reduce the risk of biofilm formation during severe infections. Our understanding of the role of P. acnes in human diseases will likely continue to increase as new associations and pathogenic mechanisms are discovered.

Does a shift in host plants trigger speciation in the Alpine leaf beetle Oreina speciosissima (Coleoptera, Chrysomelidae)?

Background: Within the Coleoptera, the largest order in the animal kingdom, the exclusively herbivorous Chrysomelidae are recognized as one of the most species rich beetle families. The evolutionary processes that have fueled radiation into the more than thirty-five thousand currently recognized leaf beetle species remain partly unresolved. The prominent role of leaf beetles in the insect world, their omnipresence across all terrestrial biomes and their economic importance as common agricultural pest organisms make this family particularly interesting for studying the mechanisms that drive diversification. Here we specifically focus on two ecotypes of the alpine leaf beetle Oreina speciosissima (Scop.), which have been shown to exhibit morphological differences in male genitalia roughly corresponding to the subspecies Oreina speciosissima sensu stricto and Oreina speciosissima troglodytes. In general the two ecotypes segregate along an elevation gradient and by host plants: Oreina speciosissima sensu stricto colonizes high forb vegetation at low altitude and Oreina speciosissima troglodytes is found in stone run vegetation at higher elevations. Both host plants and leaf beetles have a patchy geographical distribution. Through use of gene sequencing and genome fingerprinting (AFLP) we analyzed the genetic structure and habitat use of Oreina speciosissima populations from the Swiss Alps to examine whether the two ecotypes have a genetic basis. By investigating a wide range of altitudes and focusing on the structuring effect of habitat types, we aim to provide answers regarding the factors that drive adaptive radiation in this phytophagous leaf beetle.Results: While little phylogenetic resolution was observed based on the sequencing of four DNA regions, the topology and clustering resulting from AFLP genotyping grouped specimens according to their habitat, mostly defined by plant associations. A few specimens with intermediate morphologies clustered with one of the two ecotypes or formed separate clusters consistent with habitat differences. These results were discussed in an ecological speciation framework.Conclusions: The question of whether this case of ecological differentiation occurred in sympatry or allopatry remains open. Still, the observed pattern points towards ongoing divergence between the two ecotypes which is likely driven by a recent shift in host plant use.

Parental influences on pathogen resistance in brown trout embryos and effects of outcrossing within a river network.

Phenotypic plasticity can increase tolerance to heterogeneous environments but the elevations and slopes of reaction norms are often population specific. Disruption of locally adapted reaction norms through outcrossing can lower individual viability. Here, we sampled five genetically distinct populations of brown trout (Salmo trutta) from within a river network, crossed them in a full-factorial design, and challenged the embryos with the opportunistic pathogen Pseudomonas fluorescens. By virtue of our design, we were able to disentangle effects of genetic crossing distance from sire and dam effects on early life-history traits. While pathogen infection did not increase mortality, it was associated with delayed hatching of smaller larvae with reduced yolk sac reserves. We found no evidence of a relationship between genetic distance (W, FST) and the expression of early-life history traits. Moreover, hybrids did not differ in phenotypic means or reaction norms in comparison to offspring from within-population crosses. Heritable variation in early life-history traits was found to remain stable across the control and pathogen environments. Our findings show that outcrossing within a rather narrow geographical scale can have neutral effects on F1 hybrid viability at the embryonic stage, i.e. at a stage when environmental and genetic effects on phenotypes are usually large.

Amoebae as a tool to isolate new bacterial species, to discover new virulence factors and to study the host-pathogen interactions.

Amoebae are unicellular protozoan present worldwide in several environments mainly feeding on bacteria. Some of them, the amoebae-resistant bacteria (ARBs), have evolved mechanisms to survive and replicate inside amoebal species. These mainly include legionella, mycobacteria and Chlamydia-related bacteria. Amoebae can provide a replicative niche, can act as reservoir for bacteria whereas the cystic form can protect the internalized bacteria. Moreover, the amoebae represent a Trojan horse for ARBs to infect animals. The long interaction between amoebae and bacteria has likely selected for bacterial virulence traits leading to the adaptation towards an intracellular lifestyle, and some ARBs have acquired the ability to infect mammals. This review intends to highlight the important uses of amoebae in several fields in microbiology by describing the main tools developed using amoebal cells. First, amoebae such as Acanthamoeba are used to isolate and discover new intracellular bacterial species by two main techniques: the amoebal co-culture and the amoebal enrichment. In the second part, taking Waddlia chondrophila as example, we summarize some important recent applications of amoebae to discover new bacterial virulence factors, in particular thanks to the amoebal plaque assay. Finally, the genetically tractable Dictyostelium discoideum is used as a model organism to study host-pathogen interactions, in particular with the development of several approaches to manipulate its genome that allowed the creation of a wide range of mutated strains largely shared within the Dictyostelium community.

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

Résumé : Les jasmonates (JA), une famille d'hor1none végétale, jouent un rôle central dans la réponse à la blessure, et aux attaques d'insectes et de pathogènes. Les JA sont principalement dérivés d'un acide gras, l'acide linolénique. L'addition par une lipoxygénase d'une molécule d'oxygène à l'acide linolénique initie la synthèse de JA. Cependant les mécanismes régulant l'activation de la biosynthèse de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caractérisé chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activité lipoxygénase est plus élevée que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus élevés et l'activation de la synthèse de JA après blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus résistant au pathogène Botrytis cinerea et à la chenille Spodoptera littoralis. Afin de comprendre quel mécanisme chez fou2 génére ce phénotype, nous avons cloné le gène responsable du phénotype de fou2. Le mutant fou2 porte une mutation dans le gène d'un canal à deux pores transportant probablement du potassium, du lumen de la vacuole végétale vers le compartiment cytosolique. L'analyse du protéome de fou2 a permis d'identifier une expression plus élevée de sept protéines régulées par les JA ou le stress. La découverte de l'implication d'un canal dans le phénotype de fou2 renforce l'hypothèse que les flux de cations pourraient être impliqués dans les étapes précoces de la synthèse des JA. Nous avons également étudié le protéome et la physiologie d'une feuille blessée, Pour évaluer les changements d'expression protéique en réponse à la blessure et contrôlés par les JA, nous avons quantifié l'expression de 5937 protéines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synthétiser des JA. Parmi ces 5937 protéines, nous avons identifié 99 protéines régulées par la blessure chez le type sauvage. Nous avons observé pour 65% des protéines dont l'expression protéique changeait après blessure une bonne corrélation entre la quantité de transcrits et de protéines. Plusieurs enzymes de la voie des chorismates impliquées dans la biosynthèse des acides aminés phénoliques étaient induites par les JA après blessure. Une quantification des acides aminés a montré que les niveaux d'acides aminés phénoliques augmentaient significativement après blessure. La blessure induisait aussi des changements dans l'expression de protéines impliquées dans la réponse au stress et particulièrement au stress oxydatif. Nous avons quantifié l'état réduit et oxydé du glutathion, un tripeptide qui, sous sa forme réduite, est l'antioxydant majeur des cellules. Nous avons trouvé une quantité significativement plus élevée de glutathion oxydé chez le type sauvage blessé que chez la plante aus blessée. Ce résultat suggère que la génération d'un stress oxydatif et la proportion relative de glutathions réduits et oxydés sont contrôlés par les JA après blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.

Study of drug tolerance mechanisms and of the calcineurin pathway in the opportunistic pathogen "Candida albicans"

ABSTRACT :Azole antifungal drugs possess fungistatic activity in Candida albicans making this human pathogen tolerant to these agents. The conversion of azoles into fungicidal agents is of interest since their fungistatic properties increase the ability of C. albicans to develop drug resistance. In C. albicans, the phosphatase calcineurin (calcineurin) is essential for antifungal drug tolerance. Up to now, the only known target of calcineurin is Crzl, which is a transcription factor (TF) involved in responses to ionic stress. Thus, most of the components of the calcineurin signaling remain to be identified in C. albicans.In this work, the calcineurin pathway was investigated in order to i) characterize the role of calcineurin in the biology of C. albicans, ii) identify putative targets of calcineurin and iii) characterize the phenomenon of tolerance to antifungal drugs. Towards these aims, four different approaches were used.First, using C. albicans microarrays, an attempt was made to identify a set of calcineurindependent genes (CDGs). Since CDGs were highly dependent upon the external stimulus used to activate calcineurin (Ca2+ or terbinafine), this stimulus bias was bypassed by the construction of strains expressing a truncated autoactive form of calcineurin (Cmp1tr) in a doxycyclinedependent manner. The characterization of Cmpltr was undertaken and results showed that it mimicked awild-type activated calcineurin for all tested phenotypes (i.e. Cnbl-dependence, inhibition by FK506, phosphatase 2B activity, ability to dephosphorylate Crzl and to regulate Crz1-and calcineurin-dependent genes, role in antifungal drug tolerance and susceptibility, role in colony formation on Spider agar). Cmp1tr was therefore considered as a valid tool to study the calcineurin signaling pathway. In silico analysis of CDGs allowed the identification of i) a significant overlap between CDGs and genes regulated by the Cyrl signalíng pathway, ii) putative interactions between calcineurin activation and cell wall reorganization and phospholipid transport, iii) a putative interactión between calcineurin and the regulation of translation and iv) a putative relation between calcineurin and proteasome regulation. Further in silico analyses of the promoters of Crz1-independent CDGs were performed to identify TFs (other than Crz1) that were likely to regulate CDGs and therefore to be a direct target of calcineurin. The analyses revealed that Rpn4 and Mnl1 were TFs likely to be regulated by calcineurin.Second, in order to better characterize azole tolerance, an attempt was made to i) confirm the role of Hsp90 in fluconazole tolerance with a doxycycline-dependent Hsp90 expression system and ii) assess its calcineurin-dependence. Hsp90 was found to be significantly involved in fluconazole tolerance. However, results were not in agreement with the hypothesis that Hsp90 mediates fluconazole tolerance by the only downstream effector calcineurin. Rather Hsp90 is interacting with numerous components for fluconazole tolerance.Third, a collection of C. albicans TFs mutants were screened for loss of tolerance to terbinafine and fluconazole in order to identify TFs involved in antifungal drug tolerance. Out of the 265 TFs mutants screened, only the upc2Δ/Δ mutant showed a loss of fluconazole and terbinafine tolerance. Interestingly, no relation between Upc2 and calcineurin activity was found. These results suggested that the tolerance to antifungal drugs must not be only considered as a calcineurin-dependent phenomenon in C. albicans.Fourth, using FRCS analyses, an attempt was made to identify putative signs of programmed cell death (PCD) in calcineurin mutant cells upon loss of tolerance to terbinafine. A high proportion of cells died from both RO5-dependent (which is a sign of PCD) and ROS-independent (which is a sign of loss of homeostasis) processes in the calcineurin mutant. While these results suggest that calcineurin represses both loss of homeostasis and PCD, the role of calcineurin in PCD is still an open question.In conclusion, this work allowed i) the identification of several putative calcineurin targets, ii) the discovery of several links between calcineurin and signaling pathways and important biological processes and iii) the identification of novel components of calcineurin-independent mechanisms that participate in tolerance to antifungal drugs in C. albicans.RÉSUME :Les azoles sont des antifongiques qui présentent une activité fongistatique contre Candida albicans et rendent cette levure tolérante à ces agents. La conversion des azoles en agents fongicides est d'intérêts car leurs propriétés fongistatiques favorisent le développement de résistance aux drogues chez C. albicans. La calcineurine (calcineurin) est une phosphatase essentielle pour la tolérance aux antifongiques chez C. albicans. La seule cible connue de la calcineurin est Crz1, un facteur de transcription (FT) impliqué dans la réponse aux stress ionique. Ainsi, la plupart des constituants de la voie de signalisation de la calcineurin restent encore à être identifiés chez C. albicans.Dans ce travail de thèse, la voie de signalisation de la calcineurin a été étudiée de sorte à i) caractériser le rôle de la calcineurin dans la biologie de C. albicans, ii) identifier de nouvelles cibles de la calcineurin et iii) caractériser le phénomène de tolérance aux antifongiques. A ce propos, quatre approches ont été entreprises.Premièrement, des puces à ADN de C. albicans ont été utilisées afin d'identifier les gènes dépendants de la calcineurin (GDCs). Les GDCs étant étroitement dépendants du stimulus utilisé pour activer la calcineurin, le biais «stimulus» a été évité via la construction d'une souche exprimant une forme tronquée et autoactive de la calcineurin (Cmp1tr), en présence de doxycycline. La caractérisation de Cmp1tr a été entreprise et les résultats ont montré qu'elle mimait une calcineurin sauvage et activée pour la plupart des phénotypes testés (i.e. dépendance à Cnb1, inhibition par le FK506, activité phosphatase 2B, déphosphorylation de Crz1 et régulation de gènes dépendant de la calcineurin, rôle dans la tolérance et la susceptibilité aux antifongiques, rôle dans la formation des colonies sur milieu Spider). Cmp1tr a donc été considéré comme un outil pertinent pour l'étude de la voie de signalisation de la calcineurin. Les analyses in silico des GDCs ont permis l'identification i) d'un chevauchement entre les GDCs èt les gènes régulés par la voie de signalisation de Cyrl, ii) d'une interaction entre la calcineurin et la réorganisation de la paroi cellulaire ainsi que le transport des phospholipides, iii) d'une interaction entre calcineurin et la régulation de la traduction et iv) une relation entre la calcineurin et la régulation du protéasome. De plus, une analyse in silico des promoteurs des GDCs avec une régulation indépendante de Crz1 a permis d'identifier deux FTs qui pourraient être des cibles directes de la calcineurin, Rpn4 et Mnll.Deuxièmement, afin de caractériser la tolérance aux azoles, il a été entrepris i) de confirmer le rôle de Hsp90 dans la tolérance au fluconazole en utilisant un système d'expression dépendant de la doxycycline et ii) de caractériser sa dépendance à la calcineurin. Hsp90 a été montré impliqué dans la tolérance aux azoles. Cependant, les résultats n'ont pas corroboré une hypothèse expliquant le rôle d'Hsp90 dans la tolérance aux antifongiques par son unique. interaction avec la calcineurin. Il a été proposé que le rôle d'Hsp90 dans la tolérance aux antifongiques soit dû à ces multiples interactions avec le protéome de C. albicans plutôt que par son interaction avec un partenaire unique.Troisièmement, une collection de mutant pour des FTs de C. albicans a été criblée pour une perte de tolérance au fluconazole ou à la terbinafine, de sorte à identifier les FTs impliqués dans la tolérance aux antifongiques. Sur les 265 FTs passés au crible, seul le mutant upc2Δ/Δ a montré une perte de tolérance au fluconazole et à la terbinafine. Aucune relation n'a été trouvée entre la calcineurin et l'activité d'Upc2. Ces résultats suggèrent que la perte de tolérance aux antifongiques ne doit pas être considérée comme un phénomène exclusivement lié à la voie de signalisation de la calcineurin.Quatrièmement, en utilisant la cytométrie de flux, la présence de signes de mort cellulaire programmée (MCP) a été recherchée lors de la perte de tolérance du mutant calcineurin incubé avec de la terbinafine. Une grande proportion de cellules mortes incluant ou non une production de ROS (un signe de MCP) a été détectée dans le mutant calcineurin. Ces résultats préliminaires suggèrent que la calcineurin réprime autant la perte d'homéostasie qu'elle régule l'entrée en MCP. Cependant d'autres analyses sont nécessaires pour démontrer clairement le rôle de la calcineurin dans la régulation de la MCP.En conclusion, ce travail de thèse a permis i) l'identification de plusieurs cibles possibles de la calcineurine, ii) la découverte de plusieurs interactions entre la calcineurine et d'autres voies de signalisation et processus biologiques importants et iii) de démontrer la présence de voies indépendantes de la calcineurine impliquées dans la tolérance aux antifongiques chez C. albicans.

Positional scanning-synthetic peptide library-based analysis of self- and pathogen-derived peptide cross-reactivity with tumor-reactive Melan-A-specific CTL.

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.

Maternal and paternal contributions to pathogen resistance dependent on development stage in a whitefish (Salmonidae)

It is often assumed that maternal and paternal contributions to offspring phenotype change over the lifetime of an individual. However, studies on parental effects typically suffer from the problems that heritabilities and maternal environmental effects are difficult to separate, and that both may depend on environmental factors and developmental stage. In order to experimentally disentangle maternal from paternal contributions and the likely effects of developmental stage from ecological effects, we sampled a natural population of the whitefish Coregonus palaea, used gametes for full-factorial in vitro fertilizations, raised over 10000 of the resulting offspring singly at controlled conditions, and exposed them at different points during embryonic development to one of two strains of Pseudomonas fluorescens that differed in their virulence characteristics (only one caused mortality, while both delayed hatching and reduced growth). Vulnerability to infection increased markedly over embryo development. This change coincided with a distinct shift in the importance of maternal to additive genetic effects on survival. Timing of exposure also affected the variance components for hatching time and larval length, but in a less consistent direction than the variance components for mortality. No significant genetic variation was found for any reaction norms across time points of exposure, indicating a uniformity among genotypes in how susceptibility changed over development. Phenotypes were also typically correlated across time points, which could constrain the evolution of the reaction norms. Our experiment demonstrates that the relative maternal and paternal contributions to susceptibility to an infection, and hence the evolutionary potential to respond to pathogen-induced selection, depends not only on the kind of pathogenic stress but also on the timing of the challenge.

HIV infection disrupts the sympatric host-pathogen relationship in human tuberculosis.

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.

Inactivation des pathogènes des produits sanguins labiles: entre enjeux financiers et conséquences possibles à long terme [Pathogen reduction of blood components: from financial issues to possible long-term consequences].

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.

Antibiotic susceptibility of Estrella lausannensis, a potential emerging pathogen.

Estrella lausannensis is a new Chlamydia-related bacterium, belonging to the Criblamydiaceae family. As suggested by its species name, this bacterium harbors a peculiar star shape. E. lausannensis is able to infect a wide range of amoebal, fish and mammalian cell lines. Moreover, seroprevalence of 2.9% was reported in children and in women with tubal pathology, showing that humans are commonly exposed to this recently discovered strict intracellular bacteria considered as a potential pathogen. Antibiotic susceptibility was determined using two approaches: qPCR and cellular mortality assay. Antibiotics classically used against intracellular bacteria were tested, including β-lactams, fluoroquinolones, cyclines and macrolides. We showed that E. lausannensis is resistant to β-lactams and fluoroquinolones, and sensitive to cyclines. Interestingly, E. lausannensis is slightly resistant to azithromycin with a MIC of 2 μg/ml, which is 10 fold higher compared to Waddlia chondrophila and Parachlamydia acanthamoebae MIC's. A single A2059C mutation in 23S rRNA gene could be responsible for this unexpected resistance.

Pathogen-induced hatching and population-specific life-history response to water-borne cues in brown trout (Salmo trutta)

Hatching is an important niche shift, and embryos in a wide range of taxa can either accelerate or delay this life-history switch in order to avoid stage-specific risks. Such behavior can occur in response to stress itself and to chemical cues that allow anticipation of stress. We studied the genetic organization of this phenotypic plasticity and tested whether there are differences among populations and across environments in order to learn more about the evolutionary potential of stress-induced hatching. As a study species, we chose the brown trout (Salmo trutta; Salmonidae). Gametes were collected from five natural populations (within one river network) and used for full-factorial in vitro fertilizations. The resulting embryos were either directly infected with Pseudomonas fluorescens or were exposed to waterborne cues from P. fluorescens-infected conspecifics. We found that direct inoculation with P. fluorescens increased embryonic mortality and induced hatching in all host populations. Exposure to waterborne cues revealed population-specific responses. We found significant additive genetic variation for hatching time, and genetic variation in trait plasticity. In conclusion, hatching is induced in response to infection and can be affected by waterborne cues of infection, but populations and families differ in their reaction to the latter.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

 During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling.

Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.