Antifungal Resistance and New Strategies to Control Fungal Infections

Despite improvement of antifungal therapies over the last 30 years, the phenomenon of antifungal resistance is still of major concern in clinical practice. In the last 10 years the molecular mechanisms underlying this phenomenon were extensively unraveled. In this paper, after a brief overview of currently available antifungals, molecular mechanisms of antifungal resistance will be detailed. It appears that major mechanisms of resistance are essential due to the deregulation of antifungal resistance effector genes. This deregulation is a consequence of point mutations occurring in transcriptional regulators of these effector genes. Resistance can also follow the emergence of point mutations directly in the genes coding antifungal targets. In addition we further describe new strategies currently undertaken to discover alternative therapy targets and antifungals. Identification of new antifungals is essentially achieved by the screening of natural or synthetic chemical compound collections. Discovery of new putative antifungal targets is performed through genome-wide approaches for a better understanding of the human pathogenic fungi biology

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia

BACKGROUND: Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host\u27s immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. RESULTS: We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. CONCLUSION: These results suggest that, in addition to a protective role against the host\u27s immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors

Network on « Genetics of adaptation and animal welfare »

Relations between genetics and animal welfare raise numerous questions as genetic characteristics are involved in many aspects of animals’ abilities to adapt to farming conditions, whether intensive or extensive. These questions are related to the genetic mechanisms of adaptation, to the consequences on animal welfare of the selection implemented up until now, and to the future improvements of the selection process. A network on the genetics of adaptation and animal welfare was built up within the multidisciplinary project “Agri Bien-être Animal” to review current knowledge. It aims also at providing usable references for regulatory discussions, as well as promoting synergies and exchanges between research units, development organisations and breeders.Les interrogations sur les relations entre génétique et bien-être des animaux sont d'autant plus nombreuses que les caractéristiques génétiques interviennent dans de nombreuses composantes des capacités d'adaptation des animaux à leurs conditions d'élevage, que celles-ci soient intensives ou extensives. Les questions portent à la fois sur les mécanismes génétiques de l'adaptation, les conséquences en matière de bien-être des sélections opérées jusqu'à présent et la conduite à tenir dans les futurs schémas de sélection. Un réseau Génétique de l'adaptation et bien-être a donc été créé au sein du programme interdisciplinaire INRA « Agri Bien-être Animal » pour faire le point des connaissances acquises. L'objectif est de fournir ainsi des références utilisables lors des discussions réglementaires et de faciliter les synergies et les échanges entre unités de recherche, organismes de développement et professionnels de la sélection

Biological Characterization and in Vivo Assessment of the Activity of a New Synthetic Macrocyclic Antifungal Compound

We recently identified a novel family of macrocyclic amidinoureas showing potent antifungal activity against Candida spp. In this study, we demonstrate the fungicidal effect of these compounds as well as their killing activity in a dose-dependent manner. Transcriptional analysis data indicate that our molecules induce a significant change in the transcriptome involving ATP binding cassette (ABC) transporter genes. Notably, experiments against Candida albicans mutants lacking those genes showed resistance to the compound, suggesting the involvement of ABC transporters in the uptake or intracellular accumulation of the molecule. To probe the mode of action, we performed fluorescence microscopy experiments on fungal cells treated with an ad-hoc synthesized fluorescent derivative. Fluorescence microscopy images confirm the ability of the compound to cross the membrane and show a consistent accumulation within the cytoplasm. Finally, we provide data supporting the in vivo efficacy in a systemic infection murine model setup with a drug-resistant strain of C. albicans

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants

Mécanismes moléculaires de la résistance aux antifongiques chez candida glabrata

Candida glabrata, which ranks the second among the causative agents of candidiasis in all clinical forms of the disease, possesses singular traits and determines a greater mortality rate in patients with systemic candidiasis. This species is also less susceptible to azoles, the most used class of antifungals. Moreover, it seems that an acquired resistance to azole, polyene and pyrimidin analog drugs is more frequent in C. glabrata. Thus, we studied clinical isolates as well as laboratory mutants with a resistance to these three classes of antifungal and aimed at determining the molecular mechanisms responsible for their resistance. Classical molecular biology tools revealed in each case a modification in the expression of some genes involved in the metabolism of these drugs or of their target, as well as the presence of a point mutation in these genes. The haploid genome of C. glabrata, with the subsequent higher probability to express a mutated gene, and the actual therapeutic context, which favors prophylactic use of antifungals in immunocompromised patients, represent a privileged environment for the development of a candidiasis and for the simultaneous selection of resistant isolates.L'espèce Candida glabrata, qui se situe au deuxième rang parmi les agents des candidoses toutes formes cliniques confondues, possède des caractéristiques singulières et engendre notamment une plus grande mortalité chez les patients atteints de candidoses profondes ou systémiques. Cette espèce présente notamment la particularité d'être peu sensible aux azolés, les antifongiques les plus couramment utilisés en clinique. De plus, il semble que le développement d'une résistance aux antifongiques aussi bien azolés, que polyéniques ou pyrimidiques, soit plus fréquent chez C. glabrata. Nous nous sommes donc proposé d'étudier des isolats cliniques ou des mutants induits de cette espèce résistants à ces trois classes d'antifongiques et de déterminer les mécanismes moléculaires à l'origine de leur résistance. Les outils de biologie moléculaire classique ont mis en évidence dans chaque cas une dérégulation de l'expression de certains gènes impliqués dans le métabolisme de ces molécules antifongiques ou de leur cible, ainsi que des mutations sur ces mêmes gènes. Le génome haploïde de C. glabrata, et donc la plus forte probabilité d'exprimer un gène muté, ainsi que le contexte médical actuel, prônant la prophylaxie dans nombre de pathologies où l'immunité des patients est touchée, représentent un terrain favorable au développement d'une candidose et à la sélection simultanée d'isolats résistants

Génétique et adaptation chez les poissons : domestication, résistance au stress et adaptation aux conditions de milieu

National audienceThe genetic variation of the adaptation of fish to their farm environment is an important topic in the question of farm animal welfare. Three main aspects are studied: (i) the question of domestication, which is ongoing or to be done in many fish species; domesticated fish are better adapted to farming (better access to feed, less stress-sensitive), but lose some behaviours which are useful in the wild (predator avoidance); (ii) selection for stress resistance, which is efficient and seems to have some effects quite similar to those of domestication; (iii) adaptation to the environment, the genetic component of which seems quite important. From all the data studied, it seems possible to select fish which Rill be better adapted to farming. However, the high environmental sensitivity of fish will make the integration of the genotype by environmental interactions necessary when studying the effects of breeding practices on adaptation.Les variations génétiques de l’adaptation au milieu d’élevage chez les poissons sont un objet d’étude important dans le cadre des problématiques liées au bien-être en élevage. Trois aspects principaux sont distingués. Tout d’abord la question de la domestication, importante car encore en cours ou à faire sur beaucoup d’espèces : les poissons domestiqués se montrent mieux adaptés à l’élevage (plus aptes à se nourrir, moins stressables), mais perdent des comportements utiles en milieu naturel (fuite devant les prédateurs). Ensuite la sélection pour la résistance aux conditions stressantes : cette sélection est efficace et semble avoir au moins pour partie des effets comparables à la domestication. Enfin, l’adaptation aux conditions de milieu et d’élevage, dont la composante génétique semble relativement importante. Il semble possible d’après l’ensemble de ces données de sélectionner des animaux qui seront mieux adaptés à leur milieu d’élevage. Cependant, la sensibilité des poissons à leur milieu et la variabilité de ces milieux impliquent des approches intégrant les interactions génotype-milieu dans l’étude de l’effet des pratiques d’élevage sur l’adaptation des animaux

Mécanismes moléculaires de la résistance aux antifongiques chez candida glabrata

L'espèce Candida glabrata, qui se situe au deuxième rang parmi les agents des candidoses toutes formes cliniques confondues, possède des caractéristiques singulières et engendre notamment une plus grande mortalité chez les patients atteints de candidoses profondes ou systémiques. Cette espèce présente notamment la particularité d'être peu sensible aux azolés, les antifongiques les plus couramment utilisés en clinique. De plus, il semble que le développement d'une résistance aux antifongiques aussi bien azolés, que polyéniques ou pyrimidiques, soit plus fréquent chez C. glabrata. Nous nous sommes donc proposé d'étudier des isolats cliniques ou des mutants induits de cette espèce résistants à ces trois classes d'antifongiques et de déterminer les mécanismes moléculaires à l'origine de leur résistance. Les outils de biologie moléculaire classique ont mis en évidence dans chaque cas une dérégulation de l'expression de certains gènes impliqués dans le métabolisme de ces molécules antifongiques ou de leur cible, ainsi que des mutations sur ces mêmes gènes. Le génome haploïde de C. glabrata, et donc la plus forte probabilité d'exprimer un gène muté, ainsi que le contexte médical actuel, prônant la prophylaxie dans nombre de pathologies où l'immunité des patients est touchée, représentent un terrain favorable au développement d'une candidose et à la sélection simultanée d'isolats résistants.Candida glabrata, which ranks the second among the causative agents of candidiasis in all clinical forms of the disease, possesses singular traits and determines a greater mortality rate in patients with systemic candidiasis. This species is also less susceptible to azoles, the most used class of antifungals. Moreover, it seems that an acquired resistance to azole, polyene and pyrimidin analog drugs is more frequent in C. glabrata. Thus, we studied clinical isolates as well as laboratory mutants with a resistance to these three classes of antifungal and aimed at determining the molecular mechanisms responsible for their resistance. Classical molecular biology tools revealed in each case a modification in the expression of some genes involved in the metabolism of these drugs or of their target, as well as the presence of a point mutation in these genes. The haploid genome of C. glabrata, with the subsequent higher probability to express a mutated gene, and the actual therapeutic context, which favors prophylactic use of antifungals in immunocompromised patients, represent a privileged environment for the development of a candidiasis and for the simultaneous selection of resistant isolates.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Genomic Organization and Expression of Iron Metabolism Genes in the Emerging Pathogenic Mold Scedosporium apiospermum

The ubiquitous mold Scedosporium apiospermum is increasingly recognized as an emerging pathogen, especially among patients with underlying disorders such as immunodeficiency or cystic fibrosis (CF). Indeed, it ranks the second among the filamentous fungi colonizing the respiratory tract of CF patients. However, our knowledge about virulence factors of this fungus is still limited. The role of iron-uptake systems may be critical for establishment of Scedosporium infections, notably in the iron-rich environment of the CF lung. Two main strategies are employed by fungi to efficiently acquire iron from their host or from their ecological niche: siderophore production and reductive iron assimilation (RIA) systems. The aim of this study was to assess the existence of orthologous genes involved in iron metabolism in the recently sequenced genome of S. apiospermum. At first, a tBLASTn analysis using A. fumigatus iron-related proteins as query revealed orthologs of almost all relevant loci in the S. apiospermum genome. Whereas the genes putatively involved in RIA were randomly distributed, siderophore biosynthesis and transport genes were organized in two clusters, each containing a non-ribosomal peptide synthetase (NRPS) whose orthologs in A. fumigatus have been described to catalyze hydroxamate siderophore synthesis. Nevertheless, comparative genomic analysis of siderophore-related clusters showed greater similarity between S. apiospermum and phylogenetically close molds than with Aspergillus species. The expression level of these genes was then evaluated by exposing conidia to iron starvation and iron excess. The expression of several orthologs of A. fumigatus genes involved in siderophore-based iron uptake or RIA was significantly induced during iron starvation, and conversely repressed in iron excess conditions. Altogether, these results indicate that S. apiospermum possesses the genetic information required for efficient and competitive iron uptake. They also suggest an important role of the siderophore production system in iron uptake by S. apiospermum