Comparison between conventional duplex ultrasonography and the dual-gate Doppler mode for hemodynamic measurements of the carotid arteries.

PURPOSE: This study investigated the correlations of hemodynamic parameters measured to quantify stenosis between the gold-standard duplex ultrasonography and the dual-gate Doppler mode. METHODS: Patients examined due to suspicion of carotid artery stenosis or for surveillance of known stenosis were invited to participate in this prospective single-center study. Upon acceptance, the hemodynamic characteristics of the carotid arteries were determined successively in standard duplex and dual-gate Doppler modes. The correlations between the two modes were analyzed by computing Pearson coefficients (r2) and Lin concordance coefficients (ρc). The degree of agreement between the two methods was visualized using Bland-Altman graphical representations. RESULTS: The correlation between internal carotid artery peak systolic velocity measured by standard duplex ultrasonography and dual-gate Doppler mode was good (r2=0.642). The same high level of correlation was observed for the carotid ratio (r2=0.544). However, the Bland-Altman graphical representation and the Lin concordance coefficients (ρc=0.75 and ρc=0.74 for the internal carotid artery peak systolic velocity and carotid ratio, respectively) showed that a lack of precision generated some discrepancies between the two measurement methods. CONCLUSION: Although some discrepancies were observed, the hemodynamic measurements were closely correlated between the two ultrasonography modes. Therefore, the dual-gate Doppler mode may have obvious advantages over conventional ultrasonography, offering interesting development possibilities.

Draft Genome Sequence of the Environmental Fungus Scedosporium dehoogii.

Today, the genus Scedosporium comprises at least ten species with four of them, Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum and Scedosporium minutisporum capable of colonizing the lungs of patients with cystic fibrosis. Scedosporium dehoogii, which is also common in the soil, has never been reported as causing human pulmonary infections. Here we report the first genome sequence for S. dehoogii, an invaluable resource to understand the genetic bases of pathogenesis in the genus Scedosporium.

The Glycosylphosphatidylinositol-Anchored Superoxide Dismutase of Scedosporium apiospermum Protects the Conidia from Oxidative Stress.

Scedosporium species are common fungal pathogens in patients with cystic fibrosis (CF). To colonize the CF lungs, fungi must cope with the host immune response, especially the reactive oxygen species (ROS) released by phagocytic cells. To this aim, pathogens have developed various antioxidant systems, including superoxide dismutases (SODs) which constitute the first-line protection against oxidative stress. Interestingly, one of the S. apiospermum SOD-encoding genes (SODD gene) exhibits a glycosylphosphatidylinositol (GPI) anchor-binding site and encodes a conidial-specific surface SOD. In this study, a SODDΔ mutant was engineered from a non-homologous end joining-deficient strain (KU70Δ) of S. apiospermum. Compared to its parent strain, the double mutant KU70Δ/SODDΔ exhibited increased susceptibility to various oxidizing agents and triazole antifungals. In addition, the loss of SodD resulted in an increased intracellular killing of the conidia by M1 macrophages derived from human blood monocytes, suggesting the involvement of this superoxide dismutase in the evasion to the host defenses. Nevertheless, one cannot disregard an indirect role of the enzyme in the synthesis or assembly of the cell wall components since transmission electron microscopic analysis revealed a thickening of the inner cell wall layer of the conidia. Further studies are needed to confirm the role of this enzyme in the pathogenesis of Scedosporium infections, including the production of a recombinant protein and study of its protective effect against the infection in a mouse model of scedosporiosis.

Comparative transcriptome analysis unveils the adaptative mechanisms of Scedosporium apiospermum to the microenvironment encountered in the lungs of patients with cystic fibrosis.

Scedosporium species rank second among the filamentous fungi colonizing the lungs of patients with cystic fibrosis (CF). Apart from the context of immunodeficiency (lung transplantation), the colonization of the CF airways by these fungi usually remains asymptomatic. Why the colonization of the lower airways by Scedosporium species is fairly tolerated by CF patients while these fungi are able to induce a marked inflammatory reaction in other clinical contexts remains questionable. In this regards, we were interested here in exploring the transcriptional reprogramming that accompanies the adaptation of these fungi to the particular microenvironment encountered in the airways of CF patients. Cultivation of Scedosporium apiospermum in conditions mimicking the microenvironment in the CF lungs was shown to induce marked transcriptional changes. This includes notably the down-regulation of enzymes involved in the synthesis of some major components of the plasma membrane which may reflect the ability of the fungus to evade the host immune response by lowering the biosynthesis of some major antigenic determinants or inhibiting their targeting to the cell surface through alterations of the membrane fluidity. In addition, this analysis revealed that some genes encoding enzymes involved in the biosynthesis of some mycotoxins were down-regulated suggesting that, during the colonization process, S. apiospermum reduces the production of some toxic secondary metabolites to prevent exacerbation of the immune system response. Finally, a strong up-regulation of many genes encoding enzymes involved in the degradation of aromatic compounds was observed, suggesting that these catabolic properties would predispose the fungus to particular patterns of human pathogenicity. Together these data provide new insights into the adaptative mechanisms developed by S. apiospermum in the CF lungs, which should be considered for identification of potential targets for drug development, but also for the experimental conditions to be used in in vitro susceptibility testing of clinical isolates to current antifungals.

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence.

Scedosporium species rank second among the filamentous fungi capable to colonize chronically the respiratory tract of patients with cystic fibrosis (CF). Nevertheless, there is little information on the mechanisms underpinning their virulence. Iron acquisition is critical for the growth and pathogenesis of many bacterial and fungal genera that chronically inhabit the CF lungs. In a previous study, we showed the presence in the genome of Scedosporium apiospermum of several genes relevant for iron uptake, notably SAPIO_CDS2806, an ortholog of sidD, which drives the synthesis of the extracellular hydroxamate-type siderophore fusarinine C (FsC) and its derivative triacetylfusarinine C (TAFC) in Aspergillus fumigatus. Here, we demonstrate that Scedosporium apiospermum sidD gene is required for production of an excreted siderophore, namely, Nα-methylcoprogen B, which also belongs to the hydroxamate family. Blockage of the synthesis of Nα-methylcoprogen B by disruption of the sidD gene resulted in the lack of fungal growth under iron limiting conditions. Still, growth of ΔsidD mutants could be restored by supplementation of the culture medium with a culture filtrate from the parent strain, but not from the mutants. Furthermore, the use of xenosiderophores as the sole source of iron revealed that S. apiospermum can acquire the iron using the hydroxamate siderophores ferrichrome or ferrioxamine, i.e., independently of Nα-methylcoprogen B production. Conversely, Nα-methylcoprogen B is mandatory for iron acquisition from pyoverdine, a mixed catecholate-hydroxamate siderophore. Finally, the deletion of sidD resulted in the loss of virulence in a murine model of scedosporiosis. Our findings demonstrate that S. apiospermum sidD gene drives the synthesis of a unique extracellular, hydroxamate-type iron chelator, which is essential for fungal growth and virulence. This compound scavenges iron from pyoverdine, which might explain why S. apiospermum and Pseudomonas aeruginosa are rarely found simultaneously in the CF lungs.

Non-ribosomal Peptide Synthetase Gene Clusters in the Human Pathogenic Fungus Scedosporium apiospermum.

Scedosporium species are opportunistic fungi which preferentially affect patients with underlying conditions such as immunosuppression or cystic fibrosis (CF). While being the second most common molds capable to chronically colonize the CF lungs, the natural history of infection remains unclear. In filamentous fungi, a broad range of important secondary metabolites that are recognized as virulence factors are produced by multidomain non-ribosomal peptide synthetases (NRPSs). The aim of this study was to provide a global in silico analysis of NRPS-encoding genes based on the recently sequenced Scedosporium apiospermum genome. We uncovered a total of nine NRPS genes, of which six exhibited sufficient similarity scores with other fungal NRPSs to predict the class of the generated peptide: siderophores (n = 2), epidithiodioxopiperazines (n = 2), and cyclopeptides (n = 2). Phylogenetic trees based on the multiple alignments of adenylation (A) domain sequences corroborated these findings. Nevertheless, substrate prediction methods for NRPS A-domains tended to fail, thus questioning about the exact nature of the peptide produced. Further studies should be undertaken since NRPSs, which are not synthesized by human cells, could represent attractive therapeutic targets.

Tyrosine kinase inhibitor-induced carotid stenosis: A case report.

Tyrosine kinase inhibitors are considered as highly effective and relatively safe drugs for the treatment of chronic myeloid leukemia. If several side effects on short and long term are well known and described, their involvement in the development of carotid stenosis remains unclear. Here, we describe a case of carotid stenosis in a patient receiving tyrosine kinase inhibitors and discuss the current literature.

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.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Scedosporium and Lomentospora: an updated overview of underrated opportunists.

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Impact of Infection Status and Cyclosporine on Voriconazole Pharmacokinetics in an Experimental Model of Cerebral Scedosporiosis.

Cerebral Scedosporium infections usually occur in lung transplant recipients as well as in immunocompetent patients in the context of near drowning. Voriconazole is the first-line treatment. The diffusion of voriconazole through the blood-brain barrier in the context of cerebral infection and cyclosporine administration is crucial and remains a matter of debate. To address this issue, the pharmacokinetics of voriconazole was assessed in the plasma, cerebrospinal fluid (CSF), and brain in an experimental model of cerebral scedosporiosis in rats receiving or not receiving cyclosporine. A single dose of voriconazole (30 mg/kg, i.v.) was administered to six groups of rats randomized according to the infection status and the cyclosporine dosing regimen (no cyclosporine, a single dose, or three doses; 15 mg/kg each). Voriconazole concentrations in plasma, CSF, and brain samples were quantified using ultra-performance liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography UV methods and were documented up to 48 hours after administration. Pharmacokinetic parameters were estimated using a noncompartmental approach. Voriconazole pharmacokinetic profiles were similar for plasma, CSF, and brain in all groups studied. The voriconazole Cmax and area under the curve (AUC) (AUC0 ≥ 48 hours) values were significantly higher in plasma than in CSF [CSF/plasma ratio, median (range) = 0.5 (0.39-0.55) for AUC0 ≥ 48 hours and 0.47 (0.35 and 0.75) for Cmax]. Cyclosporine administration was significantly associated with an increase in voriconazole exposure in the plasma, CSF, and brain. In the plasma, but not in the brain, an interaction between the infection and cyclosporine administration reduced the positive impact of cyclosporine on voriconazole exposure. Together, these results emphasize the impact of cyclosporine on brain voriconazole exposure.

A synthetic construct for genetic engineering of the emerging pathogenic yeast Candida auris.

Candida auris has recently emerged as a global cause of severe hospital-acquired fungal infections. To enable functional genomic approaches for this prominent pathogen, we designed a synthetic construct that can be used to genetically transform the genome-sequenced strain VPCI 479/P/13 of C. auris following an efficient electroporation procedure.

Progressive loss of hybrid histidine kinase genes during the evolution of budding yeasts (Saccharomycotina).

Two-component systems (TCSs) are widely distributed cell signaling pathways used by both prokaryotic and eukaryotic organisms to cope with a wide range of environmental cues. In fungi, TCS signaling routes, that mediate perception of stimuli, correspond to a multi-step phosphorelay between three protein families including hybrid histidine kinases (HHK), histidine phosphotransfer proteins (HPt) and response regulators (RR). The best known of these fungal transduction pathways remains the Sln1(HHK)-Ypd1(HPt)-Ssk1(RR) system that governs the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway for osmo-adaptation in Saccharomyces cerevisiae. Although recent advances have provided a preliminary overview of the distribution of TCS proteins in the kingdom Fungi, underlying mechanisms that drive the remarkable diversity among HHKs and other TCS proteins in different fungal lineages remain unclear. More precisely, evolutionary paths that led to the appearance, transfer, duplication, and loss of the corresponding TCS genes in fungi have never been hitherto addressed. In the present study, we were particularly interested in studying the distribution of TCS modules across the so-called "budding yeasts clade" (Saccharomycotina) by interrogating the genome of 82 species. With the exception of the emergence of an additional RR (named Srr1) in the fungal CTG clade, TCS proteins Ypd1 (HPt), Ssk1 (RR), Skn7 (RR), and Rim15 (RR) are well conserved within the Saccharomycotina. Surprisingly, some species from the basal lineages, especially Lipomyces starkeyi, harbor several filamentous-type HHKs that appear as relict genes that have been likely retained from a common ancestor of Saccharomycotina. Overall, this analysis revealed a progressive diminution of the initial pool of HHK-encoding genes during Saccharomycotina yeast evolution.

A standardized toolkit for genetic engineering of CTG clade yeasts.

We have developed a series of synthetic constructs suitable to genetically manipulate a broad range of yeast species belonging to the fungal CTG clade. This molecular toolbox notably allows heterologous gene expression, single or dual fluorescence labeling and construction of luciferase-expressing strains for bioluminescence imaging.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

Draft Genome Sequence of the Human-Pathogenic Fungus Scedosporium boydii.

The opportunistic fungal pathogen Scedosporium boydii is the most common Scedosporium species in French patients with cystic fibrosis. Here we present the first genome report for S. boydii, providing a resource which may enable the elucidation of the pathogenic mechanisms in this species.

Microbial antioxidant defense enzymes.

Free radicals are often described as chemical compounds characterized by unpaired electrons in their outer orbital rendering them highly reactive species. In mammalians, studies on free radicals were focused on reactive oxygen species (ROS) or reactive nitrogen species (RNS) due to their relative importance in physiological as well as in pathological processes. These cellular compounds are produced by different physiological systems such as the aerobic metabolism and play a major role in cell signaling pathways but also in the host immune defenses against pathogenic microorganisms. ROS and RNS are highly reactive species with potentially harmful effects on any cellular components (lipids, proteins and nucleic acids) when produced with a high level. To maintain ROS and RNS at a non-toxic concentration, enzymatic and non-enzymatic cellular antioxidants coordinate the balance between their production and their degradation. Superoxide dismutases, catalases, glutathione system, thioredoxin system, peroxidase systems, flavohemoglobins and nitrate or nitrite reductases represent the prominent enzymatic antioxidants used to scavenge excess of internal as well as external ROS and RNS. Bacteria, fungi and parasites also display similar enzymatic activities to escape the host oxidative defenses during the immune response against infectious processes. Here we summarize current knowledge on the enzymatic systems that allow microorganisms to fight against ROS and RNS, and shed light on the role that take some of them in microbial infections. Such microbial protective systems are considered as virulence factors, and therefore represent key targets for diagnosis of the infections or development of anti-infectious drugs.

An additional Meyerozyma guilliermondii IMH3 gene confers mycophenolic acid resistance in fungal CTG clade species.

The fungal CTG clade comprises a number of well-known yeasts that impact human health or with high biotechnological potential. To further extend the set of molecular tools dedicated to these microorganisms, the initial focus of this study was to develop a mycophenolic acid (MPA) resistance cassette. Surprisingly, while we were carrying out preliminary susceptibility testing experiments in a set of yeast species, Meyerozyma guilliermondii, although not being a MPA producer, was found to be primarily resistant toward this drug, whereas a series of nine related species were susceptible to MPA. Using comparative and functional genomic approaches, we demonstrated that all MPA-susceptible CTG clade species display a single gene, referred to as IMH3.1, encoding the MPA target inosine monophosphate dehydrogenase (IMPDH) and that MPA resistance relies on the presence in the M. guilliermondii genome of an additional IMPDH-encoding gene (IMH3.2). The M. guilliermondii IMH3.2 gene displays marked differences compared to IMH3.1 including the lack of intron, a roughly 160-fold higher transcription level and a serine residue at position 251. Placed under the control of the M. guilliermondii actin 1 gene promoter, IMH3.2 was successfully used to transform Lodderomyces elongisporus, Clavispora lusitaniae, Scheffersomyces stipitis and Candida parapsilosis.

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.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.