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Background: Instances of resistant fungal infection are rising in pulmonary disease, with limited therapeutic options. Therapeutic drug monitoring of azole antifungals has been necessary to ensure safety and efficacy but is considered unnecessary for the newest triazole isavuconazole. Aims: To characterise the prevalence of isavuconazole resistance and use in a tertiary respiratory centre. Methods: A retrospective observational analysis (2016–2021) of adult respiratory patients analysing fungal culture, therapeutic drug monitoring, and outcome post-isavuconazole therapy. Results: During the study period, isavuconazole susceptibility testing was performed on 26 Aspergillus spp. isolates. A total of 80.8% of A. fumigatus isolates had isavuconazole (MIC > 1 mg/L, and 73.0% > 2 mg/L) with a good correlation to voriconazole MIC (r = 0.7, p = 0.0002). A total of 54 patients underwent isavuconazole therapy during the study period (median duration 234 days (IQR: 24–499)). A total of 67% of patients tolerated isavuconazole, despite prior azole toxicity in 61.8%, with increased age (rpb = 0.31; p = 0.021) and male sex (φc = 0.30; p = 0.027) being associated with toxicity. A total of 132 isavuconazole levels were performed with 94.8% > 1 mg/L and 72% > 2 mg/L. Dose change from manufacturer’s recommendation was, however, required in 9.3% to achieve a concentration of >2 mg/L. Conclusion: We describe the use of isavuconazole as a salvage therapy in a chronic pulmonary fungal disease setting with a high prevalence of azole resistance. Therapeutic concentrations at standard dosing were high; however, results reinforce antifungal stewardship for optimization.

Contains fulltext : 225336.pdf (Publisher’s version ) (Open Access) Triazole resistant A. fumigatus has been documented in many parts of the world. In the Netherlands, incidence is now above 10% and results in the need for long-term parenteral therapy with liposomal amphotericin B (LAmB). The long terminal half-life of LAmB suggests that intermittent dosing could be effective, making the application of outpatient antifungal therapy (OPAT) possible. Here, we report our experience with the use of OPAT for Invasive Fungal Infections (IFI). All adult patients treated with LAmB with a 2 or 3 times weekly administration via the outpatient departments in four academic tertiary care centers in the Netherlands and Belgium since January 2010 were included in our analysis. Patient characteristics were collected, as well as information about diagnostics, therapy dose and duration, toxicity, treatment history and outcome of the IFI. In total, 18 patients were included. The most frequently used regimen (67%) was 5 mg/kg 3 times weekly. A partial response to the daily treatment prior to discharge was confirmed by CT-scan in 17 (94%) of patients. A favorable outcome was achieved in 13 (72%) patients. Decrease in renal function occurred in 10 (56%) cases but was reversible in all and was treatment limiting in one patient only. The 100-day mortality and 1-year mortality after initiation of OPAT were 0% and 6%, respectively. In a selected population, and after confirmation of initial response to treatment, our data support the use of OPAT with LAmB for treatment of IFI in an intermittent dosing regimen.

An increasing number of people is affected by fungal biofilm-based infections, which are resistant to the majority of currently-used antifungal drugs. Such infections are often caused by species from the genera Candida, Aspergillus or Cryptococcus. Only a few antifungal drugs, including echinocandins and liposomal formulations of amphotericin B, are available to treat such biofilm-based fungal infections. This review discusses combination therapy as a novel antibiofilm strategy. More specifically, in vitro methods to discover new antibiofilm combinations will be discussed. Furthermore, an overview of the main modes of action of promising antibiofilm combination treatments will be provided as this knowledge may facilitate the optimization of existing antibiofilm combinations or the development of new ones with a similar mode of action. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:21 issue:22 ispartof: location:Switzerland status: published

Infections caused by invasive molds, including Aspergillus spp., can be difficult to diagnose and remain associated with high morbidity and mortality. Thus, early diagnosis and targeted systemic antifungal treatment remains the most important predictive factor for a successful outcome in immunocompromised individuals with invasive mold infections. Diagnosis remains difficult due to low sensitivities of diagnostic tests including culture and other mycological tests for mold pathogens, particularly in patients on mold-active antifungal prophylaxis. As a result, antifungal treatment is rarely targeted and reliable markers for treatment monitoring and outcome prediction are missing. Thus, there is a need for improved markers to diagnose invasive mold infections, monitor response to treatment, and assist in determining when antifungal therapy should be escalated, switched, or can be stopped. This review focuses on the role of immunologic markers and specifically cytokines in diagnosis and treatment monitoring of invasive mold infections., This research was supported in part by Gilead Sciences Grant IN-EU-131-5720.

Positron emission tomography (PET) is a powerful diagnostic nuclear medicine imaging technique. PET allows in vivo detection of a wide variety of physiologic and pathologic phenomena and it offers a noninvasive tool for the monitoring of therapy in various diseases. Invasive fungal infections (IFIs) are a global concern because of the increasing population of patients at risk of IFIs and the high morbidity and mortality. Therapy with antifungal agents is long-standing and expensive. The emerging resistant fungal strains make the management of IFIs challenging. There is an absolute need for a sensitive noninvasive biomarker capable of monitoring the disease activity of IFIs and determining the efficacy of treatment at an early time point. PET imaging with 18Ffluorodeoxyglucose (FDG) was used to detect and assess disease activity in IFI foci already over 20 years ago. At that time, it was suggested it could be a useful biomarker for monitoring antifungal therapy. However, this knowledge has still not been fully exploited for the management of IFIs. The literature reveals an increasing realization of the usefulness of PET in monitoring therapy of IFIs. In this review, we highlight the advantages of nuclear medicine techniques in the management of IFIs with emphasis of the role of PET in monitoring therapy efficacy.

Positron emission tomography (PET) is a powerful diagnostic nuclear medicine imaging technique. PET allows in vivo detection of a wide variety of physiologic and pathologic phenomena and it offers a noninvasive tool for the monitoring of therapy in various diseases. Invasive fungal infections (IFIs) are a global concern because of the increasing population of patients at risk of IFIs and the high morbidity and mortality. Therapy with antifungal agents is long-standing and expensive. The emerging resistant fungal strains make the management of IFIs challenging. There is an absolute need for a sensitive noninvasive biomarker capable of monitoring the disease activity of IFIs and determining the efficacy of treatment at an early time point. PET imaging with F-18-fluorodeoxyglucose (FDG) was used to detect and assess disease activity in IFI foci already over 20 years ago. At that time, it was suggested it could be a useful biomarker for monitoring antifungal therapy. However, this knowledge has still not been fully exploited for the management of IFIs. The literature reveals an increasing realization of the usefulness of PET in monitoring therapy of IFIs. In this review, we highlight the advantages of nuclear medicine techniques in the management of IFIs with emphasis of the role of PET in monitoring therapy efficacy.

Recently, several research groups have assigned non-vacuolar functions to the well-known Saccharomyces cerevisiae vacuolar proteases Pep4 and Prb1, which are also known as proteinases A and B. These non-vacuolar activities seem to be autophagy-independent and stress-induced and suggest an unexplored but possibly prominent role for the proteases outside the vacuole. The functions range from the involvement in programmed cell death, to protection from hazardous protein forms and regulation of gene expression. We propose that a deeper understanding of these molecular processes will provide new insights that will be important for both fungal biology as well as studies in mammalian cells, as they might open up perspectives in the search for novel drug targets. To illustrate this, we summarize the recent literature on non-vacuolar Pep4 and Prb1 functions in S. cerevisiae and review the current data on the protein homologs in pathogenic fungi. ispartof: MICROBIAL CELL vol:5 issue:10 pages:438-443 ispartof: location:Austria status: published

CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:128 issue:9 pages:3957-3975 ispartof: location:United States status: published

Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes. Availability and implementation: MARDy can be accessed at http://www.mardy.net and is free to use. The complete database can be retrieved, ordered by organism, gene and drug. Missing or new mycological antifungal resistance data can be relayed to the development team through a contribute entry form.

WOS:000443665300007; International audience; Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors. Significance: We analyzse the role in inter-kingdom communication of lipoxygenase (lox) and cyclooxygenase (cox) genes in the model fungus Podospora anserina. Through chemical analysis we define the oxylipins and volatile organic compounds (VOCs)produce by wild type and mutants for cox and lox genes, We show that the COX and LOX genes are required for the production of some eight carbon VOCs. We show that COX and LOX genes are involved in the production of chemicals repelling nematodes. This role is very different from the ones previously evidenced in other fungi.

Dark septate endophytes (DSE) are a form-group of root endophytic fungi with elusive functions. Here, the genomes of two common DSE of semiarid areas, Cadophora sp. and Periconia macrospinosa were sequenced and analyzed with another 32 ascomycetes of different lifestyles. Cadophora sp. (Helotiales) and P. macrospinosa (Pleosporales) have genomes of 70.46 Mb and 54.99 Mb with 22,766 and 18,750 gene models, respectively. The majority of DSE-specific protein clusters lack functional annotation with no similarity to characterized proteins, implying that they have evolved unique genetic innovations. Both DSE possess an expanded number of carbohydrate active enzymes (CAZymes), including plant cell wall degrading enzymes (PCWDEs). Those were similar in three other DSE, and contributed a signal for the separation of root endophytes in principal component analyses of CAZymes, indicating shared genomic traits of DSE fungi. Number of secreted proteases and lipases, aquaporins, and genes linked to melanin synthesis were also relatively high in our fungi. In spite of certain similarities between our two DSE, we observed low levels of convergence in their gene family evolution. This suggests that, despite originating from the same habitat, these two fungi evolved along different evolutionary trajectories and display considerable functional differences within the endophytic lifestyle.

Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans.

The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses. This work has been supported by grants (GIU15/36 and UFI11/25) from the UPV/EHU. AP was supported by a predoctoral fellowship from the UPV/EHU, and IB and AA were supported by predoctoral fellowships from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Contains fulltext : 182569.pdf (Publisher’s version ) (Closed access) PURPOSE: To describe concisely the current standards of care, major recent advances, common beliefs that have been contradicted by recent trials, areas of uncertainty, and clinical studies that need to be performed over the next decade and their expected outcomes with regard to Candida and Aspergillus infections in non-neutropenic patients in the ICU setting. METHODS: A systematic review of the medical literature taking account of national and international guidelines and expert opinion. RESULTS: Severe invasive fungal infections (IFIs) are becoming increasingly frequent in critically ill patients. Approximately 80% of IFIs are due to Candida spp. and 0.3-19% to Aspergillus spp. Recent observations emphasize the necessity of building a worldwide sentinel network to monitor the emergence of new fungal species and changes in susceptibility. Robust data on the attributable mortality are essential for the design of clinical studies with mortality endpoints. Although early antifungal therapy for Candida has been recommended in patients with risk factors, sepsis of unknown cause, and positive Candida serum biomarkers [beta-1 --> 3-D-glucan (BDG) and Candida albicans germ tube antibody (CAGTA)], its usefulness and influence on outcome need to be confirmed. Future studies may specifically address the optimal diagnostic and therapeutic strategies for patients with abdominal candidiasis. Better knowledge of the pharmacokinetics of antifungal molecules and tissue penetration is a key issue for intensivists. Regarding invasive aspergillosis, further investigation is needed to determine its incidence in the ICU, its relationship with influenza outbreaks, the clinical impact of rapid diagnosis, and the significance of combination treatment. CONCLUSIONS: Fundamental questions regarding IFI have to be addressed over the next decade. The clinical studies described in this research agenda should provide a template and set priorities for the clinical investigations that need to be performed. 01 september 2017

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (β-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the β-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.

Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wildtype (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. Published version

Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with increased proinflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the proinflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies. H.A.K. acknowledges the Deanship of Scientific Research at King Saud University for funding via Group No. RGP-009.

This paper discusses the importance of culture collections in plant pathology and reviews the methods currently available to store cultures. The preservation and maintenance of plant pathogenic fungi in a viable yet stable state for long periods has always been important, because isolates of these fungi can serve as standards for identification of quarantine taxa. Such isolates are also important for testing disease resistance and for plant breeding programs. The increasing use of molecular sequences analysis in the systematics of plant pathogenic fungi has meant that maintaining fungi in culture collections has become essential. Herein we discuss trends in the identification of plant and post-harvest pathogens, using Aspergillus, Colletotrichum, Phyllosticta and Mycosphaerella and its anamorphs as examples. Herbarium specimens, although still a requirement of the Botanical Code when describing new species are, perhaps, less important in providing useful information when defining a pathogenic species. Many pathogen groups consist of complexes of species and morphology alone can no longer distinguish among species. However, ex-type living cultures are essential for identification and future species comparisons that incorporate the use of molecular techniques. As such, ex-type cultures of any new species of pathogen, or when new diseases are reported or studies involving pathogenic strains are published, cultures of the taxa studied should be deposited in widely available culture collections, preferably in at least three members of World Federation for Culture Collections. Methods for the storage of fungal cultures such as water preservation, freezing, mineral oil overlay, freeze drying and lyophilization are reviewed in this paper. The main objective of culture preservation is to maintain the vigor and genetic characteristics of a pure culture. Therefore, safe long-term preservation methods are required to ensure fungal survival and retention of any valuable characteristics. To minimize the risk of any morphological, physiological, or genetic changes, several different preservation conditions should be used whenever possible. The present review also describes a complete preservation methodology that can be used for plant pathogenic fungi.

Thymus and Activation-Regulated Chemokine (TARC) may be critical in Th2 cell recruitment in allergic inflammation; however, the mechanisms of allergen-induced TARC release are unclear. Since airway epithelium is the first line of defense to inhaled allergens, we questioned whether house dust mite allergen (Der p) can induce TARC expression in bronchial epithelial cells, how this is regulated at the molecular level, and if micro-environmental cytokines augment this effect. We examined the effects of Der p and the cytokines IL-4 and TGF-beta on TARC expression in 16HBE cells and primary bronchial asthma epithelium. Real-time PCR and immunofluorescence demonstrated that Der p induces TARC expression in bronchial epithelium. Supernatants from Der p-stimulated 16HBE cells were able to induce TARC-dependent T cell trafficking. IL-4 and TGF-beta cooperatively enhanced Der p-induced TARC expression in 16HBE cells. Specific inhibitors, immunodetection, and gel-shifts revealed that these effects are mediated by phosphorylation of the epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK) signaling and subsequent nuclear factor (NF)-kappa B activation. A Disintegrin And Metalloproteinase (ADAM), a family of proteins involved in shedding of various growth factors, was shown to be responsible for EGFR activation. The increase in TARC production by direct interaction of Der p with the bronchial epithelium may be an important initial step in the generation of allergic inflammation, which is further potentiated by micro-environmental cytokines. Interference with ADAM or EGFR activity may be a novel promising target to prevent TARC release and subsequent allergic inflammation.

The number of composting sites in Europe is rapidly increasing, due to efforts to reduce the fraction of waste destined for landfill, but evidence on possible health impacts is limited. This article systematically reviews studies related to bioaerosol exposures within and near composting facilities and associated health effects in both community and occupational health settings. Six electronic databases and bibliographies from January 1960 to July 2014 were searched for studies reporting on health outcomes and/or bioaerosol emissions related to composting sites. Risk of bias was assessed using a customized score. Five hundred and thirty-six papers were identified and reviewed, and 66 articles met the inclusion criteria (48 exposure studies, 9 health studies, 9 health and exposure studies). Exposure information was limited, with most measurements taken in occupational settings and for limited time periods. Bioaerosol concentrations were highest on-site during agitation activities (turning, shredding, and screening). Six studies detected concentrations of either Aspergillus fumigatus or total bacteria above the English Environment Agency’s recommended threshold levels beyond 250 m from the site. Occupational studies of compost workers suggested elevated risks of respiratory illnesses with higher bioaerosol exposures. Elevated airway irritation was reported in residents near composting sites, but this may have been affected by reporting bias. The evidence base on health effects of bioaerosol emissions from composting facilities is still limited, although there is sufficient evidence to support a precautionary approach for regulatory purposes. While data to date are suggestive of possible respiratory effects, further study is needed to confirm this and to explore other health outcomes. Waste management policy is rapidly evolving, largely because European countries are running out of space for landfill sites. Further, poor waste management contributes to climate change and threatens environmental sustainability. It is estimated that each tonne of food waste diverted from landfill to composting reduces greenhouse gas emissions by the equivalent of 0.4 to 0.7 tonnes of carbon dioxide (Department for Environment, Food and Rural Affairs [DEFRA], 2011 Department for Environment, Food and Rural Affairs . 2011. Government Review of Waste Policy in England 2011: Action Plan 2011. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/69402/pb13542-action-plan-.pdf (accessed January 8, 2015). [Google Scholar]). Responding to the urgent need to improve waste management, the European Commission set tighter standards on Biodegradable Municipal Waste (BMW) destined for landfill (Council Directive, 1999 Council Directive . 1999. Council Directive 1999/31/EC of 26 April 1999 on the landfill of waste. The Council of the European Union. http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:31999L0031 (accessed January 8, 2015). [Google Scholar]). Under the proposal, targets were set to recycle (including composting) at least 33% of household waste by 2015. During the last decade there has been a rise in the number of large-scale composting facilities, as well as the introduction of a wide range of more advanced composting technologies. The majority of composting in the United Kingdom is completed in open windrows, and in 2007 approximately 90% of the total composting in the United Kingdom was undertaken using this method (Sykes et al., 2007 Sykes, P., K. Jones, and J. D. Wildsmith. 2007. Managing the potential public health risks from bioaerosol liberation at commercial composting sites in the UK: An analysis of the evidence base. Resources Conserv. Recycling 52: 410–424.[Crossref], [Web of Science ®], [Google Scholar]). As material is in the open air, the process generates substantial potential emissions and exposures. Newer indoor facilities known as in-vessel composting facilities (IVC) still have some open-air processes, usually as part of the final maturation phase (Smith et al., 2009 Smith, R., E. J. Gilbert, and R. Pocock . 2009. Composting and biological treatment survey/final report. In Market survey of the UK organics and recycling industry—2007/08. Wellingborough, Northamptonshire: The Association for Organics Recycling. Available from http://www.organics-recycling.org.uk/uploads/article1769/WRAP_AFOR_Report_0708_-_FINAL_AFOR__3__cg.pdf (accessed January 8, 2015). [Google Scholar]). Composting yields significant concentrations of bioaerosols (Wheeler et al., 2001 Wheeler, P. A., I. Stewart, P. Dumitrean, and B. Donovan . 2001. Health effects of composting—A study of three composting sites and review of past data. In RandD Technical Report. Bristol, UK: Environment Agency. http://warrr.org/45 (accessed January 8, 2015). [Google Scholar]; Taha et al., 2006 Taha, M. P. M., G. H. Drew, P. J. Longhurst, R. Smith, and S. J. T. Pollard. 2006. Bioaerosol releases from compost facilities: Evaluating passive and active source terms at a green waste facility for improved risk assessments. Atmos. Environ. 40: 1159–1169.[Crossref], [Web of Science ®], [Google Scholar]). Bioaerosols are airborne biological particles, microbial fragments and constituents of cells, which may be viable or nonviable, and consist of fungi, bacteria, pollen, and fragments, constituents, particulate matter (PM10), and by-products of cells (Douwes et al., 2003 Douwes, J., P. Thorne, N. Pearce, and D. Heederik. 2003. Bioaerosol health effects and exposure assessment: progress and prospects. Ann. Occup. Hyg. 47: 187–200.[Crossref], [PubMed], [Web of Science ®], [Google Scholar]; Dowd and Maier, 2000 Dowd, S.E., and R. M. Maier. 2000. Aeromicrobiology. In Environmental microbiology, ed. R. M. Maier, I. L. Pepper, and C. P. Gerba, 91–122. Toronto, Canada: Academic Press. [Google Scholar]; Viegas et al., 2014 Viegas, C., A. Q .Gomes, J. Abegão, R. Sabino, T. Graça, and S. Viegas. 2014. Assessment of fungal contamination in waste sorting plants and incineration—Case study in Portugal. J. Toxicol. Environ. Health A 77: 57–68[Taylor & Francis Online], [Web of Science ®], [Google Scholar]). A Health and Safety Executive (HSE) report in 2003 highlighted some studies where site workers demonstrated adverse health effects from occupational exposure to bioaerosols produced during composting (Swan et al., 2003 Swan, J. R. M., A. Kelsey, B. Crook, and E. J. Gilbert . 2003. Occupational and environmental exposure to bioaerosols from composts and potential health effects—A critical review of published data. Health and Safety Executive. http://www.hse.gov.uk/research/rrpdf/rr130.pdf (accessed January 8, 2015). [Google Scholar]). 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Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2 , MTI , and ERG3 , in order to obtain a rapid genotyping method for C. glabrata . One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2 , MTI , and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata . A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata . However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.

Healthy individuals are continuously exposed to fungal biomass, which includes live and dead spores and fungal debris that is entrapped in the airways. In patients with asthma and/or atopy, exposure to fungal biomass might result in age-dependent sensitization and asthmatic reactions. Interaction with Toll-like receptors (TLRs), of the innate immune defense (alveolar macrophages and epithelial cells) and protease-activated receptors (PARs) determine the effectiveness of elimination of fungal material. The association of sensitization to Alternaria with severe asthma is discussed in relation to the age-dependent sensitization, rate of release of allergens from spores and activity of its proteases. A model is described concerning the influence of polymorphic genes for airway hyperresponsiveness (AHR) and atopy, showing a cumulating influence on susceptibility for allergen-induced asthma and explaining that fungus-induced airway obstruction is mainly associated with more severe asthma.

Although augmentation of the maxillary sinus floor with autogenous bone grafts has become a well established preimplantology procedure, its effect on the function of the maxillary sinus has not been the subject of prospective human studies. In this prospective study the effects of sinus floor augmentation on maxillary sinus performance were evaluated. Seventeen consecutive patients who were to undergo augmentation of the maxillary sinus floor with an iliac crest autogenous bone graft agreed to participate in this study. All patients were subject to (i) extensive anamnestic and clinical investigation on sinusitis, (ii) conventional radiography (Waters' projection) and (iii) unilateral endoscopic inspection of the maxillary sinus. This triad of evaluations was performed preoperatively, immediately preceding the augmentation procedure (the maxillary sinus to be inspected endoscopically was randomly selected), and at 3 (at insertion of the implants) and 9 months (at uncovering of implants) postaugmentation. None of the 17 patients showed clinical or radiological signs of actual sinus pathology preoperatively, though 5 patients had a history of an impeded sinus clearance. By contrast, unilateral endoscopic evaluation revealed preexisting subclinical mucosal pathology in two out of five patients with a history of sinus clearance impairment and in one out of the other 12 patients. At 3 months' postaugmentation, clinical and radiographical examination showed chronic maxillary sinusitis in one non-compromised patient. Moreover, serial unilateral endoscopic evaluation revealed subclinical maxillary mucosal pathology in four other patients (two of whom had a history of an impeded sinus clearance), confirmed by Waters' projection in three of these four patients. At 9 months' postaugmentation, only subclinical maxillary mucosal pathology was detected endoscopically in two patients (one compromised, one non-compromised patient), confirmed by Waters' projection in this last patient. Five implants were lost during the 9-month observation period. As is obvious from this prospective evaluation, the effects of the augmentation procedure on maxillary sinus performance in patients without signs of maxillary sinusitis are of no clinical significance.

Although augmentation of the maxillary sinus floor with autogenous bone grafts has become a well established preimplantology procedure, its effect on the function of the maxillary sinus has not been the subject of prospective human studies. In this prospective study the effects of sinus floor augmentation on maxillary sinus performance were evaluated. Seventeen consecutive patients who were to undergo augmentation of the maxillary sinus floor with an iliac crest autogenous bone graft agreed to participate in this study. All patients were subject to (i) extensive anamnestic and clinical investigation on sinusitis, (ii) conventional radiography (Waters' projection) and (iii) unilateral endoscopic inspection of the maxillary sinus. This triad of evaluations was performed preoperatively, immediately preceding the augmentation procedure (the maxillary sinus to be inspected endoscopically was randomly selected), and at 3 (at insertion of the implants) and 9 months (at uncovering of implants) postaugmentation. None of the 17 patients showed clinical or radiological signs of actual sinus pathology preoperatively, though 5 patients had a history of an impeded sinus clearance. By contrast, unilateral endoscopic evaluation revealed pre-existing subclinical mucosal pathology in two out of five patients with a history of sinus clearance impairment and in one out of the other 12 patients. At 3 months' postaugmentation, clinical and radiographical examination showed chronic maxillary sinusitis in one non-compromised patient. Moreover, serial unilateral endoscopic evaluation revealed subclinical maxillary mucosal pathology in four other patients (two of whom had a history of an impeded sinus clearance), confirmed by Waters' projection in three of these four patients. At 9 months' postaugmentation, only subclinical maxillary mucosal pathology was detected endoscopically in two patients (one compromised, one non-compromised patient), confirmed by Waters' projection in this last patient. Five implants were lost during the 9-month observation period. As is obvious from this prospective evaluation, the effects of the augmentation procedure on maxillary sinus performance in patients without signs of maxillary sinusitis are of no clinical significance.

Epithelial cells in the airway wall actively interact with environmental antigens/allergens, both in healthy individuals and patients with asthma. In patients with (allergic) asthma; the epithelium is abnormal, showing damaged structures and continuous activation similar to a repair phenotype cell. Epithelial cells bind allergens by a diversity of innate receptors, similar and in part identical to the Toll-like receptor family, which can induce the release of cytokines, chemokines,and growth factors. Protease-containing extracts (house dust mite, fungi) may, additionally cause damage of the epithelial cell layer, thereby enhancing the repair phenotype of epithelial cells in patients with asthma. These interactions may result in facilitation of transport of allergens and enhanced presentation to the immune system (Th2-type response). The inflammatory response induces a second phase of Th2-type cytoknes and cytotoxic products that will enhance growth factor-mediated airway remodeling, as is found in asthma. An understanding of the largely unknown innate responses of epithelial cells with environs mental antigens/allergens may, open,view treatment modalities for asthma and other airway diseases.

Superoxide dismutase (SOD) activities of the biotrophic pathogen Claviceps purpurea, which causes the ergot disease on a wide range of host grasses, were examined in axenic and pathogenic cultures. Almost all SOD activity in axenic culture originated from a single Cu,Zn SOD; a substantial part of this activity could be separated from lyophilized intact mycelia by gentle washing, indicating that this protein is at least partially secreted. The corresponding gene (cpsod1) was cloned and characterized; like other fungal Cu,Zn SOD genes, it groups with the extracellular mammalian Cu,Zn SODs in a phylogenetic tree. Northern analyses showed that cpsod1 is strongly induced by copper and weakly induced by iron; superoxide generated by paraquat, or xanthine and xanthine oxidase, as well as hydrogen peroxide, had no effect on gene expression under axenic conditions. Analysis of the deletion mutant Deltacpsod1 showed that, although growth in axenic culture was generally slower, sensitivity to paraquat was not increased in comparison to the wild-type. Pathogenicity assays showed that this gene is not essential for parasitic growth in rye; no further soluble SOD activity is induced in the mutant.

summary Superoxide dismutase (SOD) activities of the biotrophic pathogen Claviceps purpurea, which causes the ergot disease on a wide range of host grasses, were examined in axenic and pathogenic cultures. Almost all SOD activity in axenic culture originated from a single Cu,Zn SOD; a substantial part of this activity could be separated from lyophilized intact mycelia by gentle washing, indicating that this protein is at least partially secreted. The corresponding gene (cpsod1) was cloned and characterized; like other fungal Cu,Zn SOD genes, it groups with the extracellular mammalian Cu,Zn SODs in a phylogenetic tree. Northern analyses showed that cpsod1 is strongly induced by copper and weakly induced by iron; superoxide generated by paraquat, or xanthine and xanthine oxidase, as well as hydrogen peroxide, had no effect on gene expression under axenic conditions. Analysis of the deletion mutant Deltacpsod1 showed that, although growth in axenic culture was generally slower, sensitivity to paraquat was not increased in comparison to the wild-type. Pathogenicity assays showed that this gene is not essential for parasitic growth in rye; no further soluble SOD activity is induced in the mutant.

Previous studies have suggested that endogenous protease inhibitors may participate in the mucosal host defense. Antileukoprotease (ALP) is an important protease inhibitor found on various mucosal surfaces, including those of the respiratory and genital tracts. This study reports on the antimicrobial activity of recombinant (r) ALP toward the human fungal pathogens Aspergillus fumigatus and Candida albicans. rALP expressed pronounced fungicidal activity toward metabolically active A. fumigatus conidia and C. albicans yeast cells; however, metabolically quiescent A. fumigatus conidia were totally resistant. In contrast with the protease inhibitory activity of rALP, the fungicidal activity was localized primarily in the NH2-terminal domain. On a molar base, the fungicidal activity of rALP was comparable with that of human defensins and lysozyme. In addition, rALP caused inhibition of C. albicans yeast cell growth. By exhibiting antifungal activity, ALP may play an important role in the innate mucosal defense against human pathogenic fungi.

International audience; BACKGROUND:SUN proteins are involved in yeast morphogenesis, but their function is unknown.RESULTS:SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated.CONCLUSION:Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity.SIGNIFICANCE:The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.

Actinomycetes are ubiquitous and some can be potentially pathogenic for humans when present in the air of some working areas. It's notably the case for Thermoactinomyces vulgaris in composting facilities where aerial concentrations can reach high values of more than 10 7 CFU·m − 3 . Workers exposure to these inhalable bioaerosols can be the source of various diseases. The literature reveals a lack of knowledge about risk assessment: there is neither dose–effects relationship for most agents, or threshold limit value. The objectives of this study were to develop and standardize a method to quantify workers exposure to bioaerosols. We have developed and evaluated a method to quantify airborne T . vulgaris based on DNA extraction of aerial microbial communities and qPCR. Four DNA extraction protocols were compared, and primers and a hydrolysis probe were designed for specific amplification of the target species ( gyrB gene). This method was compared to traditional methods based on viable or cultivable counting by epifluorescence microscopy or plating on selective media. The method was applied on environmental bioaerosols sampled under real exposure conditions in composting plants. We demonstrate that the method to quantify T. vulgaris in bioaerosols is specific, sensitive and repeatable. We demonstrate the occurrence and quantified T . vulgaris in the atmosphere of composting facilities with concentrations ranging from 3 × 10 2 to 3 × 10 6 × m − 3 .

A population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.0016/1,000 inhabitants. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus was Aspergillus , accounting for 86.3% of the isolates, followed by Scedosporium at 4.7%; the order Mucorales at 2.5%; Penicillium at 2.2%, and Fusarium at 1.2%. The most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. terreus (8.1%), A. tubingensis (6.8%), and A. niger (6.5%). Cryptic/sibling Aspergillus species accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those of Scedosporium and Mucorales and also among cryptic species of Aspergillus , with 40% of these isolates showing resistance to all of the antifungal compounds tested. Cryptic Aspergillus species seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.

Burkholderia terrae BS001 has previously been proposed to be a 'generalist' associate of soil fungi, but its strategies of interaction have been largely ignored. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-Delta sctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-Delta sctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-Delta sctD to Lyophyllum sp. strain Karsten and to cell envelope-extracted ceramide monohexosides (CMHs) revealed that CMHs in both conidia and hyphae could bind strain BS001 cells. As BS001-Delta sctD adhered significantly less to the CMHs than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such avid adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope, in particular of the former.

International audience; BACKGROUND: Improving the hygienic quality of forages for horse nutrition seems to be a reasonable target for decreasing the prevalence of pulmonary diseases. The aim of the experiment was to study the effects of different agricultural practices on the main aero-allergens contained in forages, including breathable dust, fungi, mycotoxins and pollens. RESULTS: Results showed that the late harvest of hay, a second crop or a haylage production provides a good alternative to increase hygienic quality by reducing fungi contamination and breathable dust content. Barn drying of hay, while having no effect on breathable dust, similarly reduced fungi contamination. In contrast, when hay was harvested at a lower dry mass content (750g DM kg(-1) versus 850 g DM kg(-1)), both breathable dust and fungi contaminations were increased, which could at least be reversed by adding propionic acid just before baling. Zearalenone was detected in different hays, and even in one case, in breathable dust. CONCLUSION: Overall, our data suggest that different approaches can be used to increase forage hygienic quality for horse feeding and thus reduce their exposure to factors involved in equine pulmonary disease. (C) 2011 Society of Chemical Industry

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml −1 , respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ( glc YM). However, when grown on sterol-supplemented glc YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ 7 -cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11 ) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glc YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glc YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glc YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml −1 , respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata .

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes oft. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants. (C) 2012 Elsevier Inc. All rights reserved.

International audience; P>Primary carbohydrate metabolism plays a special role related to carbon/nitrogen exchange, as well as metabolic support of fruiting body development, in ectomycorrhizal macrofungi. In this study, we used information retrieved from the recently sequenced Tuber melanosporum genome, together with transcriptome analysis data and targeted validation experiments, to construct the first genome-wide catalogue of the proteins supporting carbohydrate metabolism in a plant-symbiotic ascomycete. More than 100 genes coding for enzymes of the glycolysis, pentose phosphate, tricarboxylic acid, glyoxylate and methylcitrate pathways, glycogen, trehalose and mannitol metabolism and cell wall precursor were annotated. Transcriptional regulation of these pathways in different stages of the T. melanosporum lifecycle was investigated using whole-genome oligoarray expression data together with real-time reverse transcription-polymerase chain reaction analysis of selected genes. The most significant results were the identification of methylcitrate cycle genes and of an acid invertase, the first enzyme of this kind to be described in a plant-symbiotic filamentous fungus. A subset of transcripts coding for trehalose, glyoxylate and methylcitrate enzymes was up-regulated in fruiting bodies, whereas genes involved in mannitol and glycogen metabolism were preferentially expressed in mycelia and ectomycorrhizas, respectively. These data indicate a high degree of lifecycle stage specialization for particular branches of carbohydrate metabolism in T. melanosporum.

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.

Background Previous studies in Ascomycetes have shown that the function of gene families of which the size is considerably larger in extant pathogens than in non-pathogens could be related to pathogenicity traits. However, by only comparing gene inventories in extant species, no insights can be gained into the evolutionary process that gave rise to these larger family sizes in pathogens. Moreover, most studies which consider gene families in extant species only tend to explain observed differences in gene family sizes by gains rather than by losses, hereby largely underestimating the impact of gene loss during genome evolution. Results In our study we used a selection of recently published genomes of Ascomycetes to analyze how gene family gains, duplications and losses have affected the origin of pathogenic traits. By analyzing the evolutionary history of gene families we found that most gene families with an enlarged size in pathogens were present in an ancestor common to both pathogens and non-pathogens. The majority of these families were selectively maintained in pathogenic lineages, but disappeared in non-pathogens. Non-pathogen-specific losses largely outnumbered pathogen-specific losses. Conclusions We conclude that most of the proteins for pathogenicity were already present in the ancestor of the Ascomycete lineages we used in our study. Species that did not develop pathogenicity seemed to have reduced their genetic complexity compared to their ancestors. We further show that expansion of gained or already existing families in a species-specific way is important to fine-tune the specificities of the pathogenic host-fungus interaction.

The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.