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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.

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.

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

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.

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.

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.

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.

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.

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.

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.

Helicobacter pyloriis a common and persistent human pathogen of the gastric mucosa. Surfactant protein D (SP-D), a component of innate immunity, is expressed in the human gastric mucosa and is capable of aggregatingH. pylori. Wide variation in the SP-D binding affinity toH. pylorihas been observed in clinical isolates and laboratory-adapted strains. The aim of this study was to reveal potential mechanisms responsible for evading SP-D binding and establishing persistent infection. An escape variant, J178V, was generated in vitro, and the lipopolysaccharide (LPS) structure of the variant was compared to that of the parental strain, J178. The genetic basis for structural variation was explored by sequencing LPS biosynthesis genes. SP-D binding to clinical isolates was demonstrated by fluorescence-activated cell sorter analyses. Here, we show thatH. pylorievades SP-D binding through phase variation in lipopolysaccharide. This phenomenon is linked to changes in the fucosylation of the O chain, which was concomitant with slipped-strand mispairing in a poly(C) tract of the fucosyltransferase A (fucT1) gene. SP-D binding organisms are predominant in mucus in vivo (P= 0.02), suggesting that SP-D facilitates physical elimination. Phase variation to evade SP-D contributes to the persistence of this common gastric pathogen.

Helicobacterpyloriestablishes persistent infection of gastric mucosa with diverse clinical outcomes. The innate immune molecule surfactant protein D (SP-D) binds selectively to microorganisms, inducing aggregation and phagocytosis. In this study, we demonstrated the expression of SP-D in gastric mucosa by reverse transcription-PCR and immuohistochemical analysis. SP-D is present at the luminal surface and within the gastric pits, with maximal expression at the surface. Levels of expression are significantly increased inH. pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used to demonstrate binding and agglutination ofH. pyloriby SP-D in a lectin-specific manner. These activities resulted in a 50% reduction in the motility ofH. pylori, as judged on the basis of curvilinear velocity measured by using a Hobson BacTracker. Lipopolysaccharides extracted from threeH. pyloristrains were shown to bind SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding among the strains. SP-D may therefore play a significant role in the innate immune response toH. pyloriinfection.

Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1–5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.