Journal of Fungi — Open Access Mycology Journal

Online ISSN: 2309-608X
Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO2 in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study, we reported a new group of succinic acid transporters from the AceTr family, which were also described as anion channels. Here, we expressed these transporters in a succinic acid overproducing strain and compared their impact on extracellular succinic acid accumulation with that of the Dct-02 transporter. The results show that the tested transporters of the AceTr family hinder succinic acid accumulation in the extracellular medium at low pH, which is in strong contrast to Dct-02. Data suggests that the AceTr transporters prefer monovalent succinate, whereas Dct-02 prefers divalent succinate anions. In addition, the results provided deeper insights into the characteristics of Dct-02, showing its ability to act as a succinic acid importer (thus being bidirectional) and verifying its capability of exporting malate.
To promote Agaricus sinodeliciosus var. Chaidam ZJU-TP-08 growth and metabolites accumulation, a novel integrated strategy was developed by adopting high levels of metal ions coupled with light treatment. The results revealed that yellow and blue light could significantly promote biomass and exopolysaccharides production, respectively. Furthermore, the yellow–blue light shift strategy could stimulate exopolysaccharides formation. Ca2+ ions coupled with blue light mostly promoted exopolysaccharides production related to oxidative stress, which was 42.00% and 58.26% higher than that of Ca2+ ions coupled with the non-light and dark cultivation without Ca2+ ions in 5-L bioreactor. RNA-seq was performed to uncover the underlined molecular mechanism regulated by light-induced gene expressions in exopolysaccharides biosynthesis and oxidative stress. The findings of this work provide valuable insights into adopting metal ions coupled with the light-assisted method for the macrofungus submerged fermentation for exopolysaccharides production.
Nutritional constituents and biological activity of highland barley (the control groups and colonized groups with the mycelia of A. sinodeliciosus var. Chaidam ZJU-TP-08) were visualized by principal component analysis (PCA).
To enhance the nutritional value of highland barley (HB), this work investigated the effects of solid-state fermentation (SSF) by Agaricus sinodeliciosus var. Chaidam ZJU-TP-08 on nutrient content, phenolic components, antioxidant activities, and physicochemical characteristics of HB upon different pretreatments (germination, ultrasound and soaking). The results showed that germinated highland barley (GHB) exhibited higher levels of ergosterol (0.19 ± 0.01 mg/g) in all fermentation groups. The content of β-glucan was higher in the SSF-GHB, with an increase of 24.21% compared to the control. The content of total amino acids, dietary fiber, total phenols and flavonoids were higher in the fermentation HB pretreated by ultrasound, increasing respectively by 5.60%, 61.50%, 25.10% and 65.32% compared to the control group. In addition, the colonized HB exhibited excellent physicochemical characteristics, including increased water solubility index and decreased pasting characteristics. Herein, the nutritional value and the biological activities were enriched in the pretreated HB through SSF, indicating its potential application for nutrition-enriched functional foods.
Evaluation of antifungal adequacy.
The implementation of 1,3 β-d-glucan (BDG) has been proposed as a diagnostic tool in antifungal stewardship programs (ASPs). We aimed to analyze the influence of serum BDG in an ASP for oncologic patients and solid organ transplant (SOT) recipients. We conducted a pre–post study. In the initial period (PRE), the ASP was based on bedside advice, and this was complemented with BDG in the post-period (POST). Performance parameters of the BDG assay were determined. Antifungal (AF) use adequacy was evaluated using a point score. Clinical outcomes and AF costs were also compared before and after the intervention. Overall, 85 patients were included in the PRE-period and 112 in the POST-period. Probable or proven fungal infections were similar in both groups (54.1% vs. 57.1%; p = 0.67). The determination of BDG contributed to improved management in 75 of 112 patients (66.9%). The AF adequacy score improved in the POST-period (mean 7.75 vs. 9.29; p < 0.001). Median days of empiric AF treatment was reduced in the POST-period (9 vs. 5 days, p = 0.04). All-cause mortality (44.7% vs. 34.8%; p = 0.16) was similar in both periods. The cost of AF treatments was reduced in the POST-period with a difference of 779.6 €/patient. Our data suggest that the use of BDG was a cost-effective strategy that contributed to safely improving the results of an ASP for SOT and oncologic patients.
Genes related to α-1,3-glucan synthesis and degradation in Aspergillus species.
Various functions of α-1,3-glucan in fungi. (A) Swollen conidia of Aspergillus species aggregate in the presence of α-1,3-glucan, resulting in hyphal aggregates under liquid culture conditions. In contrast, germinating conidia of α-1,3-glucan-defcient mutants do not aggregate. The hyphae of these mutants are dispersed (Aspergillus nidulans) or form small pellets (Aspergillus oryzae) in liquid culture. (B) In the early stage of submerged culture of A. oryzae, TAA (Taka-amylase A) diffuses to liquid medium because α-1,3-glucan accumulates on the surface of the mycelial cell wall. In the late stage, cell wall α-1,3-glucan is degraded to be used as a carbon source, and chitin in the cell wall is exposed. Then TAA is adsorbed and retained on the cell surface. (C) In Histoplasma capsulatum chemotype II, α-1,3-glucan is found in the outer layer of the cell wall of the yeast form, where it covers β-1,3-glucan. The production of the cytokine TNFα by host macrophages is suppressed by α-1,3glucan, resulting in the suppression of immune responses and pathogen's colonization of host cells. The lack of α-1,3-glucan attenuates the ability of the pathogen to kill macrophages and to colonize 
Various functions of α-1,3-glucan in fungi. (A) Swollen conidia of Aspergillus species aggregate in the presence of α-1,3-glucan, resulting in hyphal aggregates under liquid culture conditions. In contrast, germinating conidia of α-1,3-glucan-defcient mutants do not aggregate. The hyphae of these mutants are dispersed (Aspergillus nidulans) or form small pellets (Aspergillus oryzae) in liquid culture. (B) In the early stage of submerged culture of A. oryzae, TAA (Taka-amylase A) diffuses to liquid medium because α-1,3-glucan accumulates on the surface of the mycelial cell wall. In the late stage, cell wall α-1,3-glucan is degraded to be used as a carbon source, and chitin in the cell wall is exposed. Then TAA is adsorbed and retained on the cell surface. (C) In Histoplasma capsulatum chemotype II, α-1,3-glucan is found in the outer layer of the cell wall of the yeast form, where it covers β-1,3-glucan. The production of the cytokine TNFα by host macrophages is suppressed by α-1,3-glucan, resulting in the suppression of immune responses and pathogen's colonization of host cells. The lack 
Although α-1,3-glucan is a major cell wall polysaccharide in filamentous fungi, its biological functions remain unclear, except that it acts as a virulence factor in animal and plant pathogenic fungi: it conceals cell wall β-glucan on the fungal cell surface to circumvent recognition by hosts. However, cell wall α-1,3-glucan is also present in many of non-pathogenic fungi. Recently, the universal function of α-1,3-glucan as an aggregation factor has been demonstrated. Applications of fungi with modified cell wall α-1,3-glucan in the fermentation industry and of in vitro enzymatically-synthesized α-1,3-glucan in bio-plastics have been developed. This review focuses on the recent progress in our understanding of the biological functions and biosynthetic mechanism of cell wall α-1,3-glucan in fungi. We briefly consider the history of studies on α-1,3-glucan, overview its biological functions and biosynthesis, and finally consider the industrial applications of fungi deficient in α-1,3-glucan.
Asexual sporulation of the C. graminicola WT and Δrho4 strains on PDA amended with KCl. (A) After 7 dpi the WT had formed a dark mycelium, which developed orange-colored acervuli (arrows) by 14 dpi. The Δrho4 strain developed aerial mycelium without visible acervuli. (B) Quantification of conidia formed by the WT and Δrho4 strains. (C) Shape of conidia formed by the WT and Δrho4 strains. Scale bars are 20 μm. (D) Length of conidia formed by the WT and Δrho4 strains. Error bars in B and D are SDs. Asterisks indicate statistically significant differences (p ≤ 0.05).
Chitin and β-1,3-glucan abundance in vegetative hyphae of the WT and Δrho4 mutant. (A) WT strain and Δrho4 hyphae were stained with Aniline Blue fluorochrome and Alexa Fluor 647-Wheat Germ Agglutinin conjugate to detect β-1,3-glucan and chitin, respectively. While hyphae of the WT strain showed strong β-1,3-glucan labeling (arrowheads), those of Δrho4 strains did not. By contrast, chitin was hardly observed in the WT strain, but abundant in Δrho4 hyphae, with prominent labeling at hyphal protrusions (arrows). Scale bars are 10 μm. (B) Quantification of fluorescence intensity in hyphae labelled as in (A), from the hyphal tip to subapical regions. Virtual line scans confirm strong β-1,3-glucan labeling of the WT and strong chitin labeling of Δrho4 hyphae. (C) The
Δrho4 strains are defective in infection structure differentiation on maize leaves. (A-D) DIC microscopy of the infection process of C. graminicola WT and Δrho4 strains. Conidia (A,C) of the WT strain germinate and differentiate melanized appressoria ((A), app), which invade the epidermal host cell and differentiate infection hyphae ((B), ih). The Δrho4 mutant, by contrast, exhibits bipolar germination ((C), arrows), which form long ((C), arrowheads) and often coiled hyphae ((C), dashed box) before they differentiate hyphopodia ((C), hy). Several hyphopodia of the Δrho4 mutant
Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.
Timeline of major events and drug therapy. Initial laboratory findings were as follows: QuantiFERON, EBV serology, CMV serology, and cryptococcal antigen testing were all negative, as was serum galactomannan (GM) index (0.4, range > 0.5-1.0). Serum BDG was positive (337 pg/mL, range 30-59 pg/mL). Initial lumbar puncture results showed leukocytes 465 cells/mm 3 (range 0-5 cells/mm 3 , 73% polymorphs), raised protein 122 mg/dL (CSF range: 15-45 mg/dL), and low glucose 1.6 mmol/L (range 3.5-7.7 mmol/L); no organisms were seen or cultured. A
This case highlights the use of (1,3)-beta-d glucan to direct treatment of a cervical spinal cord Aspergillus fumigatus infection in a 22-year-old woman immunocompromised due to steroid and anti-TNF therapy in the context of ulcerative colitis and interferon gamma deficiency. A 4-year treatment course requiring neurosurgical intervention on four occasions and prolonged antifungal therapy, including isavuconazole, resulted in clinical cure with a corresponding decrease in CSF beta-d-glucan to <30 pg/mL. Serum and CSF galactomannan levels were not elevated at any point during the clinical course.
Invasive aspergillosis (IA) is a severe complication among hematopoietic stem cell transplant recipients or patients with hematological malignancies and neutropenia following anti-cancer therapy. Moreover, IA is increasingly observed in other populations, such as solid-organ transplant recipients, patients with solid tumors or auto-immune diseases, and among intensive care unit patients. Frequent delay in diagnosis is associated with high mortality rates. Cultures from clinical specimens remain sterile in many cases and the diagnosis of IA often only relies on non-specific radiological signs in the presence of host risk factors. Tests for detection of galactomannan- (GM) and 1,3-β-d-glucan (BDG) are useful adjunctive tools for the early diagnosis of IA and may have a role in monitoring response to therapy. However, the sensitivity and specificity of these fungal biomarkers are not optimal and variations between patient populations are observed. This review discusses the role and interpretation of GM and BDG testing for the diagnosis of IA in different clinical samples (serum, bronchoalveolar lavage fluid, cerebrospinal fluid) and different groups of patients (onco-hematological patients, solid-organ transplant recipients, other patients at risk of IA).
Distribution of the BAL Cq values (A) and BDG titers (B) and corresponding ROC curves for BAL Cq values (C) and BDG titers (D) comparing Pneumocystis pneumonia (PCP) and Pneumocystis carriage patients (PCC). Solid lines represent calculated optimal thresholds and black/red dotted lines the manufacturer threshold of the assay (80 pg/mL for Fungitell BDG assay).
Linear (A) and categorial (B) correlation between BAL Cq value and BDG titer (pg/mL) in PCP patients. **** p < 0.0001.
Distribution of enrolment in the five centers.
Distribution of the underlying diseases.
Pneumocystis jirovecii pneumonia is a difficult invasive infection to diagnose. Apart from microscopy of respiratory specimens, two diagnostic tests are increasingly used including real-time quantitative PCR (qPCR) of respiratory specimens, mainly in bronchoalveolar lavage fluids (BAL), and serum β-1,3-d-glucan (BDG). It is still unclear how these two biomarkers can be used and interpreted in various patient populations. Here we analyzed retrospectively and multicentrically the correlation between BAL qPCR and serum BDG in various patient population, including mainly non-HIV patients. It appeared that a good correlation can be obtained in HIV patients and solid organ transplant recipients but no correlation can be observed in patients with hematologic malignancies, solid cancer, and systemic diseases. This observation reinforces recent data suggesting that BDG is not the best marker of PCP in non-HIV patients, with potential false positives due to other IFI or bacterial infections and false-negatives due to low fungal load and low BDG release.
(A) Candida glabrata cells under (a) scanning electron microscope and (b) transmission electron microscope (CW = cell wall). (B) Schematic representation of the composition and organization of the major components of the fungal cell wall in Candida spp. (Figure adapted from [6]).
Cryo-electron tomography (cryoET) revealed two ring-like structures in plasma membranes of Candida glabrata CBS138 strain. (A) Slice view of plasma membranes with clusters of loosely packed, 170 Å diameter large ring-like structures. (B) Slice view of a representative tomogram showing the small 125 Å ring-like structures. Electron-dense particles are gold fiducials. (C) Zoomed-in slice view of the large ring-like structures (blue arrows) boxed in (A). (D) Zoomed-in slice view of the small ring-like structures (pink arrows) boxed in (B).
Large ring-like structures in Candida glabrata overexpressing Fks1 strain. (A) Expression levels of Fks1. C. glabrata cells from the different strains were grown on liquid YDP with (plasmidcarrying strains) or without 100 µg/mL nourseothricin until mid-log phase, and proteins were extracted using the TCA method. pFKS1 = pCN-PDC-FKS1 and KH238 strain = 200989 Δfks1+ pFKS1. (B) Slice view of a representative tomogram showing a patch of the large ring-like structures from the wild type strain (blue arrows). (C) Slice view clusters of the large ring-like structures in membranes from the strain overexpressing Fks1 (KH238) (orange arrows). (D) Histogram showing nearest neighbor distance of the large ring-like particles within clusters from the KH238 (orange) and wild type (blue) strains. (E) 2D average of the semicrystalline array composed of the large ring-like structures.
Expression profiling of the FKS genes compared to the wild-type strains CBS138 and 200989.
Echinocandin drugs have become a front-line therapy against Candida spp. infections due to the increased incidence of infections by species with elevated azole resistance, such as Candida glabrata. Echinocandins target the fungal-specific enzyme ß-(1,3)-glucan synthase (GS), which is located in the plasma membrane and catalyzes the biosynthesis of ß-(1,3)-glucan, the major component of the fungal cell wall. However, resistance to echinocandin drugs, which results from hotspot mutations in the catalytic subunits of GS, is an emerging problem. Little structural information on GS is currently available because, thus far, the GS enzyme complex has resisted homogenous purification, limiting our understanding of GS as a major biosynthetic apparatus for cell wall assembly and an important therapeutic drug target. Here, by applying cryo-electron tomography (cryo-ET) and subtomogram analysis, we provide a preliminary structure of the putative C. glabrata GS complex as clusters of hexamers, each subunit with two notable cytosolic domains, the N-terminal and central catalytic domains. This study lays the foundation for structural and functional studies of this elusive protein complex, which will provide insight into fungal cell wall synthesis and the development of more efficacious antifungal therapeutics.
Bronchoalveolar lavage fluid (BALF) (1,3)-β-d-glucan (BDG) concentrations expressed as pg/mL in patients with Pneumocystis pneumonia characterized as proven, probable, and possible Pneumocystis infection (cases) and matched control patients. Bars represent the median BDG level for that cohort.
Receiver operating characteristic/area under the curve (ROC/AUC) analysis of bronchoalveolar lavage fluid (1,3)-β-d-glucan (BDG) concentrations to distinguish Pneumocystis pneumonia cases from matched controls, AUC 0.70 (95% C.I. 0.52-0.87).
We evaluated the performance of the (1,3)-β-d-glucan (BDG) assay on bronchoalveolar lavage fluid (BALF) as a possible aid to the diagnosis of Pneumocystis jirovecii pneumonia. BALF samples from 18 patients with well-characterized proven, probable, and possible Pneumocystis pneumonia and 18 well-matched controls were tested. We found that the best test performance was observed with a cut-off value of 128 pg/mL; receiver operating characteristic/area under the curve (ROC/AUC) was 0.70 (95% CI 0.52-0.87). Sensitivity and specificity were 78% and 56%, respectively; positive predictive value was 64%, and negative predictive value was 71%. The low specificity that we noted limits the utility of BALF BDG as a diagnostic tool for Pneumocystis pneumonia.
Plot of analytical measuring results of sera of candidemia patients (cand; full circles) and negative controls (nc; empty circles). The dotted line indicates the cut-off of both assays. For samples, which were tested positive in more than one PCR, only one result was considered for this graph. BDG concentrations below the limit of detection and negative PCR results are plotted (not to scale) in the grey shaded area.
Comparison of sensitivities (a) and specificities (b) of the Fungiplex Candida PCR and the Wako β-Glucan Test.
Bloodstream infections caused by Candida yeasts (candidemia) are associated with high morbidity and mortality. Diagnosis remains challenging, with the current gold standard—isolation from blood culture (BC)—being limited by low sensitivity and long turnaround time. This study evaluated the performance of two nonculture methods: PCR and β-1,3-D-glucan (BDG) testing. The sera of 103 patients with BC-proven candidemia and of 46 controls were analyzed with the Fungiplex Candida Real-Time PCR and the Wako β-Glucan Test. The BDG assay demonstrated higher sensitivity than the multiplex PCR (58% vs. 33%). This was particularly evident in ICU patients (60% vs. 28%) and in C. albicans candidemia (57% vs. 37%). The earlier prior to BC sampling the sera were obtained, the more the PCR sensitivity decreased (46% to 18% in the periods of 0–2 and 3–5 days before BC, respectively), while BDG testing was independent of the sampling date. No positive PCR results were obtained in sera sampled more than five days before BC. Specificities were 89% for BDG and 93% for PCR testing. In conclusion, BDG testing demonstrated several advantages over PCR testing for the diagnosis of candidemia, including higher sensitivity and earlier diagnosis. However, BC remains essential, as BDG does not allow for species differentiation.
Magnaporthe oryzae is the causal agent of rice blast outbreaks. L-ascorbic acid (ASC) is a famous antioxidant found in nature. However, while ASC is rare or absent in fungi, a five-carbon analog, D-erythroascorbic acid (EASC), seems to appear to be a substitute for ASC. Although the antioxidant function of ASC has been widely described, the specific properties and physiological functions of EASC remain poorly understood. In this study, we identified a D-arabinono-1,4-lactone oxidase (ALO) domain-containing protein, MoAlo1, and found that MoAlo1 was localized to mitochondria. Disruption of MoALO1 (ΔMoalo1) exhibited defects in vegetative growth as well as conidiogenesis. The ΔMoalo1 mutant was found to be more sensitive to exogenous H2O2. Additionally, the pathogenicity of conidia in the ΔMoalo1 null mutant was reduced deeply in rice, and defective penetration of appressorium-like structures (ALS) formed by the hyphal tips was also observed in the ΔMoalo1 null mutant. When exogenous EASC was added to the conidial suspension, the defective pathogenicity of the ΔMoalo1 mutant was restored. Collectively, MoAlo1 is essential for growth, conidiogenesis, and pathogenicity in M. oryzae.
Prevalence rates of C. haemulonii species complex clinical isolates. P1: December 2008 to June 2013; P2: July 2013 to December 2019.
Antifungal susceptibility testing of 49 C. haemulonii species complex clinical isolates.
There is worldwide concern with the increasing rates of infections due to multiresistant Candida isolates reported in tertiary medical centers. We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto (n = 21), followed by C. haemulonii var. vulnera (n = 15) and C. duobushaemulonii (n = 13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis (p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections.
Dear Friends and Colleagues,It is a great honor and pleasure for us to invite you cordially to participate in the 9th Congress on Trends in Medical Mycology (TIMM-9) [...]
(A) Distribution of human albumin gene Ct according to the extraction methods tested. The means of the Cycle threshold and their 95% confidence interval are shown. (B) Distribution of the Candida species detection rate (in percentages) according to the extraction methods tested. The detection rates (in percentages) and their 95% confidence interval are shown.
Number of positive Candida PCR test per Ct according to the 11 extraction methods. Negative results (Ct > 45 or no amplification detected) are stained orange.
The probability of obtaining at least one PCR positive result (Ct < 45) on the duplicate from the two extractions for each inoculum concentration according to the Candida species and the automated method used.
The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.
Solar radiation has been regarded as a driver of litter decomposition in arid and semiarid ecosystems. Photodegradation of litter organic carbon (C) depends on chemical composition and water availability. However, the chemical changes in organic C that respond to solar radiation interacting with water pulses remain unknown. To explain changes in the chemical components of litter organic C exposed to UV-B, UV-A, and photosynthetically active radiation (PAR) mediated by water pulses, we measured the chemistry of marcescent Lindera glauca leaf litter by solid-state 13C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR) over 494 days of litter decomposition with a microcosm experiment. Abiotic and biotic factors regulated litter decomposition via three pathways: first, photochemical mineralization of lignin methoxyl C rather than aromatic C exposed to UV radiation; second, the biological oxidation and leaching of cellulose O-alkyl C exposed to PAR and UV radiation interacts with water pulses; and third, the photopriming effect of UV radiation on lignin aromatic C rather than cellulose O-alkyl C under the interaction between radiation and water pulses. The robust decomposition index that explained the changes in the mass loss was the ratio of aromatic C to O-alkyl C (AR/OA) under radiation, but the ratio of hydrophobic to hydrophilic C (hydrophobicity), the carbohydrate C to methoxyl C ratio (CC/MC), and the alkyl C to O-alkyl C ratio (A/OA) under radiation were mediated by water pulses. Moreover, the photopriming effect and water availability promoted the potential activities of peroxidase and phenol oxidase associated with lignin degradation secreted by fungi. Our results suggest that direct photodegradation of lignin methoxyl C increases microbial accessibility to lignin aromatic C. Photo-oxidized compounds might be an additional C pool to regulate the stability of the soil C pool derived from plant litter by degrading lignin methoxyl and aromatic C.
Relative expression of abf-1, abf-3 cnc-4, npl-27 and npl-31 genes related to the production of C. elegans antimicrobial peptides when in contact with P1 (A), P2 (B) or P3 (C). Data are expressed as mean ± standard deviation of three biological replicates and three independent experiments (* p < 0.05) using Kruskal-Wallis and Dunn's.
Primers sequence of G. mellonella antimicrobial peptides.
Summary of the 14-3-3 peptides effect in vitro and invertebrate models.
Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. Results: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.
Euler diagram of the gene-based genome comparison for the completely sequenced R. solani AG1-IB isolates. The core genome of all isolates consists of 9389 genes. These genes are present in the genomes of all sequenced R. solani AG1-IB isolates: O8/2 [EMBL-EBI: CAJZAE010000001-CAJZAE010001402], 1/2/21 [EMBL-EBI: CAJZAD010000001-CAJZAD010002093] and 7/3/14 [EMBL-EBI: CDGK01000001-CDGK01018395 (Contigs); LN679100-LN679996 (Scaffolds)]. For computation of the Venn diagram, default settings of EDGAR 3.0 [34-36] were applied.
Examples of predicted unique gene products for the different R. solani AG1-IB isolates.
Identified secondary metabolite regions for the different R. solani AG1-IB isolates.
Pairwise Average Nucleotide Identity (ANI) analyses for completely sequenced R. solani AG1-IB isolates.
Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.
Total ion chromatogram of volatile compounds identified from T. asperellum T76-14. Total ion chromatogram of volatile compounds identified from T. asperellum T76-14.
Lesion development after inoculation with F. incarnatum, control without VOCs and PEA, muskmelons exposed to VOCs of Trichoderma asperellum T76-14 and phenylethyl alcohol (PEA). Arrows indicate the inoculation points.
Volatile compounds produced by Trichoderma asperellum T76-14 tentatively identified through SPME GC/MS analysis.
Comparison of phenylethyl alcohol (PEA) and other commercial volatiles against Fusar- ium incarnatum.
Postharvest fruit rot caused by Fusarium incarnatum is a destructive postharvest disease of muskmelon (Cucumis melo). Biocontrol by antagonistic microorganisms is considered an alternative to synthetic fungicide application. The aim of this study was to investigate the mechanisms of action involved in the biocontrol of postharvest fruit rot in muskmelons by Trichoderma species. Seven Trichoderma spp. isolates were selected for in vitro testing against F. incarnatum in potato dextrose agar (PDA) by dual culture assay. In other relevant works, Trichoderma asperellum T76-14 showed a significantly higher percentage of inhibition (81%) than other isolates. Through the sealed plate method, volatile organic compounds (VOCs) emitted from T. asperellum T76-14 proved effective at inhibiting the fungal growth of F. incarnatum by 62.5%. Solid-phase microextraction GC/MS analysis revealed several VOCs emitted from T. asperellum T76-14, whereas the dominant compound was tentatively identified as phenylethyl alcohol (PEA). We have tested commercial volatile (PEA) against in vitro growth of F. incarnatum; the result showed PEA at a concentration of 1.5 mg mL−1 suppressed fungal growth with 56% inhibition. Both VOCs and PEA caused abnormal changes in the fungal mycelia. In vivo testing showed that the lesion size of muskmelons exposed to VOCs from T. asperellum T76-14 was significantly smaller than that of the control. Muskmelons exposed to VOCs from T. asperellum T76-14 showed no fruit rot after incubation at seven days compared to fruit rot in the control. This study demonstrated the ability of T. asperellum T76-14 to produce volatile antifungal compounds, showing that it can be a major mechanism involved in and responsible for the successful inhibition of F. incarnatum and control of postharvest fruit rot in muskmelons.
Candida albicans is the most dominant and prevalent cause of fungal infections in humans. Azoles are considered as first-line drugs for the treatment of these infections. However, their prolonged and insistent use has led to multidrug resistance and treatment failures. To overcome this, modification or derivatization of the azole ring has led to the development of new and effective antifungal molecules. In a previous study, we reported on the development of new triazole-based molecules as potential antifungal agents against Candida auris. In this study, the most potent molecules from the previous study were docked and simulated with lanosterol 14-alpha demethylase enzyme. These compounds were further evaluated for in vitro susceptibility testing against C. albicans. In silico results revealed favorable structural dynamics of the compounds, implying that the compounds would be able to effectively bind to the target enzyme, which was further manifested by the strong interaction of the test compounds with the amino acid residues of the target enzyme. In vitro studies targeting quantification of ergosterol content revealed that pta1 was the most active compound and inhibited ergosterol production by >90% in both drug-susceptible and resistant C. albicans isolates. Furthermore, RT-qPCR results revealed downregulation of ERG11 gene when C. albicans cells were treated with the test compound, which aligns with the decreased ergosterol content. In addition, the active triazole derivatives were also found to be potent inhibitors of biofilm formation. Both in silico and in vitro results indicate that these triazole derivatives have the potential to be taken to the next level of antifungal drug development.
Lanosterol in the ligand-binding pocket (LBP) of CYP51. Relevant portions of the human HsCYP51 crystal structure (Protein Data Bank (PDB) 6UEZ) are shown. Residues within 4 Å of lanosterol (yellow) are shown with carbon atoms in green. These 16 residues are found in helix I, helix B, helix C, the B-C loop, the K-K′ internal loop and the C-terminal region. Proton channel S mutations, D231A in helix F and H314A in nearby helix I, are shown in white. Upon lanosterol binding helix C has changed conformation slightly, the K156 side chain loses its ionic interaction with the heme propionate C and becomes exposed into the enzyme surface. The K-K′ internal loop I379 main chain carbonyl hydrogen bonds with the OH of lanosterol. The main chain amides of M378 and I379 plus the main chain carbonyl of M487 in the C-terminal region form a water-mediated hydrogen bond network with the hydroxyl of lanosterol. The 14α-methyl group of lanosterol lies in proximity of the heme iron (large red ball) in a catalytically competent position.
Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel antifungals. Structural insights into drug targets and their complexes with both substrates and inhibitory ligands increase opportunity for the discovery of more effective antifungals. Implementation of this promise, which requires multiple skill sets, is beginning to yield candidates from discovery programs that could more quickly find their place in the clinic. This review will describe how structural biology is providing information for the improvement and discovery of inhibitors targeting the essential fungal enzyme sterol 14α-demethylase.
Pathogenic fungi with naturally occurring amino acid (AA) substitutions in the active site of sterol 14α-deme- thylase (SDM; CYP51 enzyme of the cytochrome P450 superfamily) and correlated azole resistance.
The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles (e.g., voriconazole). We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance.
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.
Interleukin-17 (IL-17) is a proinflammatory cytokine produced by adaptive CD4+ T helper cells and innate lymphocytes, such as γδ-T cells and TCRβ+ “natural” Th17 cells. IL-17 activates signaling through the IL-17 receptor, which induces other proinflammatory cytokines, antimicrobial peptides and neutrophil chemokines that are important for antifungal activity. The importance of IL-17 in protective antifungal immunity is evident in mice and humans, where various genetic defects related to the IL-17-signaling pathway render them highly susceptible to forms of candidiasis such oropharyngeal candidiasis (OPC) or more broadly chronic mucocutaneous candidiasis (CMC), both caused mainly by the opportunistic fungal pathogen Candida albicans. OPC is common in infants and the elderly, HIV/AIDS and patients receiving chemotherapy and/or radiotherapy for head and neck cancers. This review focuses on the role of IL-17 in protection against candidiasis, and includes a brief discussion of non-Candida albicans fungal infections, as well as how therapeutic interventions blocking IL-17-related components can affect antifungal immunity.
Invasive Candida albicans (CA) infections often arise from the intestine and cause life-threatening infections in immunocompromised individuals. The role of gut commensal microbiota, metabolites, and host factors in the regulation of CA colonization in the intestine is poorly understood. Previous findings from our lab indicate that taurocholic acid (TCA), a major bile acid present in the intestine, promotes CA colonization and dissemination. Here, we report that oral administration of TCA to CA-infected mice significantly decreased the number of mononuclear phagocytes and CD4+ IL17A+ T helper 17 cells that play a critical role in controlling CA in the intestine. Collectively, our results indicate that TCA modulates mucosal innate and adaptive immune responses to promote CA colonization in the intestine.
Characteristics and size of the GH17 proteins of A. fumigatus.
Morphology of the dormant (A) 0 h; swollen (B) 4 h; and germinated (C) 8 h conidia of the parental strain, and ∆scw4∆scw11 and ∆scw4scw11bgt3bgt2bgt1 mutants using fluorescence microscopy (×63) with UV light and Calcofluor white ® staining, in 3% Glucose medium +1% yeast extract. Green scale bars (2 µm in panel A), blue scale bars (6 µm in B and C), and yellow arrows highlight dormant conidia with abnormal size. 
Morphology of the dormant (A) 0 h; swollen (B) 4 h; and germinated (C) 8 h conidia of the parental strain, and Δscw4Δscw11 and Δscw4scw11bgt3bgt2bgt1 mutants using fluorescence microscopy (×63) with UV light and Calcofluor white ® staining, in 3% Glucose medium +1% yeast extract. Green scale bars (2 µm in panel A), blue scale bars (6 µm in B and C), and yellow arrows highlight dormant conidia with abnormal size. 
Growth kinetics of the parental strain and the GH17 mutants on RPMI agar medium at 37 • C. The diameter of the colonies was measured after 48 h. Values are mean ± SD of three independent determinations. WT, parental strain.
Percentage of hexose, N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc) in the alkali-insoluble (A) and alkali-soluble fraction (B) of the cell wall of the double, triple, and quintuple mutants of the GH17 family. Values are mean ± SD of three independent determinations. WT, parental strain.
Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3)glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17) of A. fumigatus, two β(1,3)glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p.
Composite image of haematoxylin and eosin (HE) and immunohistochemical staining of sections of chronic hyperplastic candidosis (CHC), squamous papilloma (SqP), and oral lichen planus (LP). Immunohistochemical staining was for EBI3-Epstein-Barr virus induced gene 3 (Treg, Th1), IL-12A (p35 subunit; Treg, Th1), Foxp3 (Forkhead box protein 3; Treg), and IL-17A (Th-17). Original magnification 100×, except for first row of HE stained sections (7× CHC, 4× SqP, and 7× LP).
Comparison of IL-17A producing cell numbers in tissue sections of CHC, SqP, and LP. Data are expressed as mean ± standard error. The highest numbers of IL-17A producing cells were detected in the corium (connective tissue) of CHC and LP. SqP (all with Candida co-infection) had significantly lower numbers of IL-17A + cells (Dunn's post-hoc test: p = 0.002 for CHC vs. SqP and p = 0.003 for LP vs. SqP). Few cells were positive for IL-17A in the epithelial layer of CHC, whereas almost no IL-17A-producing cells were detected in SqP and LP. * p < 0.05.
Comparison of Foxp3 + cell numbers in tissue sections of CHC, SqP and LP. Data are expressed as mean ± standard error. (A) Higher numbers of Foxp3 + cells were detected in CHC and LP tissues compared to SqP. Foxp3 + cells were detected in both the corium (connective tissue) and epithelium of CHC and LP, but not in SqP. (B) As a proportion of previously determined CD4 + cells, Foxp3 + cells were lower in the epithelium compared to the corium by a factor 2.0 for CHC (Mann−Whitney test: p = 0.340) and a factor of 4.3 for LP (Mann−Whitney test: p = 0.009). ** p < 0.01.
Comparison of EBI3 + -and IL-12A-producing cell numbers in tissue sections of CHC, SqP, and LP. Data are expressed as mean ± standard error. (A) Cells expressing EBI3 were evident in CHC but were in lower numbers in SqP and LP. Significantly higher numbers of EBI3 + cells were detected in the corium of CHC compared to SqP and LP. No EBI3 + cells were found in the epithelium of any of these conditions. (B) Numbers of IL-12A-producing cells were not significantly different in the corium of CHC and LP (Dunn's post hoc test: p = 0.481). * p < 0.05; ** p < 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
Previous research into the inflammatory cell infiltrate of chronic hyperplastic candidosis (CHC) determined that the immune response is primarily composed of T cells, the majority of which are T helper (CD4+) cells. This present investigation used immunohistochemistry to further delineate the inflammatory cell infiltrate in CHC. Cells profiled were those expressing IL-17A cytokine, EBI3 and IL-12A subunits of the IL-35 cytokine, and FoxP3+ cells. Squamous cell papilloma (with Candida infection) and oral lichen planus tissues served as comparative controls to understand the local immune responses to Candida infection. The results demonstrated that Candida-induced inflammation and immune regulation co-exist in the oral mucosa of CHC and that high prevalence of cells expressing the EBI3 cytokine subunit may play an important role in this regulation. This balance between inflammation and immune tolerance toward invading Candida in the oral mucosa may be critical in determining progress of infection.
Clinical characteristics of patients upon ICU admission.
Treatment and outcomes.
Background: COVID-19-associated fungal infections seem to be a concerning issue. The aim of this study was to assess the incidence of fungal infections, the possible risk factors, and their effect on outcomes of critically ill patients with COVID-19. Methods: A retrospective observational study was conducted in the COVID-19 ICU of the First Respiratory Department of National and Kapodistrian University of Athens in Sotiria Chest Diseases Hospital between 27 August 2020 and 10 November 2021. Results: Here, 178 patients were included in the study. Nineteen patients (10.7%) developed fungal infection, of which five had COVID-19 associated candidemia, thirteen had COVID-19 associated pulmonary aspergillosis, and one had both. Patients with fungal infection were younger, had a lower Charlson Comorbidity Index, and had a lower PaO2/FiO2 ratio upon admission. Regarding health-care factors, patients with fungal infections were treated more frequently with Tocilizumab, a high regimen of dexamethasone, continuous renal replacement treatment, and were supported more with ECMO. They also had more complications, especially infections, and subsequently developed septic shock more frequently. Finally, patients with fungal infections had a longer length of ICU stay, as well as length of mechanical ventilation, although no statistically significant difference was reported on 28-day and 90-day mortality. Conclusions: Fungal infections seem to have a high incidence in COVID-19 critically ill patients and specific risk factors are identified. However, fungal infections do not seem to burden on mortality.
HNI enhances the IL-17-related response during OPC. (A-C) Expression differences relative to GAPDH of genes related to IL-17 antifungal responses. Il17a, Defb3, S100a9 analyzed by oneway ANOVA with Tukey's post hoc. (** p < 0.01, *** p < 0.001, **** p < 0.0001) n = 3-6 mice per group per experiment. Data represent 3 experimental repeats.
IL-17RA signaling protects against OPC following HNI. (A) Quantification of toluidine blue staining by determining the surface area of the tongue positive for blue staining compared to the total surface area of tongue for each mouse (n = 3 or more mice per group) on Day 4 post-infection. Analyzed by one-way ANOVA with Tukey's post hoc. (B) Quantification of mucosal thickness on the tip, middle, and back of tongue. Measured from basal stem cell layer up to papillae using ImageJ software. Three mice were evaluated from each group, and three sections were analyzed per mouse. Investigators determining toluidine blue staining and measurement of mucosal thickness were blinded to treatment and mouse cohort. Analyzed by unpaired Student's t-test. (C) All mice were weighed daily, and % body weight (BW) loss (gram) on each day of infection is indicated. (D) WT and Il17ra −/− mice (n = 3-6 mice per group) were subject to HNI and 16 h later infected sublingually with C. albicans and on Day 4 CFU/g of tongue tissue was assessed in triplicate. Analyzed by Mann-Whitney U test. Data shown as geometric means. (** p < 0.01, *** p < 0.001) Data represent 3 experimental repeats.
IL-17RA is required to prevent dissemination of C. albicans across the radiation-damaged oral mucosa. (A) WT and ll17ra −/− (n = 3 or more mice per group) mice were subjected to HNI and 12-16 h later were infected sublingually with C. albicans. On Day 4 both kidneys were harvested, homogenized, and plated in triplicate. CFU/g of kidney tissue was determined. Analyzed by MannWhitney U test. Data shown as geometric mean. (B) On Day 4 both stomach and intestines were harvested, homogenized, and plated in duplicate. CFU/g of stomach and intestine tissue was determined (n= 3-6 mice per group). Analyzed by one-way Anova with Tukey's post hoc. (C) Representative Occludin-staining of ulcer boundary region counterstained with hematoxylin. Images taken at 10x magnification. Images representative of 3 mice per group and 2 experimental repeats. (* p < 0.05, ** p < 0.01).
HNI exposure leads to oral neutrophil defects. (A) Quantification of flow cytometry results showing phagocytosis of GFP-Candida albicans by Gr-1+ neutrophils after subjecting mice to OPC and HNI + OPC (n = 3 mice per group) (B,C) Expression differences relative to GAPDH. Analyzed by ANOVA with Tukey's post hoc. (n = 3 mice per group). (*** p < 0.001). Data representative of at least 2-3 experimental repeats.
Fungal infections caused by Candida albicans are a serious problem for immunocompromised individuals, including those undergoing radiotherapy for head and neck cancers. Targeted irradiation causes inflammatory dysregulation and damage to the oral mucosa that can be exacerbated by candidiasis. Post-irradiation the cytokine interleukin-17 (IL-17) protects the oral mucosae by promoting oral epithelial regeneration and balancing the oral immune cell populations, which leads to the eventual healing of the tissue. IL-17 signaling is also critical for the antifungal response during oropharyngeal candidiasis (OPC). Yet, the benefit of IL-17 during other forms of candidiasis, such as vulvovaginal candidiasis, is not straightforward. Therefore, it was important to determine the role of IL-17 during OPC associated with radiation-induced inflammatory damage. To answer this question, we exposed Il17ra−/− and wild-type mice to head-neck irradiation (HNI) and OPC to determine if the IL-17 signaling pathway was still protective against C. albicans. HNI increased susceptibility to OPC, and in Il17ra−/− mice, the mucosal damage and fungal burden were elevated compared to control mice. Intriguingly, neutrophil influx was increased in Il17ra−/− mice, yet these cells had reduced capacity to phagocytose C. albicans and failed to clear OPC compared to immunocompetent mice. These findings suggest that radiotherapy not only causes physical damage to the oral cavity but also skews immune mediators, leading to increased susceptibility to oropharyngeal candidiasis.
The use of biological control agents (BCAs) is a promising alternative control measure for Fusarium crown rot (FCR) of wheat caused by Fusarium pseudograminearum. A bacterial strain, YB-185, was isolated from the soil of wheat plants with FCR and identified as Bacillus velezensis. YB-185 exhibited strong inhibition of F. pseudograminearum mycelial growth and conidial germination in culture. Seed treatment with YB-185 in greenhouse and field resulted in reductions in disease by 66.1% and 57.6%, respectively, along with increased grain yield. Microscopy of infected root tissues confirmed that YB-185 reduced root invasion by F. pseudograminearum. RNA-seq of F. pseudograminearum during co-cultivation with B. velezensis YB-185 revealed 5086 differentially expressed genes (DEGs) compared to the control. Down-regulated DEGs included genes for glucan synthesis, fatty acid synthesis, mechanosensitive ion channels, superoxide dismutase, peroxiredoxin, thioredoxin, and plant-cell-wall-degrading enzymes, whereas up-regulated DEGs included genes for chitin synthesis, ergosterol synthesis, glutathione S-transferase, catalase, and ABC transporters. In addition, fungal cell apoptosis increased significantly, as indicated by TUNEL staining, and the scavenging rate of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS·+) in the fungus significantly decreased. Thus, F. pseudograminearum may be trying to maintain normal cell functions by increasing cell wall and membrane synthesis, antioxidant and anti-stress responses, detoxification of bacterial antimicrobial compounds, and transportation of damaging compounds from its cells. However, cell death and free radical accumulation still occurred, indicating that the responses were insufficient to prevent cell damage. Bacillus velezensis YB-185 is a promising BCA against FCR that acts by directly damaging F. pseudograminearum, thus reducing its ability to colonize roots and produce symptoms.
Due to its acetate content, the pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw could provide an inexpensive substrate for microbial fermentation. However, PAC also contains several inhibitors that make its detoxification inevitable. In our study, we examined the transcriptional response of Aspergillus oryzae to cultivation on 20% detoxified PAC, pure acetate and glucose using RNA-seq analysis. Functional enrichment analysis of 3463 significantly differentially expressed (log2FC > 2 & FDR < 0.05) genes revealed similar metabolic tendencies for both acetate and PAC, as upregulated genes in these cultures were mainly associated with ribosomes and RNA processing, whereas transmembrane transport was downregulated. Unsurprisingly, metabolic pathway analysis revealed that glycolysis/gluconeogenesis and starch and sucrose metabolism were upregulated for glucose, whereas glyoxylate and the tricarboxylic acid (TCA) cycle were important carbon utilization pathways for acetate and PAC, respectively. Moreover, genes involved in the biosynthesis of various amino acids such as arginine, serine, cysteine and tryptophan showed higher expression in the acetate-containing cultures. Direct comparison of the transcriptome profiles of acetate and PAC revealed that pyruvate metabolism was the only significantly different metabolic pathway and was overexpressed in the PAC cultures. Upregulated genes included those for methylglyoxal degradation and alcohol dehydrogenases, which thus represent potential targets for the further improvement of fungal PAC tolerance.
Despite increasing associated mortality and morbidity, the diagnosis of fungal infections, especially with Aspergillus fumigatus (A. fumigatus), remains challenging. Based on known ability of Aspergillus species to utilize sorbitol, we evaluated 2-[18F]-fluorodeoxysorbitol (FDS), a recently described Enterobacterales imaging ligand, in animal models of A. fumigatus infection, in comparison with 2-[18F]-fluorodeoxyglucose (FDG). In vitro assays showed slightly higher 3H-sorbitol uptake by live compared with heat-killed A. fumigatus. However, this was 10.6-fold lower than E. coli uptake. FDS positron emission tomography (PET) imaging of A. fumigatus pneumonia showed low uptake in infected lungs compared with FDG (0.290 ± 0.030 vs. 8.416 ± 0.964 %ID/mL). This uptake was higher than controls (0.098 ± 0.008 %ID/mL) and minimally higher than lung inflammation (0.167 ± 0.007 %ID/mL). In the myositis models, FDS uptake was highest in live E. coli infections. Uptake was low in A. fumigatus myositis model and only slightly higher in live compared with the heat-killed side. In conclusion, we found low uptake of 3H-sorbitol and FDS by A. fumigatus cultures and infection models compared with E. coli, likely due to the need for induction of sorbitol dehydrogenase by sorbitol. Our findings do not support FDS as an Aspergillus imaging agent. At this point, FDS remains more selective for imaging Gram-negative Enterobacterales.
Chest CT-scan of LAPA and CAPA patients. Chest CT scans in the axial plane (lung window: W1600/L-500 HU) of three patients with LAPA (A-C) and three patients with CAPA (D-F). Typical CT findings in LAPA are unilateral or bilateral areas of consolidation with air bronchogram (A,B), cavity formation (asterisks), tree in bud (white arrowheads), bronchial wall thickening (white arrow), or occasionally nodules with halo signs (C, black arrows). Patients with CAPA may exhibit non-specific CT findings, such as bilateral areas of ground-glass opacity and/or crazy paving (D), extensive consolidations areas associated with peripheral traction bronchiectasis (E, black arrowheads), or, more rarely, unilateral consolidation areas (F). Despite not being very specific for a SARS COV2 infection, findings observed in (D,E) pictures can be seen frequently in severe COVID-19 patients.
Characteristics of patients according to aspergillosis status.
Summary of similarities and differences between CAPA and IAPA.
Invasive pulmonary aspergillosis (IPA) in intensive care unit patients is a major concern. Influenza-associated acute respiratory distress syndrome (ARDS) and severe COVID-19 patients are both at risk of developing invasive fungal diseases. We used the new international definitions of influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) to compare the demographic, clinical, biological, and radiological aspects of IAPA and CAPA in a monocentric retrospective study. A total of 120 patients were included, 71 with influenza and 49 with COVID-19-associated ARDS. Among them, 27 fulfilled the newly published criteria of IPA: 17/71 IAPA (23.9%) and 10/49 CAPA (20.4%). Kaplan–Meier curves showed significantly higher 90-day mortality for IPA patients overall (p = 0.032), whereas mortality did not differ between CAPA and IAPA patients. Radiological findings showed differences between IAPA and CAPA, with a higher proportion of features suggestive of IPA during IAPA. Lastly, a wide proportion of IPA patients had low plasma voriconazole concentrations with a higher delay to reach concentrations > 2 mg/L in CAPA vs. IAPA patients (p = 0.045). Severe COVID-19 and influenza patients appeared very similar in terms of prevalence of IPA and outcome. The dramatic consequences on the patients’ prognosis emphasize the need for a better awareness in these particular populations.
Mucormycosis (MCR) has been increasingly described in patients with coronavirus disease 2019 (COVID-19) but the epidemiological factors, presentation, diagnostic certainty, and outcome of such patients are not well described. We review the published COVID-19-associated mucormycosis (CAMCR) cases (total 41) to identify risk factors, clinical features, and outcomes. CAMCR was typically seen in patients with diabetes mellitus (DM) (94%) especially the ones with poorly controlled DM (67%) and severe or critical COVID-19 (95%). Its presentation was typical of MCR seen in diabetic patients (mostly rhino-orbital and rhino-orbital-cerebral presentation). In sharp contrast to reported COVID-associated aspergillosis (CAPA) cases, nearly all CAMCR infections were proven (93%). Treating physicians should have a high suspicion for CAMCR in patients with uncontrolled diabetes mellitus and severe COVID-19 presenting with rhino-orbital or rhino-cerebral syndromes. CAMR is the convergence of two storms, one of DM and the other of COVID-19.
UPGMA phylogenetic tree generated with MEGA X software [8]. There were 20 nucleotide sequences and 299 nucleotide positions in the final dataset. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of number of base substitutions per site. The AST of the two strains showed minimal inhibitory concentrations of 1 µg/mL, 2 µg/mL, 0.03 µg/L, and 0.06 µg/mL for amphotericin B, fluconazole, voriconazole, and anidulafungin, respectively. The microsatellite analyses revealed that the Brazilian strains were clonal and closely related to the South Asian clade (Figure 2). Despite being related to the South Asian clade, the Brazilian strains showed distinct alleles for the markers M3-I a, M3-II b, and M3-II b (Figure 2).
Short tandem repeat (STR) typing of C. auris isolates from Brazil. UPGMA dendrogram of the Brazilian (L1537/2020 and L1685/2020) and representative strains from South Asian and other four clades are shown. The scale in the upper left corner represents similarity (%).
In December 2020, Candida auris emerged in Brazil in the city of Salvador. The first two C. auris colonized patients were in the same COVID-19 intensive care unit. Antifungal susceptibility testing showed low minimal inhibitory concentrations of 1 µg/mL, 2 µg/mL, 0.03 µg/L, and 0.06 µg/mL for amphotericin B, fluconazole, voriconazole, and anidulafungin, respectively. Microsatellite typing revealed that the strains are clonal and belong to the South Asian clade C. auris. The travel restrictions during the COVID-19 pandemic and the absence of travel history among the colonized patients lead to the hypothesis that this species was introduced several months before the recognition of the first case and/or emerged locally in the coastline Salvador area.
Regional variation in number of inclusions and CAPA incidence in studies with observational data. Bar chart on the left shows the number of patients included in studies with observational data on CAPA, either per continent or per country. The maps on the right show the pooled CAPA incidence per country in studies with observational data. Only studies with exact information on the countries where patients were recruited were included in this figure [2,9,15,16,18,20-33,35-38,40,41,43-45,47-49,51-59]. CAPA incidence is calculated per study for the combination of proven, probable, putative and possible CAPA cases.
Comparison between IAPA and CAPA. Infograph on the similarities and differences between CAPA and IAPA. Both co-infections occur in ICU-admitted patients. Corticosteroids and anti-IL-6 therapy have been implicated in CAPA pathophysiology, while this is the case for corticosteroids and oseltamivir in IAPA. IAPA tends to occur earlier, more frequently and with more frequent angioinvasion than CAPA. Both lead to an increased mortality. Created with the aid of
CAPA incidence in observational studies.
Coronavirus disease 19 (COVID-19)-associated pulmonary aspergillosis (CAPA) is a severe fungal infection complicating critically ill COVID-19 patients. Numerous retrospective and prospective studies have been performed to get a better grasp on this lethal co-infection. We performed a qualitative review and summarized data from 48 studies in which 7047 patients had been included, of whom 820 had CAPA. The pooled incidence of proven, probable or putative CAPA was 15.1% among 2953 ICU-admitted COVID-19 patients included in 18 prospective studies. Incidences showed great variability due to multiple factors such as discrepancies in the rate and depth of the fungal work-up. The pathophysiology and risk factors for CAPA are ill-defined, but therapy with corticosteroids and anti-interleukin-6 therapy potentially confer the biggest risk. Sampling for mycological work-up using bronchoscopy is the cornerstone for diagnosis, as imaging is often aspecific. CAPA is associated with an increased mortality, but we do not have conclusive data whether therapy contributes to an increased survival in these patients. We conclude our review with a comparison between influenza-associated pulmonary aspergillosis (IAPA) and CAPA.
Coinfections with bacteria or fungi may be a frequent complication of COVID-19, but coinfections with Candida species in COVID-19 patients remain rare. We report the 53-day clinical course of a complicated type-2 diabetes patient diagnosed with COVID-19, who developed bloodstream infections initially due to methicillin-resistant Staphylococcus aureus, secondly due to multidrug-resistant Gram-negative bacteria, and lastly due to a possibly fatal Candida glabrata. The development of FKS-associated pan-echinocandin resistance in the C. glabrata isolated from the patient after 13 days of caspofungin treatment aggravated the situation. The patient died of septic shock shortly before the prospect of receiving potentially effective antifungal therapy. This case emphasizes the importance of early diagnosis and monitoring for antimicrobial drug-resistant coinfections to reduce their unfavorable outcomes in COVID-19 patients.
(1) Background: The diagnosis of invasive aspergillosis (IA) in an intensive care unit (ICU)remains a challenge and the COVID-19 epidemic makes it even harder. Here, we evaluatedAspergillus PCR input to help classifying IA in SARS-CoV-2-infected patients. (2) Methods: 45COVID-19 patients were prospectively monitored twice weekly for Aspergillus markers and anti-Aspergillus serology. We evaluated the concordance between (Ι) Aspergillus PCR and culture inrespiratory samples, and (ΙΙ) blood PCR and serum galactomannan. Patients were classified asputative/proven/colonized using AspICU algorithm and two other methods. (3) Results: Theconcordance of techniques applied on respiratory and blood samples was moderate (kappa = 0.58and kappa = 0.63, respectively), with a higher sensitivity of PCR. According to AspICU, 9/45 patientswere classified as putative IA. When incorporating PCR results, 15 were putative IA because theymet all criteria, probably with a lack of specificity in the context of COVID-19. Using a modifiedAspICU algorithm, eight patients were classified as colonized and seven as putative IA. (4)Conclusion: An appreciation of the fungal burden using PCR and Aspergillus serology was addedto propose a modified AspICU algorithm. This proof of concept seemed relevant, as it was inagreement with the outcome of patients, but will need validation in larger cohorts.
Fungal or bacterial co-infections in patients with H1N1 influenza have already been reported in many studies. However, information on the risk factors, complications, and prognosis of mortality cases with coronavirus disease 2019 (COVID-19) are limited. We aimed to assess 36 mortality cases of 178 hospitalized patients among 339 patients confirmed to have had SARS-CoV-2 infections in a medical center in the Wenshan District of Taipei, Taiwan, between January 2020 and September 2021. Of these 36 mortality cases, 20 (60%) were men, 28 (77.7%) were aged >65 years, and the median age was 76 (54–99) years. Comorbidities such as hypertension, coronary artery disease, and chronic kidney disease were more likely to be found in the group with length of stay (LOS) > 7 d. In addition, the laboratory data indicating elevated creatinine-phosphate-kinase (CPK) (p < 0.001) and lactic acid dehydrogenase (LDH) (p = 0.05), and low albumin (p < 0.01) levels were significantly related to poor prognosis and mortality. The respiratory pathogens of early co-infections (LOS < 7 d) in the rapid progression to death group (n = 7 patients) were two bacteria (22.2%) and seven Candida species (77.8.7%). In contrast, pathogens of late co-infections (LOS > 7 d) (n = 27 patients) were 20 bacterial (54.1%), 16 Candida (43.2%), and only 1 Aspergillus (2.7%) species. In conclusion, the risk factors related to COVID-19 mortality in the Wenshan District of Taipei, Taiwan, were old age, comorbidities, and abnormal biomarkers such as low albumin level and elevated CPK and LDH levels. Bacterial co-infections are more common with Gram-negative pathogens. However, fungal co-infections are relatively more common with Candida spp. than Aspergillus in mortality cases of COVID-19.
Clinical characteristics and initial presentations of six patients with COVID-19 complicated by invasive aspergillosis who received tocilizumab.
Tocilizumab administration, diagnostic time, treatment of invasive aspergillosis, and outcomes.
Coronavirus disease-2019 (COVID-19) causes severe pneumonia and acute respiratory distress syndrome. According to the current consensus, immunosuppressants, such as dexamethasone and anti-interleukin-6 receptor monoclonal antibodies, are therapeutic medications in the early stages of infection. However, in critically ill patients, viral, fungal, and bacterial coinfection results in higher mortality. We conducted a single-center, retrospective analysis of 29 mechanically ventilated patients with artificial airways. Patients were adults with confirmed COVID-19 infection and severe pneumonia. Acute respiratory distress syndrome was diagnosed according to the Kigali modification of the Berlin definition. Six patients had invasive pulmonary aspergillosis coinfection based on elevated serum galactomannan levels and/or bronchoalveolar lavage fluid. We present two cases with brief histories and available clinical data. We also conducted a literature review to determine whether immunosuppressants, such as tocilizumab, increase infection risk or invasive aspergillosis in patients with COVID-19. There is no conclusive evidence to suggest that tocilizumab increases coinfection risk. However, further studies are needed to determine the optimal dose, between-dose interval, and timing of tocilizumab administration in patients with COVID-19.
Clinical symptoms, site, and comorbidities among COVID-19 patients (N = 124).
This study aimed to determine the patient demographics, risk factors, which include comorbidities, medications used to treat COVID-19, and presenting symptoms and signs, and the management outcome of COVID-19-associated invasive fungal sinusitis. A retrospective, propensity score-matched, comparative study was conducted at a tertiary care center, involving 124 patients with invasive fungal sinusitis admitted between April 2021 and September 2021, suffering from or having a history of COVID-19 infection. Among the 124 patients, 87 were male, and 37 were female. A total of 72.6% of patients received steroids, while 73.4% received antibiotics, and 55.6% received oxygen during COVID-19 management. The most common comorbidities were diabetes mellitus (83.9%) and hypertension (30.6%). A total of 92.2% had mucor, 16.9% had aspergillus, 12.9% had both, and one patient had hyalohyphomycosis on fungal smear and culture. The comparative study showed the significant role of serum ferritin, glycemic control, steroid use, and duration in COVID-19-associated invasive fungal disease (p < 0.001). Headache and facial pain (68, 54.8%) were the most common symptoms. The most involved sinonasal site was the maxillary sinus (90, 72.6%). The overall survival rate at the three-month follow-up was 79.9%. COVID-19-related aggressive inflammatory response, uncontrolled glycemic level, and rampant use of steroids are the most important predisposing factors in developing COVID-19-associated invasive fungal sinusitis.
Severe cases of coronavirus disease 2019 (COVID-19) managed in the intensive care unit are prone to complications, including secondary infections with opportunistic fungal pathogens. Systemic fungal co-infections in hospitalized COVID-19 patients may exacerbate COVID-19 disease severity, hamper treatment effectiveness and increase mortality. Here, we reiterate the role of fungal co-infections in exacerbating COVID-19 disease severity as well as highlight emerging trends related to fungal disease burden in COVID-19 patients. Furthermore, we provide perspectives on the risk factors for fungal co-infections in hospitalized COVID-19 patients and highlight the potential role of prolonged immunomodulatory treatments in driving fungal co-infections, including COVID-19-associated pulmonary aspergillosis (CAPA), COVID-19-associated candidiasis (CAC) and mucormycosis. We reiterate the need for early diagnosis of suspected COVID-19-associated systemic mycoses in the hospital setting.
Aspergillus co-infection in patients with severe coronavirus disease 2019 (COVID-19) pneumonia, leading to acute respiratory distress syndrome, has recently been reported. To date, 38 cases have been reported, with other cases most likely undiagnosed mainly due to a lack of clinical awareness and diagnostic screening. Importantly, there is currently no agreed case definition of COVID-19 associated invasive pulmonary aspergillosis (CAPA) that could aid in the early detection of this co-infection. Additionally, with the global emergence of triazole resistance, we emphasize the importance of antifungal susceptibility testing in order to ensure appropriate antifungal therapy. Herein is a review of 38 published CAPA cases, which highlights the diagnostic and therapeutic challenges posed by this novel fungal co-infection.
Cases of COVID-19 and Pneumocystis jirovecii co-infection.
Cases of COVID-19 and aspergillosis co-infection.
Cases of COVID-19 and mucormycosis co-infection.
Cases of COVID-19 and endemic mycosis co-infection.
The physiopathologic characteristics of COVID-19 (high levels of inflammatory cytokines and T-cell reduction) promote fungal colonization and infection, which can go unnoticed because the symptoms in both diseases are very similar. The objective of this work was to study the current epidemiology of systemic mycosis in COVID-19 times. A literature search on the subject (January 2020–February 2021) was performed in PubMed, Embase, Cochrane Library, and LILACS without language restrictions. Demographic data, etiological agent, risk factors, diagnostic methods, antifungal treatment, and fatality rate were considered. Eighty nine publications were found on co-infection by COVID-19 and pneumocystosis, candidiasis, aspergillosis, mucormycosis, coccidioidomycosis, or histoplasmosis. In general, the co-infections occurred in males over the age of 40 with immunosuppression caused by various conditions. Several species were identified in candidiasis and aspergillosis co-infections. For diagnosis, diverse methods were used, from microbiological to molecular. Most patients received antifungals; however, the fatality rates were 11–100%. The latter may result because the clinical picture is usually attributed exclusively to SARS-CoV-2, preventing a clinical suspicion for mycosis. Diagnostic tests also have limitations beginning with sampling. Therefore, in the remainder of the pandemic, these diagnostic limitations must be overcome to achieve a better patient prognosis.
Characteristics of the patients included in the study.
Aspergillus species isolated in CAPA patients.
Diagnostic methods.
Factors associated with CAPA diagnosis. Univariate analysis.
Factors associated with CAPA diagnosis. Multivariate analysis.
Our study aims to assess the prevalence of CAPA (COVID-19-associated pulmonary aspergillosis) and describe the associated risk factors and their impact on mortality. A prospective study was conducted. We included patients with COVID-19 disease who were admitted to the ICU with a diagnosis of respiratory failur. Mycological culture and other biomarkers (calcofluor staining, LFD, LFA, PCR, GM, and B-D-glucan) were performed. A total of 300 patients were included in the study. Thirty-five patients were diagnosed with CAPA (prevalence 11.7%). During admission, 57 patients died (19%), and, in the group of CAPA patients, mortality was 31.4%. In multivariate analysis, independent risk factors associated with CAPA diagnosis were age (OR: 1.05; 95% CI 1.01–1.09; p = 0.037), chronic lung disease (OR: 3.85; 95% CI 1.02–14.9; p = 0.049) and treatment with tocilizumab during admission (OR: 14.5; 95% 6.1–34.9; p = 0.001). Factors independently associated with mortality were age (OR: 1.06; 95% CI 1.01–1.11; p = 0.014) and CAPA diagnosis during admission (OR: 3.34; 95% CI 1.38–8.08; p = 0.007). CAPA is an infection that appears in many patients with COVID-19 disease. CAPA is associated with high mortality rates, which may be reduced by early diagnosis and initiation of appropriate antifungal therapy, so screening of COVID-19 ARDS (acute respiratory distress syndrome) patients for CAPA is essential.
Top-cited authors
David Denning
  • The University of Manchester
Felix Bongomin
  • Gulu University (GU)
Rita Oladele
  • College of Medicine University of Lagos
Sara Gago
  • The University of Manchester
Martin Hoenigl
  • University of California, San Diego