Major Fungal Species Research Articles

Integrated Pest Management Decision Support System (IPM-DSS) a tool to support management of tree diseases in Kenya

Tree diseases have been noted as one of the major causes of abnormalities and malformations on perennial plants. Tree disease research in Kenya has resulted in a list of diseases identified to cause significant losses on forest plantations. The need for management of such diseases have been on the increase. The Decision Support System (DSS) was envisioned to help stakeholders make decisions to manage tree diseases on farm and in tree plantations. Information was collected across Kenya on diseases affecting seed, seedlings, plantations, natural forests and post-harvest products in sawmills. The data was entered into excel sheets and organized for database administration on MySQL software. Over 20 major fungal species were identified and their biology, distribution and management determined. This information was further fed into a query system to allow different groups of stakeholders to access the information for diagnosis and control of tree diseases. Results showed that major tree diseases in Kenya were caused by fungi belonging to the families; Botryosphaeriaceae, Nectriaceae, and Amphisphaeriaceae. The data and information availed through this query system is being used in trend analysis for proactive control and management of tree diseases in Kenya. It will also be used for farmer advisory services through closest match selection of symptom appearance based on the tree species. The query system is an innovation that will also be used for Citizen Science Data Capture to aid Kenya Forestry Research Institute in detecting emerging diseases in real-time for phytosanitary measures. The identification of commonly occurring pathogens in commercial forestry species in the country has led to research towards their control and tree improvement for resistance to tree diseases. Future plantation establishment should be based on resistant species and high-quality germplasm for maximum survival and improved yields. Key words: Advisory services, Botryosphaeriaceae, citizen science, Decision Support System, Integrated Pest Management, tree diseases.

The buoyancy of cryptococcal cells and its implications for transport and persistence of Cryptococcus in aqueous environments.

Cryptococcus is a genus of saprophytic fungi with global distribution. Two species complexes, C. neoformans and C. gattii, pose health risks to humans and animals. Cryptococcal infections result from inhalation of aerosolized spores and/or desiccated yeasts from terrestrial reservoirs such as soil, trees, and avian guano. More recently, C. gattii has been implicated in infections in marine mammals, suggesting that inhalation of liquid droplets or aerosols from the air-water interface is also an important, yet understudied, mode of respiratory exposure. Water transport has also been suggested to play a role in the spread of C. gattii from tropical to temperate environments. However, the dynamics of fungal survival, persistence, and transport via water have not been fully studied. The size of the cryptococcal capsule was previously shown to reduce cell density and increase buoyancy. Here, we demonstrate that cell buoyancy is also impacted by the salinity of the media in which cells are suspended, with formation of a halocline interface significantly slowing the rate of settling of cryptococcal cells through water, resulting in persistence of C. neoformans within 1 cm of the air-water interface for over 60 min and C. gattii for 4-6 h. Our data also showed that during culture in yeast peptone dextrose media (YPD), polysaccharide accumulating in the supernatant formed a raft that augmented buoyancy and further slowed settling of cryptococcal cells. These findings illustrate new mechanisms by which cryptococcal cells may persist in aquatic environments, with important implications for aqueous transport and pathogen exposure.

Entomophthoralean and hypocrealean fungal pathogens of the sugarcane aphid, Melanaphis sacchari (Hemiptera: Aphididae), on sorghum in Georgia

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.

Development of a Taxon-Specific Real-Time Polymerase Chain Reaction Method to Detect Trichoderma reesei Contaminations in Fermentation Products

Recently, a genetically modified microorganism (GMM) detection strategy using real-time PCR technology was developed to control fermentation products commercialized in the food and feed chain, allowing several unexpected GMM contaminations to be highlighted. Currently, only bacterial strains are targeted by this strategy. Given that fungal strains, like Trichoderma reesei, are also frequently used by the food industry to produce fermentation products, a novel real-time PCR method specific to this fungal species was developed and validated in this study to reinforce the GMM detection strategy. Designed to cover a sequence of 130 bp from the translation elongation factor alpha 1 (Tef1) gene of T. reesei, this real-time PCR method, namely TR, allows for the screening of commercial fermentation products contaminated with T. reesei, genetically modified or not, which is one of the major fungal species used as an industrial platform for the manufacturing of fermentation products. The developed real-time PCR TR method was assessed as specific and sensitive (LOD95% = eight copies). In addition, the developed real-time PCR TR method performance was confirmed to be in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. The validated real-time PCR TR method was also demonstrated to be applicable to commercial microbial fermentation products. Based on all these results, the novel real-time PCR TR method was assessed as valuable for strengthening the current GMM detection strategy regarding major fungal species used by the food industry to produce microbial fermentation products.

P063 Host-mycobiome interactions during the resolution of inflammation in experimental colitis

Abstract Background Recent studies have documented the complexity of the intestinal fungal community (‘mycobiome’) in mice, and clinical and experimental observations have shown that the mycobiome influences both gut health and disease, e.g., IBD. In fact, prior studies have shown that CD patients, compared to healthy controls, harbor higher levels of intestinal Candida tropicalis (Ct), which is the major fungal species detected in the colons of colitic mice after dextran sodium sulfate (DSS) challenge. Moreover, proteins encoded by genes within IBD susceptibility loci, such as the pattern recognition receptor (PRR), NOD2, are known not only to recognize bacterial components, but also a fungal cell wall element, chitin. In fact, the role of PRRs in regulating immunity against intestinal fungi, and how fungi influence IBD remains poorly defined. Methods We challenged DSS colitic wildtype (WT) and Nod2-/- mice with Ct 2 days before DSS administration and subsequently on day 0, 3 and 6. Bone marrow-derived macrophages (BMDMs) from untreated mice were administered chitin in vitro. Results Our data confirms previous studies that Ct challenge does not exacerbate colitis in DSS-treated C57BL/6 WT mice, however, Ct-infected Nod2-/- mice possess a higher fungal burden and exhibit worse colitis symptoms, such as weight loss, decreased stool consistency, and presence of blood in stools, vs. Ct-infected WT mice, indicating an essential and protective role for NOD2 during colitis recovery after Ct challenge. Our results also show that Ct-infected Nod2-/- mice display a marked reduction in colonic Il22 and Il17, which are cytokines previously reported to be important in maintaining epithelial barrier integrity during DSS colitis. These data were confirmed in colons of Ct infected DSS challenged, ileitis prone SAMP1/YitFc (SAMP) mice NOD2 deficient. Moreover, our in vitro data show, a decrease in Il1ß and Il23 in BMDM from SAMP Nod2-/- mice compared to WT after 2h of exposure to chitin. IL-17+ innate lymphoid cells (ILCs) are also known to control fungal burden during opportunistic fungal infections, and interestingly, our findings indicate that Ct infection of Nod2-/- vs. WT mice results in a decreased frequency of mesenteric lymph node–derived type 3 ILCs (ILC3s), suggesting that delay in fungal clearance and recovery in Nod2-/-mice may be due to the inability to mount protective type 3 immune responses. Conclusion Taken together, the data collected suggests that NOD2 is essential to maintain gut mycobiome homeostasis and drives protective innate immune responses, via a macrophage-mediated ILC3 recruitment and IL-17 mechanism, by preventing the overgrowth of opportunistic fungi that may contribute to chronic intestinal inflammation, such as that observed in CD.

MYCOCENOSIS OF GRAPE BERRIES CULTIVATED IN THE CENTRAL SLOVAK WINE REGION

Eleven samples of grapes from the Central Slovak wine-growing region were examined at the time of harvest in 2021 using the plating method and the plating method with surface disinfection. Plates were incubated aerobically at 25±1 °C in the dark for one week. Identification of fungi was based on morphological and microscopic characteristics. Of the 574 strains detected and identified in the exogenous mycobiota, the most common genus was Alternaria (90.9%), followed by Aspergillus (72.7%), Cladosporium, Penicillium, and Rhizopus (63.6%, each). The most abundant genera found by descending order were Alternaria (53.3%), Rhizopus (9.4%), Aspergillus (5.5%) and only in minor percentage by Penicillium (3%), among others. Of the 512 strains detected and identified in the endogenous mycobiota, the most common genus was Alternaria (100%), followed by Rhizopus (81.8%), Penicillium (72.7%), Aspergillus (63.6%), Cladosporium (45.5%), and Epicoccum (27.3%). The most abundant genera were Alternaria (61.1%), Cladosporium (13.1%), Aspergillus (11.7%), and Rhizopus (9%). The major fungal species isolated from the genera Aspergillus and Penicillium were Aspergillus section Nigri and Penicillium glabrum. Potentially toxigenic Aspergillus and Penicillium species were evaluated for toxinogenicity by thin-layer chromatography. Positive toxinogenity was found for all strains of Aspergillus clavatus tested and for almost all strains of Penicillium expansum and P. crustosum. Out of the two Aspergillus flavus isolates tested, none produced AFB1, AFG1, and CPA. Ochratoxigenic microfungi did not produce OTA.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Milk provides the basis for an eco-friendly shorter process for skin preservation and leather manufacture

The traditional beamhouse method for tanning skins to produce leather uses copious amounts of water, large quantities of chemicals deemed to be dangerous by the public and produces large quantities of liquid waste (90 kg/ton of raw hide) that have become a significant problem due to environmental concerns. In this work, we chart the development of the use of a milk product that both preserves unwashed sheepskins and depilates them, removing the need for salt, sulfide and lime and reduces the copious amounts of water used in the traditional process. This method can completely replace salting and the first five steps of the traditional beamhouse process and the depilated skins can be taken straight to degreasing and tanning. Evaluation of the depilated skins by both eye and high-resolution microscopy showed no apparent damage to the grain or fraying of the collagen bundles. The leather produced from them had physical properties that were either identical or better than those of leather made using the traditional methods. To try and understand the process, microbial culturing of the depilation liquid routinely identified two major bacterial species ( Lactococcus lactis and Lactiplantibacillus plantarum ) and two major fungal species ( Geotrichum candidum and Yarrowia lipolytica ) among other minor species. We predict that the secretion of an unidentified mix of antimicrobial substances and extracellular enzymes from these microorganisms are responsible for the preservation and depilation of sheepskin. This procedure shows potential for further development and could result in a sustainable green beamhouse operation for the leather industry. • A completely sulfide free protocol for sheepskin depilation has been developed. • It uses dairy by-products whey or permeate to preserve and depilate over 3–4 days. • No need to lime, delime bate or pickle so water use is considerably reduced. • The resulting leather has an enhanced appearance compared to normal leather. • The leather's physical properties are the same as those of the traditional product.

Isoeugenol affects expression pattern of conidial hydrophobin gene RodA and transcriptional regulators MedA and SomA responsible for adherence and biofilm formation in Aspergillus fumigatus.

Aspergillus fumigatus is one of the major pathogenic fungal species, causing life-threatening infections. Due to a limited spectrum of available antifungals, exploration of new drug targets as well as potential antifungal molecules has become pertinent. Rodlet layer plays an important role in adherence of fungal conidia to hydrophobic cell surfaces in host, which also leads to A. fumigatus biofilm formation, contributing factor to fungal pathogenicity. From decades, natural sources have been known for the development of new active molecules. The present study investigates effect of isoeugenol on genes responsible for hydrophobins (RodA), adhesion as well as biofilm formation (MedA and SomA) of A. fumigatus. Minimum inhibitory concentrations (MIC and IC50) of isoeugenol against A. fumigatus were determined using broth microdilution assay. The IC50 results showed reduced hydrophobicity and biofilm formation as well as eradication after treatment with the compound and electron micrograph data corroborated these findings. The qRT-PCR showed a significant downregulation of genes RodA, MedA, SomA and pksP involved in hydrophobicity and biofilm formation. SwissADME studies potentiated drug-like propensity for isoeugenol which formed four hydrogen bonds with low binding energy (− 4.54 kcal/mol) at the catalytic site of RodA protein studied via AutoDock4. Hence, the findings conclude that isoeugenol inhibits conidial hydrophobicity and biofilm formation of A. fumigatus and further investigations are warranted in this direction.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00203-022-02817-w.

NOD2 PROMOTES HOST DEFENSE AND RECOVERY INDUCING MACROPHAGE-MEDIATED INNATE LYMPHOID CELLS TYPE 3 ACTIVATION FOLLOWING FUNGAL INFECTION IN EXPERIMENTAL COLITIS

Abstract Recent studies have documented the complexity of the intestinal fungal community (‘mycobiome’) in mice, and clinical and experimental observations have shown that the mycobiome influences both gut health and disease, e.g., inflammatory bowel disease (IBD). In fact, prior studies have shown that Crohn’s disease (CD) patients, compared to healthy controls, harbor higher levels of intestinal Candida tropicalis (Ct), which is the major fungal species detected in the colons of colitic mice after DSS challenge. More recently, Debaryomyces hansenii (also known as Candida famata) has been found in intestinal wounds of mice and inflamed mucosal tissues of CD patients. Moreover, proteins encoded by genes within IBD susceptibility loci, such as the pattern recognition receptor (PRR), NOD2, are known not only to recognize bacterial components, but also a fungal cell wall element, chitin. In fact, the role of PRRs in regulating immunity against intestinal fungi, and how fungi influence IBD remains poorly defined. We challenged DSS colitic WT and Nod2-/- mice with Ct 2 days before DSS administration and subsequently on day (d)0, 3 and 6. Our data confirms previous studies that Ct challenge does not exacerbate colitis in DSS-treated C57BL/6 wildtype (WT) mice, however, Ct-infected Nod2-/- mice possess a higher fungal burden and exhibit worse colitis symptoms, such as weight loss, decreased stool consistency, and presence of blood in stools, vs. Ct-infected WT mice, indicating an essential and protective role for NOD2 during colitis recovery after Ct challenge. Our results also show that Ct-infected Nod2-/- mice display a marked reduction in colonic Il22 and Il17, which are cytokines previously reported to be important in maintaining epithelial barrier integrity during DSS colitis, These data were confirmed in colons of Ct infected DSS challenged, ileitis-prone SAMP1/YitFc (SAMP) mice that were deficient in NOD2. Moreover, Our in vitro data show, a decrease in Il1b and Il23 in bone marrow-derived macrophages from SAMP Nod2-/- mice compared to WT after 2h of exposure to chitin. IL-17+ innate lymphoid cells (ILCs) are also known to control fungal burden during opportunistic fungal infections, and interestingly, our findings indicate that Ct infection of Nod2-/- vs. WT mice results in a decreased frequency of mesenteric lymph node–derived type 3 ILCs (ILC3s), suggesting that delay in fungal clearance and recovery in Nod2-/- mice may be due to the inability to mount protective type 3 immune responses. Taken together, the data so far collected suggests that NOD2 is essential to maintain gut mycobiome homeostasis and drives protective innate immune responses, via a macrophage-mediated ILC3 recruitment and IL-17 mechanism, by preventing the overgrowth of opportunistic fungi that may contribute to chronic intestinal inflammation, such as that observed in CD.

NOD2 PROMOTES HOST DEFENSE AND RECOVERY INDUCING MACROPHAGE-MEDIATED INNATE LYMPHOID CELLS TYPE 3 ACTIVATION FOLLOWING FUNGAL INFECTION IN EXPERIMENTAL COLITIS

Recent studies have documented the complexity of the intestinal fungal community (‘mycobiome’) in mice, and clinical and experimental observations have shown that the mycobiome influences both gut health and disease, e.g., inflammatory bowel disease (IBD). In fact, prior studies have shown that Crohn’s disease (CD) patients, compared to healthy controls, harbor higher levels of intestinal Candida tropicalis (Ct), which is the major fungal species detected in the colons of colitic mice after DSS challenge.

Comparative Study on the Antimicrobial Activity in Various Solvent Extracts of Garcinia gummi-gutta (L.) Roxb and Solanum betaceum Cav. Fruits Against Major Fish Pathogens

The present study aimed to evaluate the antimicrobial activity of the extracts from the fruits of Garcinia gummi-gutta and Solanum betaceum using the standard Agar well diffusion method. Three concentrations (25, 50,and 75 µl/ml) of various extracts such as aqueous, ethanol, petroleum ether, and chloroform were tested against six fish pathogens such as Aeromonas hydrophila, Vibrio cholerae, Streptococcus agalactia, Streptococcus iniae Pseudomonas aeruginosa, and Klebsiella pneumoniae. Amoxiclav 10 mcg was used as the positive control (Disc). Three major fungal species Aspergillus niger, Aspergillus flavus, and Candida albicans were used to determine the fruit extracts' antifungal activity at three different concentrations (25, 50, 100 µl/ml). Amphotericin B (100 units) was used as a positive control. Both plant extracts showed dose-dependent antimicrobial activity against the tested pathogens. The crude ethanol and petroleum ether extracts of G. gummi-gutta and S. betaceum displayed more potential against all strains at higher concentrations. However, G. gummi-gutta showed better efficacy against microbes when compared to S. betaceum. The findings of present study will be valuable for future identification, quality control, and pharmacological studies of natural chemicals derived from these plants especially in aquaculture.

Microbial Diversity in the Phyllosphere and Rhizosphere of an Apple Orchard Managed under Prolonged "Natural Farming" Practices.

Microbial diversity in an apple orchard cultivated with natural farming practices for over 30 years was compared with conventionally farmed orchards to analyze differences in disease suppression. In this long-term naturally farmed orchard, major apple diseases were more severe than in conventional orchards but milder than in a short-term natural farming orchard. Among major fungal species in the phyllosphere, we found that Aureobasidium pullulans and Cryptococcus victoriae were significantly less abundant in long-term natural farming, while Cladosporium tenuissimum predominated. However, diversity of fungal species in the phyllosphere was not necessarily the main determinant in the disease suppression observed in natural farming; instead, the maintenance of a balanced, constant selection of fungal species under a suitable predominant species such as C. tenuissimum seemed to be the important factors. Analysis of bacteria in the phyllosphere revealed Pseudomonas graminis, a potential inducer of plant defenses, predominated in long-term natural farming in August. Rhizosphere metagenome analysis showed that Cordyceps and Arthrobotrys, fungal genera are known to include insect- or nematode-infecting species, were found only in long-term natural farming. Among soil bacteria, the genus Nitrospira was most abundant, and its level in long-term natural farming was more than double that in the conventionally farmed orchard.

Indigenous arbuscular mycorrhizal fungi enhance resistance of apple rootstock ‘M9T337’ to apple replant disease

Apple replant disease is common in apple cultivation, leading to decay and necrosis of the root tip, growth retardation, and even death of the plant. Biological control, inducing activation of the plant's defense systems, is a promising alternative strategy for controlling the disease. In this study, we used the common rootstock ‘M9T337’ as the test material and harvested the plants at 60 and 120 days after transplantation. We investigated the physiological effects of inoculating the soil with indigenous arbuscular mycorrhizal fungi (AMF) to improve the resistance of the apple rootstock under replant conditions. The Glomus sp. and Paraglomus sp. acted as the major fungal species in QX and LZ. The results showed that inoculation of indigenous AMF increased the biomass of the plants and promoted root growth under replanting conditions. The antioxidant and defense-related enzyme activities in the plants were significantly raised, including the activities of the enzymes superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase. In the seedling root system, the H2O2 content and O2·− production were reduced, resulting in decreased lipid oxidation. Indigenous AMF treatment also induced the activity of chitinase and β-1,3-glucanase. This suggests that inoculation of indigenous AMF enhances the resistance of apple rootstock by regulating root reactive oxygen species levels and the antioxidant system under replanting conditions. This provides a theoretical basis for the prevention and treatment of apple replant disease.

Evaluation of Chitin as a Biomarker of Pathogenic Fungal Isolates

Chitin is a polysaccharide component of the inner cell wall of fungi that has been used to estimate fungal invasion in plant products. However, its detection in major pathogenic fungal species has not been investigated. The present study aimed to determine the chitin contents of pathogenic fungal species in order to evaluate its diagnostic potential as a biomarker for fungal infections. High performance liquid chromatography (HPLC) was used to measure chitin content. Pure chitin was acid hydrolyzed and the fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine produced were measured. The chitin contents of ten pathogenic fungal isolates were determined per mycelial dry weight. They varied from 18.61 (± 0.09) to 47.12 (± 0.50) μg/mg dry mycelial weight. Candida albicans and Cryptococcus neoformans exhibited the highest and lowest levels of chitin, respectively. Based upon relative amounts of chitin produced, three groups namely: high (Candida albicans, Cryptococcus gattii, Aspergillus niger and Penicilliumat 47.12, 46.98, 46.05, and 44.15 μg/mg respectively), medium (Rhizopus, Aspergillus fumigatus, Fusarium solani, andMucor at 36.61, 36.30, 35.03, and 34.84 μg/mg, respectively), and low (Candida tropicalis and Cryptococcus neoformans at 20.78 and 18.61 μg/mg, respectively), were identified. Chitin was not detectable in bacterial isolates used as controls. The chitin detection method offers a sensitive and specific tool for the quantification of chitin in pathogenic fungal isolates. The detection of chitin may be a useful assay for the diagnosis of fungal infections in clinical samples.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Background aimsThe genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8+ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). ResultsGXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-ς signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR+ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosis ConclusionsThus, these findings reveal that GXMR-CAR+ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR+ T cells in an animal model of cryptococcosis.

Efficient recovery of metal tolerant fungi from the soil of industrial area and determination of their biosorption capacity

Due to various anthropogenic activities, different heavy metals like arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), mercury (Hg) and nickel (Ni) accumulate in the soils of industrial areas. In the current study, contaminated soil was collected from Yuepu industrial area of Shanghai, China. Soil analysis revealed the presence of Pb and Cu in high concentrations, ranging from 125.9–128.0 mg/kg and 98.4–100.2 mg/kg, respectively. These soil samples were further used to isolate metal tolerant microbes. Subsequent screening resulted in the isolation of four major fungal species including Fusarium fujikuroi, Fusarium solani, Trichoderma citronoviridae and Trichoderma reesei. Molecular characterization of isolated fungi was performed and amplified sequences were deposited in GenBank NCBI database. Metal tolerance and biosorption capacity of these fungal strains towards lead and copper were tested. Fungal strains were exposed to increasing concentrations (100–1000 ppm) of chlorides of lead and copper and the tolerance of the selected fungi was evaluated by measuring the minimum inhibitory concentrations (MIC). The range of MIC values was found to be 400 ppm to 1000 ppm. The tolerance index of all the tested fungi was calculated and T. citronoviridae was observed to be tolerant at 1 mM concentration of lead while F. solani was the most tolerant species at 1 mM concentration of copper. The highest biosorption capacity of Pb was exhibited by T. citronoviridae, while T. reesei showed the best absorption capacity of Cu followed by F. solani. Scanning electron microscopy (SEM) showed visible adsorption of metal on fungal mycelia and suggested it to be the mechanism of metal removal. On the basis of these findings, it could be concluded that T. citronoviridae and F. solani are the potential mycoremediation microbes in Pb and Cu contaminated soils, respectively.

Broad-Spectrum Antimicrobial and Antibiofilm Activity of a Natural Clay Mineral from British Columbia, Canada.

Worldwide increases in antibiotic resistance and the dearth of new antibiotics have created a global crisis in the treatment of infectious diseases. These concerns highlight the pressing need for novel antimicrobial agents. Natural clay minerals have a long history of therapeutic and biomedical applications and have lately received specific attention for their potent antimicrobial properties. In particular, Kisameet clay (KC) has strong antibacterial activity against a variety of multidrug-resistant (MDR) bacterial pathogens in vitro Here, we have extended the known spectrum of activity of KC by demonstrating its efficacy against two major fungal pathogens, Candida albicans and Cryptococcus neoformans In addition, KC also exhibits potent activity against the opportunistic bacterial pathogen Mycobacterium marinum, a model organism for M. ulcerans infection. Moreover, aqueous KC leachates (KC-L) exhibited broad-spectrum antibacterial activity, eradicated Gram-negative and Gram-positive biofilms, and prevented their formation. The mechanism(s) underlying KC antibacterial activity appears to be complex. Adjusting KC-L to neutral pH rendered it inactive, indicating a contribution of pH, although low pH alone was insufficient for its antibacterial activity. Treatment of KC minerals with cation-chelating agents such as EDTA, 2,2'-bipyridyl, and deferoxamine reduced the antibacterial activity, while supplementation of KC-L with these chelating agents eliminated the inhibitory activity. Together, the data suggest a positive role for divalent and trivalent cations, including iron and aluminum, in bacterial inhibition by KC. Collectively, these studies demonstrate the range of KC bioactivity and provide a better understanding of the mechanism underlying its antibacterial effects.IMPORTANCE The escalating emergence of multidrug-resistant (MDR) bacteria, together with the paucity of novel antimicrobial agents in antibiotic development, is recognized as a worldwide public health crisis. Kisameet clay (KC), found in British Columbia (BC), Canada, is a clay mineral with a long history of therapeutic applications among people of the First Nations. We previously reported the antibacterial activity of KC against a group of MDR clinical pathogens. Here, we demonstrate its activity against two major human-pathogenic fungal species, as well as against bacterial biofilms, which underlie many recalcitrant bacterial infections. In these studies, we also identified several geochemical characteristics of KC, such as metal ions and low pH, which are involved in its antibacterial activity. These findings provide a better understanding of the components of KC antibacterial activity and a basis for developing defined preparations of this clay mineral for therapeutic applications.

Epidemiological and Molecular Investigation of Ocular Fungal Infection in Equine from Egypt.

Diagnosis and treatment of ocular fungal infection in equine seems very challenging for owners and clinicians. The present study aimed to identify and characterize fungal species isolated from the eyes of clinically healthy and diseased equines (N = 100) from Dakahlia Governorate, Egypt. The work also involved morphological and molecular characterization of the major fungal species. In addition, correlations between the occurrence of isolated fungi and some of the potential risk factors were also investigated. Interestingly, the prevalence rate of ocular mycosis in all examined equines in the study was 28% and there were major clinical signs associated with ocular fungal infection. Moreover, the identified fungal species included Aspergillus flavus, A. fumigatus, A. niger, Penicillium spp., Mucor spp., and Alternari spp. with a corresponding prevalence rate of 63.9%, 27.8%, 15.3%, 18.1%, 13.9%, and 4.2%, respectively, in healthy equine eyes, while their prevalence in diseased equine eyes was 57.1%, 32.1%, 21.4%, 7.1%, 3.6%, and 0%. Furthermore, a statistical significant association (p < 0.05) was found between the frequency of isolation of A. fumigatus and Penicillium and several risk factors (breed, sex, and ground type), while the remaining risk factors and occurrence of fungi were not statistically correlated. A subset of the Aspergillus species samples positive by polymerase chain reaction (PCR) were sequenced and their phylogenetic analysis identified three species of Aspergillus. Taken together, our study provides novel data related to the occurrence of ocular mycosis in equine in Egypt. Given the zoonotic potential of some identified fungi, our data may be helpful for implementation of novel diagnostic and therapeutic strategies for combating this sight-threatening infection in equine.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR+ T-cell therapy

Abstract The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: C. gattii and C. neoformans. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection’s mortality rate ranges from 20% to 70%. To combat the cryptococcosis, we proposed the redirection of CD8+ T cells to target glucuronoxylomannan (GXM), a sugar present in the Cryptococcus species capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-zeta and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined using bright-field microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed, while no clusters were present in the control cells. The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by flow cytometry and fluorescent microscopy, but no binding was detected in mock-transduced control T cell (NoDNA T cells). Furthermore, when GXMR-CAR+ T cells were administered to immunocompromised NSG mice infected with C. neoformans their C. neoformans burden was significantly lower than mock-transduced control T cell treated mice as shown via immunofluorescence using an anti-GXM antibody and Gomori methenamine-silver (GMS) staining of Titan cells in lung tissue.