Fungal Particles Research Articles

Fabrication of nanocomposites and hybrid materials using microbial biotemplates

Microbes are important part of life that vary in sizes and shapes, diverse surface chemistry and biology, and porous nature of their cell walls. Besides their importance in industrial processes such as fermentation, these serve as biotemplates and provide a biomimetic approach for fabrication of multifarious complex constructs with predefined features, ordered composites and hybrid nanomaterials, microdevices, and micro/nanorobots through various strategies. The template or building blocks for such approaches can be bacterial, algal, and fungal cells or virus particles. Here, we have summarized recent advancements in biofabrication based on live microbes. Using engineering approaches and suitable methods, live microbes can be manipulated as functional “micro/nanodevices and -robots” to further perform biological functions such as replication, distribution, motility, formation of colonies, and secretion of metabolites at will. Biofabrication based on microbes provides effective methods to control and manipulate microbes as functional live building blocks to create micro/nanodevices and -robots for biomedical and energy applications.

Effect of electrical charges on potential of fibers for transport of microbial particles in dry and humid air

Coexistence of natural, synthetic or semi-synthetic fibers and microbial particles in aerosol state has not received so far a proper attention. In the scientific literature, there is almost no research devoted to fibrous aerosols as carriers of microbial particulates. The aim of this study was to assess the potential of natural and man-made fibers for airborne transport of electrically charged bacterial and fungal particles in different humidity conditions. The abilities of 4 natural (cotton, hemp, dog and horse hair) and 2 man-made (polyester and viscose) fibers to transport 7 microbial strains (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptomyces albus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium melinii) in dry (RH < 30%) and humid (RH > 60%) air were analyzed in laboratory conditions using newly built aerosolization chamber. Each tested microbial aerosol was generated (for 10min) into the chamber from water suspension by Collison nebulizer (bacteria) or from contaminated agar surface using tube generator (fungi) and electrically charged or neutralized (–4500V, −3000V, −1500V, −500V, 0V, +500V, +1500V, +3000V, +4500V) using Topas electrostatic aerosol neutralizer. Bioaerosol concentrations were measured with Grimm optical particle counter. In the chamber, each tested microbial strain was mixed (for 10min) with respective studied fibers (their concentrations were controlled using 7400AD real-time fiber monitor) and such mixture was sampled with IOM cassette (for 5min at 5Lmin−1) on polycarbonate filter. After sampling, randomly selected samples were checked using scanning electron microscope to confirm the microbial presence on fibers. Then, all collected fibers carrying microbial particles were eluted by vortexing from the filter using 0.9% NaCl with Tween 80. Spread plate method was applied to assess culturable bacterial or fungal concentration in the resulted suspension.The quantitative assessment of electrically charged culturable microbial particles revealed that fibers were able to transport in the air up to 100 bacterial vegetative cells/spores or fungal conidia per 1 fiber. The most effective in this case were animal (dog and horse) and plant (cotton) fibers. Both tested man-made fibers were significantly less efficient than natural ones (p < 0.05). The study also showed that fraction of culturable microorganisms generated into the air and subsequently transported on fibers may reach 50% of total bioaerosol particles. In the environment of low air humidity (RH < 30%), the percentage of culturable microorganisms was always higher than those in high (RH > 60%) humidity conditions (p < 0.05). Moreover, the highest percentage of culturable microorganisms was always noted when the particles (bacterial vegetative cells or spores as well as fungal conidia) were electrically neutralized (0V). Each time when microbial particles were electrically charged (± 500–4500V), such process decreased the percentage of culturable bacteria and fungi (p < 0.05). This phenomenon can be explained by a disruptive influence of electrical conduction on viable microorganisms in the environment in which water is relatively easily available.

Biological synthesis of metallic nanoparticles: plants, animals and microbial aspects

The green synthesis (GS) of different metallic nanoparticles (MNPs) has re-evaluated plants, animals and microorganisms for their natural potential to reduce metallic ions into neutral atoms at no expense of toxic and hazardous chemicals. Contrary to chemically synthesized MNPs, GS offers advantages of enhanced biocompatibility and thus has better scope for biomedical applications. Plant, animals and microorganisms belonging to lower and higher taxonomic groups have been experimented for GS of MNPs, such as gold (Au), silver (Ag), copper oxide (CuO), zinc oxide (ZnO), iron (Fe2O3), palladium (Pd), platinum (Pt), nickel oxide (NiO) and magnesium oxide (MgO). Among the different plant groups used for GS, angiosperms and algae have been explored the most with great success. GS with animal-derived biomaterials, such as chitin, silk (sericin, fibroin and spider silk) or cell extract of invertebrates have also been reported. Gram positive and gram negative bacteria, different fungal species and virus particles have also shown their abilities in the reduction of metal ions. However, not a thumb rule, most of the reducing agents sourced from living world also act as capping agents and render MNPs less toxic or more biocompatible. The most unexplored area so far in GS is the mechanism studies for different natural reducing agents expect for few of them, such as tea and neem plants. This review encompasses the recent advances in the GS of MNPs using plants, animals and microorganisms and analyzes the key points and further discusses the pros and cons of GS in respect of chemical synthesis.

Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA

Mycobacterium tuberculosis' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although Mtuberculosis activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to Mtuberculosis control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but Mtuberculosis fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for Mtuberculosis to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the ΔcpsA mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the ΔcpsA mutant was largely restored in macrophages missing LAP components (Nox2, Rubicon, Beclin, Atg5, Atg7, or Atg16L1) but not in macrophages defective in a related, canonical autophagy pathway (Atg14, Ulk1, or cGAS). The ΔcpsA mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, Atg5, or Atg7, demonstrating that CpsA makes a significant contribution to the resistance of Mtuberculosis to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.

Effectiveness of a shielded ultraviolet C air disinfection system in an inpatient pharmacy of a tertiary care children's hospital

Viable air particles pose a risk in areas where sterile preparations are compounded. This study investigated the efficacy of an innovative air purification technology that uses a shielded ultraviolet C light lamp to continuously purify the air in an inpatient pharmacy. Mean airborne fungal and bacterial colony forming units were obtained preinstallation and again in 6 months. A statistically significant decrease of 78% and 62% was observed for fungal and bacterial particles, respectively. This study demonstrates a potential role for this novel technology in decreasing the spread of airborne pathogens.

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine‐palmitoyltransferase (SPT) and ceramide synthase‐enzymes involved in sphingolipid biosynthesis‐by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9‐mediated genome editing to generate Sptlc2‐deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2−/− DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2−/− DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2−/− DC2.4 cells express reduced levels of the pattern recognition receptors Dectin‐1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.Support or Funding InformationThis work was funded by The Netherlands Organization for Scientific Research (FGT), and the National Institute of Health grant AI087879 (HLP).

Characteristics of airborne bacteria and fungi in some Polish wastewater treatment plants

The aim of this study was to characterize both the viable airborne bacteria and fungi collected in different types of wastewater treatment plants in Poland. Bacterial and fungal aerosols in five different wastewater treatment plants in Poland were studied, including the pioneering antibiotic resistance testing of airborne bacteria. Concentration level was measured using the six-stage Andersen impactor. After incubation, bioaerosol particles captured on nutrient media on Petri dishes were quantitatively evaluated and qualitatively identified. It was found that the concentration levels of both airborne bacteria and fungi ranged from 10² to 10³ colony-forming units/m³, in all stages of the wastewater treatment plants. The patterns of size distributions of airborne bacteria and fungi were very similar, showing dominant peaks typically in the size range between 3.3 and 4.7 μm. The majority fraction of small bacterial and fungal particles (less than 4.7 μm) in the studied areas indicates that the bioaerosol is relatively fresh, and mostly of local origin. The Gram-positive cocci and nonsporing Gram-positive rods were the dominating forms in the studied wastewater treatment plants environment. The existing differences in the concentration levels between sampling sites enabled identification of the main bioaerosol sources. The highest concentration of bacterial aerosol appeared in sections, where activated sludge post-processing and mechanical purifying are conducted. The most frequently occurring species in the sampled bacterial aerosol were Gram-positive cocci and nonsporing Gram-positive rods. Multi-antibiotic resistance testing showed that among the isolated airborne bacteria, the most antibiotic-resistant features were present among Bacillus species (especially Bacillus mycoides).

Liquid impinger BioSampler's performance for size-resolved viable bioaerosol particles

This study aimed at studying the collection efficiencies of the widely used SKC BioSampler when sampling size-resolved biological viable aerosol particles using an Ultraviolet Aerodynamic Particle Sizer (UV-APS) unit. The test bioaerosols include aerosolized bacterial species and those from indoor air. For optimal performance, different flow rates (Qsampl) (5, 12.5 and 20L/min) and different volumes of collection liquids (Vcl) (5, 10 and 20mL) were tested for bioaerosol particles from 0.5 to 10µm. In addition, the DNA stain method-the LIVE/DEAD BacLight dye and culturing were also utilized to confirm the viable bioaerosol collection efficiencies of the BioSampler.Experimental data showed that when sampling aerosolized Bacillus subtilis at the flow rate of 12.5L/min, the collection efficiency of the BioSampler decreased from 82.7% (at Vcl=20mL) to 24.8% (at Vcl=5mL). The results from the BacLight dye and culturing performed for the samples collected by the BioSampler achieved similar conclusions. When sampling indoor air particles, the overall collection efficiencies of all viable biological particles were 95.3%, 87.7% and 65.5%, respectively, for Vcl=20mL, 10mL and 5mL at the flow rate of 12.5L/min. When Qsampl was 20L/min, the BioSampler performed better with Vcl= 5mL than other volumes. The results from this work suggest that the sampling flow rate plays a dominant role in the overall physical collection efficiency while the collection volume is crucial in preserving the viability of bioaerosol particles. Different from general practice, the operating parameters for the BioSampler should thus be adjusted respectively for target viral, bacterial and fungal aerosol particles.

Autocrine Type I IFN Signaling in Dendritic Cells Stimulated with Fungal β-Glucans or Lipopolysaccharide Promotes CD8 T Cell Activation.

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection, but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor, but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation, activation marker (CD44 and CD69) expression, and production of IFN-γ, IL-2, and granzyme B. Moreover, we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically, type I IFNs promote Ag presentation on MHC I molecules, CD86 and CD40 expression, and the production of IL-12 p70, IL-2, IL-6, and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation, although, in the context of LPS stimulation, this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection, which may be useful in the rational design of vaccines directed against pathogens and tumors.

Assessment of multi-contaminant exposure in a cancer treatment center: a 2-year monitoring of molds, mycotoxins, endotoxins, and glucans in bioaerosols.

Indoor air quality in health care facilities is a major public health concern, particularly for immunocompromised patients who may be exposed to microbiological contaminants such as molds, mycotoxins, endotoxins, and (1,3)-ß-D-glucans. Over 2years, bioaerosols were collected on a monthly basis in a cancer treatment center (Centre F. Baclesse, Normandy, France), characterized from areas where there was no any particular air treatment. Results showed the complexity of mycoflora in bioaerosols with more than 100 fungal species identified. A list of major strains in hospital environments could be put forward due to the frequency, the concentration level, and/or the capacity to produce mycotoxins in vitro: Aspergillus fumigatus, Aspergillus melleus, Aspergillus niger, Aspergillus versicolor, Cladosporium herbarum, Purpureocillium lilacinum, and Penicillium brevicompactum. The mean levels of viable airborne fungal particles were less than 30.530CFU per m3 of air and were correlated to the total number of 0.30 to 20μm particles. Seasonal variations were observed with fungal particle peaks during the summer and autumn. Statistical analysis showed that airborne fungal particle levels depended on the relative humidity level which could be a useful indicator of fungal contamination. Finally, the exposure to airborne mycotoxins was very low (only 3 positive samples), and no mutagenic activity was found in bioaerosols. Nevertheless, some fungal strains such as Aspergillus versicolor or Penicillium brevicompactum showed toxigenic potential in vitro.

Assessment of indoor bioaerosols using a lab-made virtual impactor

ABSTRACTTo assess indoor bioaerosols, a virtual impactor having 1 µm cutoff diameter was designed, fabricated, and evaluated with computational fluid dynamics simulation and also with laboratory test using polystyrene latex particles. Two other cutoff diameters of 635 nm and 1.5 µm were obtained by changing the inlet flow rate and the ratio of minor channel-to-inlet flow rates. In field test, the virtual impactor was operated with varying cutoff diameter and field-emission scanning electron microscope (FE-SEM) analysis was performed for each cutoff diameter to observe morphologies of indoor aerosol particles sampled at the major and minor outlet channels. Particles were sampled at both outlet channels using the SKC Button Aerosol sampler and subsequently cultured. By colony counting, it was found that 56% of cultured fungal particles and 63% of cultured bacterial particles had aerodynamic sizes smaller than 1 µm. MALDI-TOF analysis and visual inspection of culture samples were used to identify indoor bact...

Quantification of the fungal fraction released from various preloaded fibrous filters during a simulated ventilation restart.

This study aimed to demonstrate that particles, especially those associated with fungi, could be released from fibrous filters used in the air-handling unit (AHU) of heating, ventilation and air-conditioning (HVAC) systems during ventilation restarts. Quantification of the water retention capacity and SEM pictures of the filters was used to show the potential for fungal proliferation in unused or preloaded filters. Five fibrous filters with various particle collection efficiencies were studied: classes G4, M5, M6, F7, and combined F7 according to European standard EN779:2012. Filters were clogged with micronized rice particles containing the fungus Penicillium chrysogenum and then incubated for three weeks at 25°C and 90% relative humidity. The results indicated that the five clogged tested filters had various fungal growth capacities depending on their water retention capacity. Preloaded filters were subjected to a simulated ventilation restart in a controlled filtration device to quantify that the fraction of particles released was around 1% for the G4, 0.1% for the M5 and the M6, and 0.001% for the F7 and the combined F7 filter. The results indicate that the likelihood of fungal particle release by low efficiency filters is significantly higher than by high efficiency filters.

Annotating public fungal ITS sequences from the built environment according to the MIxS-Built Environment standard – a report from a May 23-24, 2016 workshop (Gothenburg, Sweden)

Recent molecular studies have identified substantial fungal diversity in indoor environments. Fungi and fungal particles have been linked to a range of potentially unwanted effects in the built environment, including asthma, decay of building materials, and food spoilage. The study of the built mycobiome is hampered by a number of constraints, one of which is the poor state of the metadata annotation of fungal DNA sequences from the built environment in public databases. In order to enable precise interrogation of such data – for example, “retrieve all fungal sequences recovered from bathrooms” – a workshop was organized at the University of Gothenburg (May 23-24, 2016) to annotate public fungal barcode (ITS) sequences according to the MIxS-Built Environment annotation standard (http://gensc.org/mixs/). The 36 participants assembled a total of 45,488 data points from the published literature, including the addition of 8,430 instances of countries of collection from a total of 83 countries, 5,801 instances of building types, and 3,876 instances of surface-air contaminants. The results were implemented in the UNITE database for molecular identification of fungi (http://unite.ut.ee) and were shared with other online resources. Data obtained from human/animal pathogenic fungi will furthermore be verified on culture based metadata for subsequent inclusion in the ISHAM-ITS database (http://its.mycologylab.org).

(1→3)-β-d-Glucan and Galactomannan for Differentiating Chemical "Black Particles" and Fungal Particles Inside Peritoneal Dialysis Tubing.

♦ Aseptic, sheet-like foreign bodies observed inside Tenckhoff (TK) catheter lumens (referred to as "black particles") are, on gross morphology, hardly distinguishable from fungal colonization because these contaminants adhere tightly to the catheter. Detection of fungal cell wall components using (1→3)-β-d-glucan (BG) and galactomannan index (GMI) might be an alternative method for differentiating the particles. ♦ Foreign particles retrieved from TK catheters in 19 peritoneal dialysis patients were examined microscopically and cultured for fungi and bacteria. Simultaneously, a Fungitell test (Associates of Cape Cod, Falmouth, MA, USA) and a Platelia Aspergillus ELISA assay (Bio-Rad Laboratories, Marnes-La-Coquette, France) were used to test the spent dialysate for BG and GMI respectively. ♦ Of the 19 patients, 9 had aseptic black particles and 10 had fungal particles in their tubing. The fungal particles looked grainy, were tightly bound to the catheter, and appeared more "colorful" than the black particles, which looked sheet-like and could easily be removed by milking the tubing. Compared with effluent from patients having aseptic particles, effluent from patients with fungal particles had significantly higher levels of BG (501 ± 70 pg/mL vs. 46 ± 10 pg/mL) and GMI (10.98 ± 2.17 vs. 0.25 ± 0.05). Most of the fungi that formed colonies inside the catheter lumen were molds not usually found in clinical practice, but likely from water or soil, suggesting environmental contamination. Interestingly, in all 10 patients with fungal colonization, visualization of black particles preceded a peritonitis episode and TK catheter removal by approximately 1-3 weeks; in patients with aseptic particles, a 17-week onset to peritonitis was observed. ♦ In all patients with particle-coated peritoneal dialysis tubing, spent dialysate should be screened for BG and GMI. Manipulation of the TK catheter by squeezing, hard flushing, or even brushing to dislodge black particles should be avoided. Replacement of the TK catheter should be suspended until a cause for the particles is determined.

Fully Automated Field-Deployable Bioaerosol Monitoring System Using Carbon Nanotube-Based Biosensors.

Much progress has been made in the field of automated monitoring systems of airborne pathogens. However, they still lack the robustness and stability necessary for field deployment. Here, we demonstrate a bioaerosol automonitoring instrument (BAMI) specifically designed for the in situ capturing and continuous monitoring of airborne fungal particles. This was possible by developing highly sensitive and selective fungi sensors based on two-channel carbon nanotube field-effect transistors (CNT-FETs), followed by integration with a bioaerosol sampler, a Peltier cooler for receptor lifetime enhancement, and a pumping assembly for fluidic control. These four main components collectively cooperated with each other to enable the real-time monitoring of fungi. The two-channel CNT-FETs can detect two different fungal species simultaneously. The Peltier cooler effectively lowers the working temperature of the sensor device, resulting in extended sensor lifetime and receptor stability. The system performance was verified in both laboratory conditions and real residential areas. The system response was in accordance with reported fungal species distribution in the environment. Our system is versatile enough that it can be easily modified for the monitoring of other airborne pathogens. We expect that our system will expedite the development of hand-held and portable systems for airborne bioaerosol monitoring.

Intact Immunotaxis Comprises an Intricate Spatiotemporal Hierarchy of Distinct Chemotactic Processes - A New Paradigm

A close look at cytokine-directed chemotaxis reveals an intriguing mechanistic dilemma. If cytokine gradients provided the only directional cue for chemotaxing immune cells, these cells would ultimately target the cytokine-producing cells of their own host. Such friendly fire may well be an inherent part of an indiscriminate mop-up duty of immune cells in the inflammatory response. However, it would preclude these cells from partaking in any pathogen-specific defenses requiring pre-contact recognition of invaders. Evidently, effective immune-cell recruitment requires additional directional guidance via chemical signals that emanate from the surfaces of microbes rather than host cells. To detect minuscule amounts of chemoattractant produced at the surfaces of bacteria, fungi, and surrogate particles, we have developed a single-cell, pure-chemotaxis assay that employs human neutrophils as ultrasensitive biodetectors. We use micropipette manipulation and/or optical tweezers to maneuver target particles into the proximity of non-adherent, initially quiescent neutrophils. Microscopic inspection of the resulting neutrophil morphology provides a clear readout of the chemotactic activity of the cells. In all cases where neutrophils extended chemotactic protrusions toward nearby targets, this cell response required serum. For bacterial targets such as Salmonella Typhimurium and Escherichia coli, and for zymosan particles as well as fungal particles of Coccidioides posadasii and Candida albicans, this type of microbe-guided chemotaxis was predominantly mediated by complement, in particular the anaphylatoxin C5a. The pre-contact neutrophil response to these targets was generally vigorous but short range, consistent with our analytical solution of a theoretical diffusion-reaction problem that takes into account the deactivation of anaphylatoxins by carboxypeptidases. Together, these findings establish complement-mediated chemotaxis as a universal homing mechanism by which chemotaxing immune cells implement a last-minute course correction toward bacterial, fungal, and model pathogens.

Release and characteristics of fungal fragments in various conditions

Intact spores and submicrometer size fragments are released from moldy building materials during growth and sporulation. It is unclear whether all fragments originate from fungal growth or if small pieces of building materials are also aerosolized as a result of microbial decomposition. In addition, particles may be formed through nucleation from secondary metabolites of fungi, such as microbial volatile organic compounds (MVOCs). In this study, we used the elemental composition of particles to characterize the origin of submicrometer fragments released from materials contaminated by fungi.Particles from three fungal species (Aspergillus versicolor, Cladosporium cladosporioides and Penicillium brevicompactum), grown on agar, wood and gypsum board were aerosolized using the Fungal Spore Source Strength Tester (FSSST) at three air velocities (5, 16 and 27m/s). Released spores (optical size, dp≥0.8μm) and fragments (dp≤0.8μm) were counted using direct-reading optical aerosol instruments. Particles were also collected on filters, and their morphology and elemental composition analyzed using scanning electron microscopes (SEMs) coupled with an Energy-Dispersive X-ray spectroscopy (EDX).Among the studied factors, air velocity resulted in the most consistent trends in the release of fungal particles. Total concentrations of both fragments and spores increased with an increase in air velocity for all species whereas fragment–spore (F/S) ratios decreased. EDX analysis showed common elements, such as C, O, Mg and Ca, for blank material samples and fungal growth. However, N and P were exclusive to the fungal growth, and therefore were used to differentiate biological fragments from non-biological ones. Our results indicated that majority of fragments contained N and P.Because we observed increased release of fragments with increased air velocities, nucleation of MVOCs was likely not a relevant process in the formation of fungal fragments. Based on elemental composition, most fragments originated from fungi, but also fragments from growth material were detected.

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

A Ca-alginate particle co-immobilized with Phanerochaete chrysosporium cells and the combined cross-linked enzyme aggregates from Trametes versicolor

For improving stability of immobilized white-rot fungus to treat various effluents, Phanerochaete chrysosporium cells and the combined cross-link enzyme aggregates (combi-CLEAs) prepared from Trametes versicolor were co-immobilized into the Ca-alginate gel particles in this paper. The activity yields of obtained combi-CLEAs were 42.7% for lignin peroxidases (LiPs), 31.4% for manganese peroxidases (MnPs) and 40.4% for laccase (Lac), respectively. And their specific activities were 30.2U/g as combi-CLEAs-LiPs, 9.5U/g as combi-CLEAs-MnPs and 28.4U/g as combi-CLEAs-Lac. Further, the present of the combi-CLEAs in the particles extremely improved their ability to degrade the dyes. Compared to the immobilized Ph. chrysosporium without the combi-CLEAs, the co-immobilized particles enhanced the decolorized rate of Acid Violet 7 (from 45.2% to 93.4%) and Basic Fuchsin (from 12.1% to 67.9%). In addition, the addition of the combi-CLEAs improved the adaptability of the white-rot fungal particles to adverse environmental conditions.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.