Rim Pathway-Mediated Alterations in the Fungal Cell Wall Influence Immune Recognition and Inflammation

ACKNOWLEDGMENTS We acknowledge Jennifer Lodge, Woei Lam, and Rajendra Upadhya for developing and sharing the chitin and chitosan MTBH assay. We thank Todd Brennan of Duke University for providing MyD88-deficient mice. We acknowledge Neil Gow for providing access to the Dionex HPAEC-PAD instrumentation. We also acknowledge Connie Nichols for critical reading of the manuscript. These experiments were supported by an NIH grant to J.A.A. and F.L.W., Jr. (R01 AI074677). C.M.L.W. was supported by a fellowship provided through the Army Research Office of the Department of Defense (no. W911NF-11-1-0136 f) (F.L.W., Jr.). J.W., L.W., and C.M. were supported by the Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377) and the MRC, Centre for Medical Mycology (MR/N006364/1). FUNDING INFORMATION MRC Centre for Medical MycologyMR/N006364/1 Carol A. Munro HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060R01 AI074677J. Andrew Alspaugh Wellcome https://doi.org/10.13039/100010269097377 Carol A. Munro DOD | United States Army | RDECOM | Army Research Office (ARO) https://doi.org/10.13039/100000183W911NF-11-1-0136 f Chrissy M. Leopold WagerPeer reviewe

Relationship between mycelial morphology, cell wall composition and product formation of rhizopus arrhizus

Pelleting of hyphae of Rhizopus arrhizus commences 9-10 h after inoculation, and not at a pregermination stage as for other fungi. The anionic polymers carboxymethylcellulose (CMC) and Carbopol-934 prevent pelleting, causing dispersal of growth of most species of Rhizopus, including R. arrhizus, when used as media supplements. The non-ionic polymer methylcellulose also dispersed growth, but less effectively. The specific growth rate of mycelia in control (unsupplemented) and in polymer-supplemented media was similar until 24 h, when growth became restricted in control medium. Polymer supplementation promoted increased biomass production, but in the case of Carbopol, this increase may be partly due to the tightly bound polymer, as indicated by scanning electron microscopy and biomass studies, particularly at low pH's. Hyphal extension zones and branch points of mycelia from Carbopol-supplemented media at low pH did not fluoresce when treated with Calcofluor White, unlike those from control, CMC- or Carbopol-supplemented media at higher pH values. The cell walls of dispersed mycelia from viscous media had a higher concentration of hexosamines (34-35%) and a lower concentration of protein (7-6%) compared to the cell walls of pelleted (24% hexosamines/18% protein) or less finely dispersed mycelia (23% hexosamines/13% protein). Results from acid/alkali extraction indicate that a greater proportion of glucuronan is held in non-glucosamine linkages in walls from pelleted than from dispersed mycelia. The analysis of the chitin component of the cell walls by X-ray diffraction demonstrated less crystallinity in the cell walls isolated from Carbopol-supplemented media compared to the other cell wall types examined. The presence of solid or semi-solid medium components were necessary to promote production of glucoamylase by R. arrhizus. The presence of Carbopol inhibited glucoamylase production. It was also shown to inhibit fumaric acid production when mycelia were grown in' its presence and when used in the biotransformation media using pre-grown mycelia. Dispersed filamentous mycelia from CMC-supplemented medium produced the highest yields of fumaric acid in batch culture and in biotransformation studies. However in pH controlled fermenters, where higher aeration/agitation rates prevented densley-packed pelleting, the control mycelia produced the highest yields. Dispersed mycelia from CMC-supplemented medium also had the highest specific capacity to convert progesterone to 1la-hydroxyprogesterone. 48 h congealed mycelia from control medium manifested a low metabolic rate and poor rates of biotransformation

Effects of Quorum Quenchers on Aspergillus fumigatus Conidia Aggregation, Adhesion to Surfaces, and Biofilm Formation

Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with biofilm features under static growth conditions. Microbial quorum sensing (QS) system regulates genetic competence and biofilm formation. Notwithstanding, triclosan, as a synthetic antimicrobial, disrupts QS signal in some bacteria, yeasts, and dermatophytes through blocking the biosynthesis of amino acid and fatty acids in the microbes. In this study, triclosan quorum quenching role as well as the effects of microbial quorum quenchers, furanone, farnesol and tyrosol were investigated against A. fumigatus for the first time. Amphotericin B (AMB), a common antifungal agent used against A. fumigatus infection, carries adverse effects against human health. So, triclosan’s antifungal effect in combination with AMB was also studied. Microbial cells’ detrimental attachment and biofilm formation on the indwelling medical implants, healthcare and hospital facilities, prompts interests to introduce feasible and cost-effective methods for eliminating/reducing microbial attachment to the surfaces. Using water contact angle (WCA) measurements, hydrophobicity of some hydrophilic surfaces (glass, acrylic, HDPE, nylon 6 and UPVC), and hydrophobic surfaces (PTFE and silicone) was measured following coating them with the mentioned antimicrobial agents. A. fumigatus conidia were exposed to the agents and the subsequent changes in the conidial viability, biofilm biomass production, total and EPS-related protein/ polysaccharide/ and nucleic acid were studied. The effect of AMB was weaker than the other agents on mitigating the biofilm formation. eDNA release was observed from A. fumigatus mycelia treated with triclosan, tyrosol and farnesol as compared with untreated control group. This eDNA release appeared to be due to necrosis occurrence in the treated samples verified by gel electrophoresis and protein quantity analysis. As a novel finding, the necrosis induced by triclosan followed by apoptosis induced by AMB resulted in a synergistic interaction to reduce the conidia viability even at the sub-MIC doses of triclosan and AMB. Confocal microscopy of the A. fumigatus exposed to triclosan-AMB combination confirmed the synergistic interaction between them against the fungus biofilm. Assaying for hydrophobicity of the conidia cell wall revealed a change from hydrophobic to hydrophilic property. ags3 down-regulation approved with real-time PCR assay showed triclosan’s quorum sensing inhibitory role against A. fumigatus. This was through inhibiting conidia aggregation, and hence prohibiting initiation of quorum sensing signalling pathway. Through SDS-PAGE assay it was established that the conidia cell wall hydrophobic rodlet layer was absent in farnesol-treated sample. This study revealed some of the physio-chemical properties that are involved in A. fumigatus conidia attachment to the surfaces. This work for the first time established that hydrophobic surfaces, PTFE and silicone, coated with farnesol showed hydrophilicity, while AMB changed UPVC surface charge from hydrophilic to hydrophobic. Taken together, besides influencing the conidia cell wall hydrophobicity, farnesol and AMB can also be used to coat the surfaces in clinical healthcare settings to diminish hydrophobic/hydrophilic microbial cells attachment to the surfaces

Fungi exploit natural defence to induce neuronal and glial loss in Drosophila brain

Parasites have evolved behaviour manipulation strategies to exploit their host for nutrition and reproduction. In Drosophila, the fungus Enthomophthora muscae affects the nervous system altering behaviour (Carolyn et al., 2018). Furthermore, volatile compounds produced by fungi can cause dopaminergic neuron loss and locomotion deficits, reminiscent of Parkinsonism (Inamdar et al., 2013). In humans, genetics accounts for a small fraction of Parkinson’s disease onset and environmental triggers are likely a common cause. How fungi affect the brain and the causal links between cellular phenotypes and behaviour are poorly understood. Innate immune response is the first line of defence against a pathogenic infection which is mediated by Toll signalling. Tolls are evolutionary conserved and have varied functions from immunity to regulation of cell number plasticity regulated via different pathways. The fungus Beauveria bassiana is a known activator of Toll signalling in Drosophila and Tolls are present throughout the brain. Thus, the aim of the thesis is to investigate whether B. bassiana alters the fruit-fly brain by exploiting Toll signalling. Here, I provide evidence that upon natural exposure to B. bassiana, the spores enter the brain. B. bassiana spores could enter into the brain through feeding and damage the blood brain barrier. Exposure causes neuronal and glial loss, loss of the dopaminergic neurons and a decrease in brain size over time. Expression of anti-microbial peptides is upregulated in the brain upon B. bassiana exposure, demonstrating activation of Toll signalling. Importantly, expression of the pro-apoptotic Toll signalling adaptors sarm and wek (Foldi et al., 2017) is also upregulated. These neurodegeneration phenotypes correlate with impaired locomotion and shorter lifespan. Furthermore, upon downregulating the expression of Toll-1, wek or sarm, B. bassiana cannot induce cells and dopaminergic neurons loss in the adult Drosophila brain. This indicates the non-canonical Toll signalling Wek and Sarm could have a role in B.bassiana induced neuronal and glial cell loss in the adult Drosophila brain. Together these findings reveal that fungi exploited an evolutionary opportunity to bypass defence to their advantage

Microalgae for the biochemical conversion of CO2 and production of biodiesel

As the global population rises to an estimated 9.4bn by 2050, the pressure for food, fuel and freshwater will continue to increase. Current renewable energy technologies are not widely applicable to the transport sector, which requires energy dense liquid fuels that drop into our existing infrastructure. Algal biofuels promise significantly higher yields than plants, without the displacement of valuable agricultural resources and have the potential to meet the global demand for transport fuel. Fossil fuel energy is largely ‘a legacy of algal photosynthesis’, with algae accounting for ~50% of global CO2 fixation today. In addition, these curious organisms show remarkable diversity in form, behaviour and composition. Recently there has been a global resurgence of interest in microalgae as a resource of biomass and novel products. With the present level of technology, knowledge and experience in commercial scale aquaculture, the capital cost and energy investment for algal biomass production is high. Culturing, harvesting and disrupting microalgal cells account for the largest energy inputs with more positive energy balances requiring low energy designs for culture, dewatering and extraction, efficient water and nutrient recycling with minimal waste. Little is known about the variable cell wall of microalgae, which presents a formidable barrier to the extraction of microalgal products. Staining, transmission electron microscopy (TEM) and enzymatic digestion were all utilised in an attempt to visualise, digest and characterise the cell wall of stock strains of Chlorella spp. and Pseudochoricystis ellipsoidea. The presence of algaenan, a highly resistant biopolymer, rendered staining and enzymatic digestion techniques ineffective. TEM revealed that algaenan is present in the outer walls of microalgae in a variety of conformations which appeared to impart strength to cells. A preliminary investigation utilising Fusarium oxysporum f.sp. elaeidis as a novel source of enzymes for the digestion of algaenan has also been described. Methods were developed for the mutagenesis of Chlorella emersonii and P. ellipsoidea using EMS and UV with the intent of generating cell-wall mutants. Although no viable cell wall mutants were produced, a viable pale mutant of C. emersonii was recovered 5 from UV mutagenesis. Growth rates of the pale mutant were significantly slower than the wild type, yet FAME profile was largely unaffected. Fluorescence activated cell sorting (FACS) was also investigated as a means for the rapid screening of mutagenized cells for cell wall mutants. In an attempt to reduce cooling costs of closed-culture systems, temperature tolerant species of microalgae were sought by bioprospecting the thermal waters of the Roman Baths. Numerous methods for isolation and purification of microalgae from the Baths were employed, ultimately yielding seven diverse isolates including cyanobacterial, eukaryotic, filamentous and single celled species. Despite some species possessing an increased tolerance to higher temperatures, none showed marked temperature tolerance coupled with high productivity. Further improvements to the culture conditions may have improved the productivity at higher temperatures. All seven isolates were deposited to the Culture Collection of Algae and Protozoa (CCAP). A variety of extraction methods including soxhlet, beadbeating, sonication and microwaving was investigated for efficacy of extracting fatty acid methyl esters (FAMEs) from C. emersonii. Beadbeating proved most effective in the extraction of FAMEs from C. emersonii. Microwaving showed potential as a rapid method of extraction yet was coupled with degradation of FAMEs, requiring further method development to resolve this issue. Method development has been a significant component of the work described in this thesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo