Fungal Carbohydrate Research Articles

Structural, Biochemical, and Phylogenetic Analysis of Bacterial and Fungal Carbohydrate Esterase Family 15 Glucuronoyl Esterases in the Rumen

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Study of fungal cell wall evolution through its monosaccharide composition: An insight into fungal species interacting with plants

Every fungal cell is encapsulated in a cell wall, essential for cell viability, morphogenesis, and pathogenesis. Most knowledge of the cell wall composition in fungi has focused on ascomycetes, especially human pathogens, but considerably less is known about early divergent fungal groups, such as species in the Zoopagomycota and Mucoromycota phyla. To shed light on evolutionary changes in the fungal cell wall, we studied the monosaccharide composition of the cell wall of 18 species including early diverging fungi and species in the Basidiomycota and Ascomycota phyla with a focus on those with pathogenic lifestyles and interactions with plants. Our data revealed that chitin is the most characteristic component of the fungal cell wall, and was found to be in a higher proportion in the early divergent groups. The Mucoromycota species possess few glucans, but instead have other monosaccharides such as fucose and glucuronic acid that are almost exclusively found in their cell walls. Additionally, we observed that hexoses (glucose, mannose and galactose) accumulate in much higher proportions in species belonging to Dikarya. Our data demonstrate a clear relationship between phylogenetic position and fungal cell wall carbohydrate composition and lay the foundation for a better understanding of their evolution and their role in plant interactions.

Pseudomonas aeruginosa increases the susceptibility of Candida albicans to amphotericin B in dual-species biofilms.

Biofilms are the leading cause of nosocomial infections and are hard to eradicate due to their inherent antimicrobial resistance. Candida albicans is the leading cause of nosocomial fungal infections and is frequently co-isolated with the bacterium Pseudomonas aeruginosa from biofilms in the cystic fibrosis lung and severe burn wounds. The presence of C. albicans in multispecies biofilms is associated with enhanced antibacterial resistance, which is largely mediated through fungal extracellular carbohydrates sequestering the antibiotics. However, significantly less is known regarding the impact of polymicrobial biofilms on antifungal resistance. Here we show that, in dual-species biofilms, P. aeruginosa enhances the susceptibility of C. albicans to amphotericin B, an effect that was biofilm specific. Transcriptional analysis combined with gene ontology enrichment analysis identified several C. albicans processes associated with oxidative stress to be differentially regulated in dual-species biofilms, suggesting that P. aeruginosa exerts oxidative stress on C. albicans, likely through the secretion of phenazines. However, the mitochondrial superoxide dismutase SOD2 was significantly down-regulated in the presence of P. aeruginosa. Monospecies biofilms of the sod2Δ mutant were more susceptible to amphotericin B, and the susceptibility of these biofilms was further enhanced by exogenous phenazines. We propose that in dual-species biofilms, P. aeruginosa simultaneously induces mitochondrial oxidative stress, while down-regulating key detoxification enzymes, which prevents C. albicans mounting an appropriate oxidative stress response to amphotericin B, leading to fungal cell death. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Beauveria bassiana Growth and Development in Various Liquid Media

Beauveria bassiana is entomopathogenic fungi that can be cultivated on solid or liquid media. The cultivation of B. bassiana on cracked corn medium leaves unwanted waste. Therefore, cultivating B. bassiana on liquid media is expected to shorten the cultivation time and leave no waste. The purpose of the research was to analyze the physical characteristics of the B. bassiana colony, nutritional changes of the liquid media, and the pH level changes of the liquid media. This research used an experimental complete randomized design with six treatments and 4 times repetitions. The main phases of this research were the subculture of B. bassiana isolate; the preparation of liquid media consisting of distilled water, Potato Dextrose Broth (PDB), Potato Sugar extract (PSE), PSE+NPK 1%, PSE+coconut water, PSE+NPK 1%+coconut water each for 150 ml; B. bassisana inoculation into the liquid media; incubation process for 30 days; and measurement of fungal blastospore density, medium pH, and carbohydrate, protein, and lipid content. The results obtained from the morphological observation of B.bassiana on liquid media showed that the colonies are white, growing on the surface of the media with a powdery texture. There are some differences in the nutrient content of the media after 30 days of B. Bassiana incubation. After the incubation, there is an increase in carbohydrates and a decrease in protein and lipid content, as well as an increase in the pH level of the media.

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.

Source files of the Carbohydrate Structure Database: the way to sophisticated analysis of natural glycans

The Carbohydrate Structure Database (CSDB, http://csdb.glycoscience.ru/) is a free curated repository storing various data on glycans of bacterial, fungal and plant origins. Currently, it maintains a close-to-full coverage on bacterial and fungal carbohydrates up to the year 2020. The CSDB web-interface provides free access to the database content and dedicated tools. Still, the number of these tools and the types of the corresponding analyses is limited, whereas the database itself contains data that can be used in a broader scope of analytical studies. In this paper, we present CSDB source data files and a self-contained SQL dump, and exemplify their possible application in glycan-related studies. By using CSDB in an SQL format, the user can gain access to the chain length distribution or charge distribution (as an example) in a given set of glycans defined according to specific structural, taxonomic, or other parameters, whereas the source text dump files can be imported to any dedicated database with a specific internal architecture differing from that of CSDB.

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases.

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

The yapsin family of aspartyl proteases regulate glucose homeostasis in Candida glabrata

Invasive candidiasis poses a major healthcare threat. The human opportunistic fungal pathogen Candida glabrata, which causes mucosal and deep-seated infections, is armed with distinct virulence attributes, including a family of 11 glycosylphosphatidylinositol-linked aspartyl proteases, CgYapsins. Here, we have profiled total membrane proteomes of the C. glabrata wildtype and 11 proteases-deficient strain, Cgyps1-11Δ, by mass spectrometry analysis and uncovered a novel role for fungal yapsins in glucose sensing and homeostasis. Furthermore, through label-free quantitative membrane proteome analysis, we showed differential abundance of 42% of identified membrane proteins, with electron transport chain and glycolysis proteins displaying lower and higher abundance in Cgyps1-11Δ cells, compared with wildtype cells, respectively. We also demonstrated elevated glucose uptake and upregulation of genes that code for the low-glucose sensor CgSnf3, transcriptional regulators CgMig1 and CgRgt1, and hexose transporter CgHxt2/10 in the Cgyps1-11Δ mutant. We further elucidated a potential underlying mechanism through genetic and transcript measurement analysis under low- and high-glucose conditions and found CgSNF3 deletion to rescue high glucose uptake and attenuated growth of the Cgyps1-11Δ mutant in YPD medium, thereby linking CgYapsins with regulation of the CgSnf3-dependent low-glucose sensing pathway. Last, high ethanol production, diminished mitochondrial membrane potential, and elevated susceptibility to oxidative phosphorylation inhibitors point toward increased fermentative and decreased respiratory metabolism in the Cgyps1-11Δ mutant. Altogether, our findings revealed new possible glucose metabolism-regulatory roles for putative cell surface-associated CgYapsins and advanced our understanding of fungal carbohydrate homeostasis mechanisms.

Integrative Study Supports the Role of Trehalose in Carbon Transfer From Fungi to Mycotrophic Orchid.

Orchids rely on mycorrhizal symbiosis, especially in the stage of mycoheterotrophic protocorms, which depend on carbon and energy supply from fungi. The transfer of carbon from fungi to orchids is well-documented, but the identity of compounds ensuring this transfer remains elusive. Some evidence has been obtained for the role of amino acids, but there is also vague and neglected evidence for the role of soluble carbohydrates, probably trehalose, which is an abundant fungal carbohydrate. We therefore focused on the possible role of trehalose in carbon and energy transfer. We investigated the common marsh orchid (Dactylorhiza majalis) and its symbiotic fungus Ceratobasidium sp. using a combination of cultivation approaches, high-performance liquid chromatography, application of a specific inhibitor of the enzyme trehalase, and histochemical localization of trehalase activity. We found that axenically grown orchid protocorms possess an efficient, trehalase-dependent, metabolic pathway for utilizing exogenous trehalose, which can be as good a source of carbon and energy as their major endogenous soluble carbohydrates. This is in contrast to non-orchid plants that cannot utilize trehalose to such an extent. In symbiotically grown protocorms and roots of adult orchids, trehalase activity was tightly colocalized with mycorrhizal structures indicating its pronounced role in the mycorrhizal interface. Inhibition of trehalase activity arrested the growth of both symbiotically grown protocorms and trehalose-supported axenic protocorms. Since trehalose constitutes only an inconsiderable part of the endogenous saccharide spectrum of orchids, degradation of fungal trehalose likely takes place in orchid mycorrhiza. Our results strongly support the neglected view of the fungal trehalose, or the glucose produced by its cleavage as compounds transported from fungi to orchids to ensure carbon and energy flow. Therefore, we suggest that not only amino acids, but also soluble carbohydrates are transported. We may propose that the soluble carbohydrates would be a better source of energy for plant metabolism than amino acids, which is partially supported by our finding of the essential role of trehalase.

A "Bridge-Building" Glycan Scaffold Mimicking Microbial Invasion for In Situ Endothelialization.

The globally high prevalence of peripheral artery diseases poses a pressing need for biomaterials grafts to rebuild vasculature. When implanted, they should promote endothelial cells (ECs) adhesion both profoundly and selectively-but the latter expectation remains unfulfilled. Here, this work is inspired by fungi that invade blood vessels via the "bridge" of galectins that, secreted by ECs, can simultaneously bind carbohydrates on fungal surface and integrin receptors on ECs. A glucomannan decanoate (GMDE) substrate mimicking fungal carbohydrates that highly and preferentially supports ECs adhesion while rejecting several other cell types is designed. Electrospun GMDE scaffolds efficiently sequester endogenous galectin-1-which bridges ECs to the scaffolds as it functions in fungal invasions-and promote blood perfusion in a murine limb ischemic model. Meanwhile, the application of GMDE requires no exogenous pro-angiogenic agents and causes no organ toxicity or adverse inflammation in mice, highlighting its high safety of potential translation. This glycan material, uniquely mimicking a microbial action and harnessing a secreted protein as a "bridge," represents an effective, safe, and different strategy for ischemic vascular therapy.

Anti-glycan antibodies: roles in human disease.

Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.

Cinnamon Oil Inhibits Penicillium expansum Growth by Disturbing the Carbohydrate Metabolic Process.

Penicillium expansum is a major postharvest pathogen that mainly threatens the global pome fruit industry and causes great economic losses annually. In the present study, the antifungal effects and potential mechanism of cinnamon oil against P. expansum were investigated. Results indicated that 0.25 mg L−1 cinnamon oil could efficiently inhibit the spore germination, conidial production, mycelial accumulation, and expansion of P. expansum. In addition, it could effectively control blue mold rots induced by P. expansum in apples. Cinnamon oil could also reduce the expression of genes involved in patulin biosynthesis. Through a proteomic quantitative analysis, a total of 146 differentially expressed proteins (DEPs) involved in the carbohydrate metabolic process, most of which were down-regulated, were noticed for their large number and functional significance. Meanwhile, the expressions of 14 candidate genes corresponding to DEPs and the activities of six key regulatory enzymes (involving in cellulose hydrolyzation, Krebs circle, glycolysis, and pentose phosphate pathway) showed a similar trend in protein levels. In addition, extracellular carbohydrate consumption, intracellular carbohydrate accumulation, and ATP production of P. expansum under cinnamon oil stress were significantly decreased. Basing on the correlated and mutually authenticated results, we speculated that disturbing the fungal carbohydrate metabolic process would be partly responsible for the inhibitory effects of cinnamon oil on P. expansum growth. The findings would provide new insights into the antimicrobial mode of cinnamon oil.

Combined and single effects of elevated CO2 and temperatures on rice bakanae disease under controlled conditions in phytotrons

AbstractBakanae disease, caused by Fusarium fujikuroi, was investigated under different CO2 and temperature environments in order to simulate climate changes in the F. fujikuroi–rice pathosystem. F. fujikuroi‐infected plants were grown under six phytotron conditions: low (18/22 °C night/day), medium (22/26 °C), and high (26/30 °C) temperature, at either ambient (450 ppm) or elevated (850 ppm) CO2 concentrations. Bakanae disease index (DI), seedling death incidence, fungal DNA quantity, and chlorophyll and carbohydrate contents varied significantly in infected plants as a consequence of changes in both CO2 and temperature. Plant height and dry weight were only influenced by single factors (temperature for height, and temperature or CO2 for dry weight), and not by the CO2 × temperature interaction. Medium and high temperatures (irrespective of the CO2 level) increased the DI significantly (range from 67.5% to 95.8%) compared to low temperatures (range from 45.8% to 47.5%). Under elevated CO2 levels, noticeable differences in the expression of four rice defence‐related genes and fungal DNA quantity were observed between those plants grown at higher temperatures and those at lower temperatures. Overall, combined and single effects of elevated CO2 and high temperatures seem to be favourable for bakanae disease development in the Mediterranean basin.

Quantifying Receptor-Mediated Phagocytosis and Inflammatory Responses to Fungi in Immune Cells.

Phagocytosis and cytokine production are important processes by which innate immune cells, especially professional phagocytes such as neutrophils and macrophages, control and regulate immunity to fungi. These cellular responses are initiated when conserved pathogen components, known as pathogen-associated molecular patterns (PAMPs), are recognized by pattern-recognition receptors (PRRs), which include members of the C-type lectin receptor (CLR) family that are able to bind to fungal cell wall-derived carbohydrates. Phagocytosis and cytokine production can be quantitatively examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, using in vitro based assays with primary-derived murine cells and cell lines. Here, we describe a flow cytometry-based method using transduced cell lines to assess the ability of CLRs to mediate internalization, using A. fumigatus conidia and the β-1,3 glucan receptor, Dectin-1 (CLEC7A), as an example. The use of ELISA-based assays to measure cytokine production by immune cells that are induced in response to fungi and methods for isolating and culturing primary macrophages from various murine tissues are described.

Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines.

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-d-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.

Abstract IA13: Regulation of pancreatic oncogenesis by pathogens

Abstract Pancreatic ductal adenocarcinoma (PDA) is a devastating disease in which the mortality rate approaches the incidence rate. PDA is characterized by immune suppression. Specifically, the PDA tumor microenvironment in the majority of patients and in animal models of disease is associated with a modest T-cell infiltrate, dominated by Th2- and Treg-polarized CD4+ T cells and a paucity of cytotoxic CD8+ T cells. We and others have shown that the tolerogenic T-cell environment in PDA is programmed by suppressive myeloid cell elements. M2-polarized tumor-associated macrophages (TAMs) promote the differentiation of immune-suppressive Th2 cells and Tregs. Further, myeloid-derived suppressor cells (MDSC) veto cytotoxic CD8+ T-cell responses. As a consequence of the resultant immune anergy, immunotherapy has not been efficacious in this disease. Underlying all this, we found that the suppressive myeloid cell programming in PDA is dictated by the distinctive PDA-associated microbiome, which promotes accumulation of MDSC and M2-polarized TAMs via the release of bacterial-derived motifs that ligate diverse pattern recognition receptors in myeloid cells, leading to tumor-promoting myeloid cell differentiation and inflammation, which leads to adaptive immune collapse. In parallel to our interest in the role of the bacterial microbiome in oncogenesis, we developed an interest in the role of the fungal mycobiome. As we became interested in characterizing immune-modulatory sterile ligands for C-type lectin receptors (CLRs), which are cousins to TLRs, we identified Galectin-9 as a binding partner for Dectin-1 and SAP130 as a binding partner for Mincle in the tumor microenvironment. While our identification of sterile ligands for CLRs in cancer is interesting, the “natural” ligands for CLRs are fungal wall carbohydrate polymers. As such, we postulated that fungi may modulate the intratumoral inflammation and influence oncogenesis. We found that human pancreatic tumors and mouse models of this cancer displayed an increase in fungi of about 3,000-fold compared to normal pancreatic tissue. The composition of the mycobiome of PDA tumors was distinct from that of the gut or normal pancreas. Specifically, the fungal community that infiltrated PDA tumors was markedly enriched for Malassezia spp. in both mice and humans. Ablation of the mycobiome was protective against tumor growth, whereas repopulation with a Malassezia species—but not other species—accelerated oncogenesis. We also discovered that ligation of the CLR mannose-binding lectin (MBL), which binds to glycans of the fungal wall to activate the complement cascade, was required for oncogenic progression, whereas deletion of MBL or C3 in the extratumoral compartment—and knockdown of C3aR in tumor cells—were both protective against tumor growth. Collectively, our work revealed that pathogenic fungi promote PDA by driving the complement cascade through the activation of MBL. Citation Format: George Miller. Regulation of pancreatic oncogenesis by pathogens [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA13.

Genomic and proteomic biases inform metabolic engineering strategies for anaerobic fungi

Anaerobic fungi (Neocallimastigomycota) are emerging non-model hosts for biotechnology due to their wealth of biomass-degrading enzymes, yet tools to engineer these fungi have not yet been established. Here, we show that the anaerobic gut fungi have the most GC depleted genomes among 443 sequenced organisms in the fungal kingdom, which has ramifications for heterologous expression of genes as well as for emerging CRISPR-based genome engineering approaches. Comparative genomic analyses suggest that anaerobic fungi may contain cellular machinery to aid in sexual reproduction, yet a complete mating pathway was not identified. Predicted proteomes of the anaerobic fungi also contain an unusually large fraction of proteins with homopolymeric amino acid runs consisting of five or more identical consecutive amino acids. In particular, threonine runs are especially enriched in anaerobic fungal carbohydrate active enzymes (CAZymes) and this, together with a high abundance of predicted N-glycosylation motifs, suggests that gut fungal CAZymes are heavily glycosylated, which may impact heterologous production of these biotechnologically useful enzymes. Finally, we present a codon optimization strategy to aid in the development of genetic engineering tools tailored to these early-branching anaerobic fungi.

CD4+ T Cell Regulation of Antibodies Cross-Reactive with Fungal Cell Wall-Associated Carbohydrates after Pneumocystis murina Infection.

Pneumocystis pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. Molecules with the ability to bind β-glucan and signal at Fcγ receptors enhance defense against Pneumocystis f. sp. murina, though it is unclear whether antibodies reactive with fungal cell wall carbohydrates are induced during Pneumocystis infection. We observed that systemic and lung mucosal immunoglobulins cross-reactive with β-glucan and chitosan/chitin are generated after Pneumocystis infection, with increased quantities within the lung mucosal fluid after challenge. While IgG responses against Pneumocystis protein antigens are markedly CD4+ T cell dependent, CD4+ T cell depletion did not impact quantities of IgG cross-reactive with β-glucan or chitosan/chitin in the serum or mucosa after challenge. Notably, lung mucosal quantities of IgA cross-reactive with β-glucan or chitosan/chitin are decreased in the setting of CD4+ T cell deficiency, occurring in the setting of concurrent reduced quantities of active transforming growth factor β, while mucosal IgM is significantly increased in the setting of CD4+ T cell deficiency. Interleukin-21 receptor deficiency does not lead to reduction in mucosal IgA reactive with fungal carbohydrate antigens after Pneumocystis challenge. These studies demonstrate differential CD4+ T cell-dependent regulation of mucosal antibody responses against β-glucan and chitosan/chitin after Pneumocystis challenge, suggesting that different B cell subsets may be responsible for the generation of these antibody responses, and suggest a potential immune response against fungi that may be operative in the setting of CD4+ T cell-related immunodeficiency.

Tetraspanin CD82 Organizes Dectin-1 into Signaling Domains to Mediate Cellular Responses to Candida albicans.

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.