Antifungal drug resistance (click to expand)

Recent annual global estimates of fungal infections in humans have indicated 3M cases of chronic pulmonary aspergillosis, ~220K cases of cryptococcal meningitis associated with HIV/AIDS, ~700K cases of invasive candidiasis, ~500K cases of Pneumocystis jirovecii-caused pneumonia, ~250K cases of invasive aspergillosis, ~100K cases of disseminated histoplasmosis, more than 10M cases of fungal-triggered asthma and ~1M cases of fungal keratitis 1-3. Fungal diseases have been leading to major social and economic costs in food systems 4,5, pushing animal species to the brink of extinction 6,7, and are on the rise due to global warming 8. A restricted number of therapeutic options against human fungal infections are available, including polyene (amphotericin B), echinocandin and azole compounds 9. In agriculture, fungicides include ethyl benzimidazole carbamates, succinate dehydrogenase inhibitors, anilinopyrimidines, quinone outside inhibitors, morpholines and azoles 10. Antifungal drug resistance due to the long term exposure to fungicides and concomitant cellular adaptation occurs because of the acquisition of genetic instability, target modification, overexpression of efflux pump genes, detoxification by metabolic enzymes, or hot spot amino acid substitutions 11,12.

1 Brown, G. D. et al. Hidden killers: human fungal infections. Sci Transl Med 4, 165rv113 (2012).

2 Vallabhaneni, S., Mody, R. K., Walker, T. & Chiller, T. The Global Burden of Fungal Diseases. Infect Dis Clin North Am 30, 1-11 (2016).

3 Bongomin, F., Gago, S., Oladele, R. O. & Denning, D. W. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. J Fungi (Basel) 3 (2017).

4 Avery, S. V., Singleton, I., Magan, N. & Goldman, G. H. The fungal threat to global food security. Fungal Biol 123, 555-557 (2019).

5 Kettles, G. J. & Luna, E. Food security in 2044: How do we control the fungal threat? Fungal Biol 123, 558-564 (2019).

6 Scheele, B. C. et al. Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 363, 1459-1463 (2019).

7 Cameron, S. A. et al. Patterns of widespread decline in North American bumble bees. Proceedings of the National Academy of Sciences of the United States of America 108, 662-667 (2011).

8 Cavicchioli, R. et al. Scientists' warning to humanity: microorganisms and climate change. Nat Rev Microbiol 17, 569-586 (2019).

9 Nett, J. E. & Andes, D. R. Antifungal Agents: Spectrum of Activity, Pharmacology, and Clinical Indications. Infect Dis Clin North Am 30, 51-83 (2016).

10 Brauer, V. S. et al. Antifungal Agents in Agriculture: Friends and Foes of Public Health. Biomolecules 9, 521 (2019).

11 Perlin, D. S., Rautemaa-Richardson, R. & Alastruey-Izquierdo, A. The global problem of antifungal resistance: prevalence, mechanisms, and management. Lancet Infect Dis 17, e383-e392 (2017).

12 Shor, E. & Perlin, D. S. Coping with stress and the emergence of multidrug resistance in fungi. PLoS Pathog 11, e1004668 (2015).

Our project intends to explore the natural diversity in antifungal drug resistance using natural populations of the microbial eukaryote Neurospora crassa. We are also interested in studying transcription factors that regulate fungal sensitivity to drugs. We anticipate that our research will lead us to relevant data towards the understanding of antifungal drug resistance, a dramatic public health problem with worldwide ramifications.

Publications by Pedro Gonçalves and colleagues that are relevant to this project:

Goncalves, A. P., McCluskey, K., Glass, N. L. & Videira, A. The Fungal Cell Death Regulator czt-1 Is Allelic to acr-3. J Fungi (Basel) 5 (2019). Free full text available

Goncalves, A. P., Heller, J., Daskalov, A., Videira, A. & Glass, N. L. Regulated Forms of Cell Death in Fungi. Front Microbiol 8, 1837 (2017). Free full text available

Goncalves, A. P. et al. Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa. Biochim Biophys Acta 1847, 1064-1074 (2015). Free full text available

Goncalves, A. P. et al. Transcription profiling of the Neurospora crassa response to a group of synthetic (thio)xanthones and a natural acetophenone. Genom Data 4, 26-32 (2015). Free full text available

Goncalves, A. P. et al. Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death. J Cell Sci 127, 3817-3829 (2014). Free full text available

Goncalves, A. P., Hall, C., Kowbel, D. J., Glass, N. L. & Videira, A. CZT-1 is a novel transcription factor controlling cell death and natural drug resistance in Neurospora crassa. G3 (Bethesda) 4, 1091-1102 (2014). Free full text available

Goncalves, A. P. et al. Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa. Microbial cell 1, 289-302 (2014). Free full text available

Fernandes, A. S. et al. Modulation of fungal sensitivity to staurosporine by targeting proteins identified by transcriptional profiling. Fungal genetics and biology : FG & B 48, 1130-1138 (2011). Free full text available

Evolution of fungal allorecognition (click to expand)

Sociality is not restricted to the so-called ‘higher organisms’. In some filamentous fungi, somatic cell-cell fusion typifies a cooperative behavior that is essential for the formation of the multicellular hyphal network 1,2. It is the syncytial nature of the fungal mycelium that allows these organisms to translocate nutrients and organelles across the colony and efficiently grow and colonize substrates 3; hence, it is unsurprising that somatic cell fusion is beneficial for fitness in Neurospora crassa 4. Intercellular cooperation in N. crassa depends greatly on genetic similarity and conflict can arise from inopportune fusion between genetically nonidentical partners due to the transmission of infectious elements, assimilation of harmful genotypes or selection of cheaters, individuals that gain the benefit of social traits (e.g., enjoy public goods such as nutrients) while contributing less to their production 5. As microbes are evidently unable to use visual, olfactory or tactile cues to recognize their kin and socially evaluate potential fusion partners, cooperation versus competition is dictated by genetic relatedness at certain genomic loci, a phenomenon known as allorecognition. In multicellular colonies, hyphal fusion of strains carrying dissimilar het loci triggers a rapid response that leads to cell death 6. Importantly, this allorecognition reaction, known as heterokaryon incompatibility, is accompanied by septal plugging of the incompatible fusion compartment in a way that cell death is restricted to that particular area 6. This phenomenon has, thus, been portrayed as having an altruistic character, since part of the mycelium perishes for the benefit of the colonial community. Notably, heterokaryon incompatibility is inactive in germinated spores (germlings). However, genetic analyses of germling fusion in a wild population of N. crassa revealed that nonself recognition can occur at distance 7, during cell-cell adhesion 8 and after cytoplasmic mixing 9,10, resulting in various phenotypes that hinder the success of incompatible cell fusion events 11,12.

1 Glass, N. L., Rasmussen, C., Roca, M. G. & Read, N. D. Hyphal homing, fusion and mycelial interconnectedness. Trends Microbiol 12, 135-141 (2004).

2 Goncalves, A. P., Chow, K. M., Cea-Sanchez, S. & Glass, N. L. WHI-2 Regulates Intercellular Communication via a MAP Kinase Signaling Complex. Front Microbiol 10, 3162 (2019). Free full text available

3 Roper, M., Lee, C., Hickey, P. C. & Gladfelter, A. S. Life as a moving fluid: fate of cytoplasmic macromolecules in dynamic fungal syncytia. Curr Opin Microbiol 26, 116-122 (2015).

4 Aanen, D. K., Debets, A. J., de Visser, J. A. & Hoekstra, R. F. The social evolution of somatic fusion. BioEssays : news and reviews in molecular, cellular and developmental biology 30, 1193-1203 (2008).

5 Bastiaans, E., Debets, A. J. & Aanen, D. K. Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters. Nat Commun 7, 11435 (2016).

6 Goncalves, A. P., Heller, J., Daskalov, A., Videira, A. & Glass, N. L. Regulated Forms of Cell Death in Fungi. Front Microbiol 8, 1837 (2017). Free full text available

7 Heller, J. et al. Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote. PLoS Biol 14, e1002431 (2016).

8 Goncalves, A. P. et al. Allorecognition upon Fungal Cell-Cell Contact Determines Social Cooperation and Impacts the Acquisition of Multicellularity. Curr Biol 29, 3006-3017 e3003 (2019). Free full text available

9 Heller, J., Clave, C., Gladieux, P., Saupe, S. J. & Glass, N. L. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi. Proceedings of the National Academy of Sciences of the United States of America 115, E2292-E2301 (2018).

10 Daskalov, A., Gladieux, P., Heller, J. & Glass, N. L. Programmed Cell Death in Neurospora crassa Is Controlled by the Allorecognition Determinant rcd-1. Genetics 213, 1387-1400 (2019).

11 Gonçalves, A. P. et al. Conflict, Competition, and Cooperation Regulate Social Interactions in Filamentous Fungi. Annu Rev Microbiol 74 (2020). Full text

12 Goncalves, A. P. & Glass, N. L. Fungal social barriers: to fuse, or not to fuse, that is the question. Commun Integr Biol 13, 39-42 (2020). Free full text available

Since all of these allorecognition checkpoints are active and suppressed during asexual and sexual reproduction, respectively, our hypothesis is that genetic diversification in N. crassa (and potentially other heterothallic fungi) occurs preferentially via outbreeding. Our laboratory aims to analyze the evolution of fungal self/nonself recognition by undertaking an in silico approach. We intend to integrate our findings in the context of species within the Neurospora genus and their diverse sexual and asexual lifestyles.

Fungal biotechnology (click to expand)

While a number of fungal pathogens can lead to devastating consequences to plants, humans and other animals 1, many fungi play a major beneficial role in the bioeconomy 2,3. Firstly, some fungi are the base of multiple food products that rely on fermentative processes, including bread, beer, wine, cheese, soy sauce, tempeh and stinky tofu, while other species may be directly eaten -- the edible mushroom industry was worth USD 50 billion in 2019 and is projected to continue on an expansion trend 4. Furthermore, several features of some fungal species, such as the vigorous growth on inexpensive substrates, amenability for genetic engineering and large-scale production in submerged fermentations, and intrinsic ability to secrete large amounts of protein directly into the culture medium, has turned them into useful instruments for protein and chemical production. This includes essential compounds such as citric acid, β-lactam antibiotics, statins, a wide range of industrially-relevant enzymes and even some antifungals 2,3. Fungi used in this manner, for research or industrial purposes, include Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa, Saccharomyces cerevisiae, Pichia pastoris, and others 5.

1 Fisher, M. C. et al. Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture. mBio 11, e00449-20 (2020).

2 Hyde, K. D. et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Diversity 9, 1-136 (2019).

3 Meyer, V. et al. Growing a circular economy with fungal biotechnology: a white paper. Fungal Biol Biotechnol 7, 5 (2020).

4 Mushroom Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast 2020-2025. 1-108 (Research and Markets, 2020).

5 Cairns, T. C., Zheng, X., Zheng, P., Sun, J. & Meyer, V. Moulding the mould: understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories. Biotechnology for Biofuels 12 (2019).

Fungal biotechnology has come a long way in the last 20 years, propelled by the development of novel tools and resources. In our group, we are interested in applying an integrated approach encompassing genetics, molecular biology and bioinformatics to develop a sustainable and innovative mode of production for certain molecules currently used in folk medicine.