Secretion of virulence factors by the fungal pathogen Cryptococcus neoformans
Final Report Abstract
The fungal pathogen Cryptococcus neoformans is a major driver of fungal-associated deaths in humans. C. neoformans is acquired from the environment by inhalation of spores or desiccated yeast cells and establishes an infection in the host lungs. In persons with strongly impaired immune systems, like, e.g. in HIV/AIDS-patients, the fungus disseminates from the lung tissue to the brain where it causes severe infections (meningoencephalitis), ultimately leading to organ failure and death. The current anti-cryptococcal armamentarium is scarce, relying on just a handful of drugs that need to be used in combination, display considerable toxicity to human host cells and require lifelong administration. Hence, understanding the biology of this major human pathogen, and specifically the virulence mechanisms that are important for infection, is crucial in order to identify novel drug targets that may lead to the development of new, potent, and ideally less host cell cytotoxic antifungal drugs. Three main virulence factors have been described in C. neoformans: production of a polysaccharide capsule which protects the fungus from the attack of host immune cells, synthesis of the black anti-oxidant pigment melanin, and more generally, the capacity to grow at human body temperature. The exact mechanisms of how the capsule polysaccharide components are translocated from the inner cell compartment to the outside during capsule biosynthesis has remained elusive. However, a specialized secretion system is likely to be involved in this process. The cAMP/PKA nutrient sensing pathway was identified several years ago as a crucial regulatory axis of capsule elaboration. Deletion of the key protein kinase A gene PKA1 resulted in a cryptococcal mutant unable to form capsule. This mutant was also shown to be hypersensitive towards elevated concentrations of lithium chloride. Indeed, lithium has recently been shown to directly inhibit the cAMP/PKA pathway in yeast and human cells. Furthermore, it has previously been shown that lithium inhibits capsule formation in wild type (Wt) C. neoformans cells. Based on these observations, it was hypothesized that identifying novel C. neoformans mutants with similarly enhanced sensitivity to lithium as the pka1Δ mutant may reveal novel factors contributing to capsule formation. In this project, a total of 3,401 C. neoformans mutant strains were screened for enhanced sensitivity towards lithium chloride (LiCl). The results established a role for seven new factors required for Wt LiCl-tolerance and for normal capsule formation and/or melanin biosynthesis. One of the factors, Sec5, for example, is part of the exocyst complex, a specialized machinery within cells which mediates docking of intracellular vesicles (which may contain capsule polysaccharides) with the plasma membrane. These newly discovered proteins contributing to C. neoformans virulence factor elaboration may represent promising novel targets for treatment of cryptococcosis. Furthermore, a potential role of the endoplasmic reticulum - plasma membrane (ER-PM) tether machinery in capsule biosynthesis was identified. One of the targets of the cAMP/PKA pathway is the mannoprotein Ova1 which negatively affects capsule size. As part of this project, a total of 20,000 Agrobacterium tumefaciens TDNA insertion mutants were constructed in an ova1Δ mutant background. Screening of this large collection for mutants with altered capsule size and/or lithium chloride sensitivity has been initiated and may reveal novel PKA-regulated target proteins. Unlike pathogens obligately associated with the host, the natural habitat of C. neoformans and the related species C. gattii is the environment. Both fungi primarily occur in soil, on trees and plants, and in bird droppings. In a further project, it was hence hypothesized that natural microbes co-existing in these natural cryptococcal habitats may have evolved anti-fungal mechanisms for spatial and nutritional competition. Following isolation of a set of 60 environmental microbes, a gram-positive bacterium was found to specifically inhibit capsule and melanin formation in C. neoformans, while only moderately affecting overall fungal growth. Dissection of the mechanism-of-action through inter-kingdom co-cultivation experiments and genetic screens established the fungal cell wall as a likely target. These results may lead to the development of targeted anti-virulence strategies and/or products for the treatment of soils with high Cryptococcus prevalence using live bacteria.
Publications
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28th Fungal Genetics Conference, Pacific Grove, CA, USA, March 17-22, 2015. Screening mutants for enhanced lithium sensitivity identifies novel factors contributing to capsule elaboration and melanization in Cryptococcus neoformans
François Mayer
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13th European Conference on Fungal Genetics, Paris, France, April 3-6, 2016. Natural Bacterium-Mediated Repression of Virulence Factor Production by Human Fungal Pathogens
François Mayer
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Networks of fibers and factors: regulation of capsule formation in Cryptococcus neoformans. F1000Res, Vol. 5. 2016, F1000 Faculty Rev-1786.
Ding H., Mayer F.L., Sánchez-León E., de S. Araújo G.R., Frases S., Kronstad J.W.
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Disarming Fungal Pathogens: Bacillus safensis Inhibits Virulence Factor Production and Biofilm Formation by Cryptococcus neoformans and Candida albicans. mBio, vol. 8. 2017, no. 5: e01537-17.
Mayer FL & Kronstad JW
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Discovery of a Novel Antifungal Agent in the Pathogen Box. mSphere, Vol. 2. 2017,Issue 2: e00120-17.
Mayer F.L., Kronstad J.W.
