Intracellular Action of a Secreted Peptide Required for Fungal Virulence

Homer, Christina M.; Summers, Diana; Goranov, Alexi I.; Clarke, Starlynn; Wiesner, Darin L.; Diedrich, Jolene K.; Moresco, James J.; Toffaletti, Dena L.; Upadhya, Rajendra; Caradonna, Ippolito; Petnic, Sarah; Pessino, Veronica; Cuomo, Christina A.; Lodge, Jennifer K.; Perfect, John R.; Yates, John R.; Nielsen, Kirsten; Craik, Charles S.; Madhani, Hiten D.

doi:10.1016/j.chom.2016.05.001

Cited by 87 publications

(123 citation statements)

References 72 publications

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“…Only one protein, CNAG_05312, had both an Nterminal signal peptide and a GPI-anchor indicative of plasma membrane association. These secreted peptides were previously reported to play critical roles in C. neoformans virulence (61)(62)(63)(64)(65). Taken together, these results revealed that C. neoformans melanin granules are indeed intimately associated with proteins that employ both a conventional and non-conventional secretory pathway for regulation, and which may be involved with structural and/or functional properties of the cryptococcal eumelanin.…”

Section: Neoformans Melanin Is Molecularly Associated With Non-polsupporting

confidence: 64%

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

Camacho

Vij

Chrissian

et al. 2019

Preprint

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Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-asociated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans. We observed that melanin is assembled into the cryptococcal cell wall in spherical structures of ~200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres of ~30 nm in diameter, the fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.

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Section: Neoformans Melanin Is Molecularly Associated With Non-polsupporting

confidence: 64%

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

Camacho

Vij

Chrissian

et al. 2019

Preprint

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“…Bone-marrow derived macrophages (BMDMs) were isolated from C56Bl/6 mice and used for phagocytosis assays as described previously [80]. Briefly, BMDMs were plated in a 96-well plate (10,000/well) and simulated with 100 ng/ml Interferon-γ (Roche) starting 24 hr prior to assay initiation and continuing throughout.…”

Section: Methodsmentioning

confidence: 99%

“…Cryptococcus accumulation within macrophages was assessed as described previously [80]. Briefly, BMDMs were plated in 24 well plates at a concentration of 100,000 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence

et al. 2016

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The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

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“…On the other hand, Ustilago maydis Rep1 protein is also cleaved by kexin-protease and has a structural role in the fungal cell wall (Wödsten et al, 1996;Teertstra et al, 2006). An 11 amino acid long peptide processed by KEX2 in Cryptococcus neoformans is required for virulence and to activate the sexual program (Homer et al, 2016;Tian et al, 2018). Since MiSSP8-RNAi lines are not impaired in hyphal growth or fungal cell-wall sensitivity, it is unlikely that MiSSP8 or its derived peptides are involved in fungal cell wall structure.…”

Section: Discussionmentioning

confidence: 99%

Laccaria bicolor MiSSP8 is a small‐secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

Pellegrin

Daguerre

Ruytinx

et al. 2019

Environmental Microbiology

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Summary The ectomycorrhizal symbiosis is a predominant tree–microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long‐term and intimate relationship between the soil‐borne fungi and the roots of trees. Mycorrhiza‐induced Small‐Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8‐RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C‐terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small‐secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

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Intracellular Action of a Secreted Peptide Required for Fungal Virulence

Cited by 87 publications

References 72 publications

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence

Laccaria bicolor MiSSP8 is a small‐secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

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