Systematic analysis of signaling networks associated with melanin production in Cryptococcus neoformans

다국어 초록 (Multilingual Abstract)

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Among its virulence factors, melanin is an antioxidant polyphenol pigment important for protection against reactive oxygen species (ROS)-mediated cellular damages and phagocytosis by host immune cells. Melanin production is known to be regulated by several signaling pathways, including the cAMP and HOG pathway. However, the comprehensive overview of signaling networks regulating melanin production still remains elusive. To systematically analyze melanin-regulating signaling pathways in C. neoformans, I focused on the melanin production capability in 155 transcription factor mutant libraries that were previously constructed. For 26 transcription factor mutants, which exhibit altered melanin production level in Niger seed medium. To examine which genes are fundamental for melanin production, I first check melanin production level of 26 transcription factor mutants on L-DOPA and epinephrine-containing medium. The bzp4Δ, usv101Δ, hob1Δ and mbs1Δ mutants exhibited melanin production defect on all melanin-inducing medium. Supporting this, their LAC1 induction was reduced in carbon starvation condition than wild type strain. Also, epistatic analysis and their mutual expression levels indicated that Bzp4, Usv101 and Mbs1 independently regulate melanin production and Hob1 controls Bzp4 and Usv101. To find out the upstream regulator of this network, I confirmed that the cAMP and HOG pathways regulate them. Only BZP4 was negatively regulated by HOG pathway, but not cAMP pathway. To determine other potential upstream regulator candidates, I additionally check melanin production level of 129 kinase mutants which exhibit altered melanin production in Niger seed medium. The 49 kinase mutants show altered melanin production, we additionally tested melanin production on L-DOPA and epinephrine-containing medium. The nine kinase mutants exhibited defective melanin production on all medium and their LAC1 induction was reduced in carbon starvation condition. Expression levels of four transcription factor genes in the nine kinase mutants under carbon starvation condition indicated that several kinases regulate this transcription factor networks. Especially, Gsk3 may controls all TF networks under melanin-inducing condition. It means that Gsk3 is fundamental melanin-regulating kinase. In transcriptomic analysis using four transcription factor mutants revealed that several melanin-regulating signaling factors are regulated by TF networks as upstream regulator. The SKS1 kinase is thought to be controlled by all TF networks. In conclusion, this study will provide insights into the regulatory mechanism of melanin regulating signaling pathways in C. neoformans. Furthermore, it will be able to expand understanding of melanin regulation in pathogenic fungi.