Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.

bProtein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1-and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.

Melanin is a fungal extracellular redox buffer which, in principle, can neutralize antimicrobial oxidants generated by immunologic effector cells, but its source of reducing equivalents is not known. We wondered whether Fe(II) generated by the external ferric reductase of fungi might have the physiologic function of reducing fungal melanin and thereby promoting pathogenesis. We observed that exposure of a melanin film electrode to reductants decreased the open-circuit potential (OCP) and reduced the area of a cyclic voltammetric reduction wave whereas exposure to oxidants produced the opposite effects. Exposure to 10, 100, 1,000 or 10,000 M Fe(II) decreased the OCP of melanin by 0.015, 0.038, 0.100, and 0.120 V, respectively, relative to a silver-silver chloride standard, and decreased the area of the cyclic voltammetric reduction wave by 27, 35, 50, and 83%, respectively. Moreover, exposure to Fe(II) increased the buffering capacity by 44%, while exposure to millimolar dithionite did not increase the buffering capacity. The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffering. Light absorption by melanin suspensions was decreased 14% by treatment with Fe(II), consistent with reduction of melanin. Light absorption by suspensions of melanized Cryptococcus neoformans was decreased 1.3% by treatment with Fe(II) (P < 0.05). Cultures of C. neoformans generated between 2 and 160 M Fe(II) in culture supernatant, depending upon the strain and the conditions [the higher values were achieved by a constitutive ferric reductase mutant in high concentrations of Fe(III)]. We infer that Fe(II) can reduce melanin under physiologic conditions; moreover, it binds to melanin and cooperatively increases redox buffering. The data support a model for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular Fe(II) maintain the reducing capacity of the extracellular redox buffer.Leukocytes attack pathogens with a flux of secreted strong oxidants (1). Thus, any extracellular microbial product which neutralizes oxidants is likely to protect the pathogen and promote invasive disease. Melanin, an extracellular redox buffer composed of polymerized catechols (5,14,15,21), is a virulence factor for certain pathogenic fungi (3, 11). Its ability to neutralize strong oxidants is supported by several types of evidence: mutants of Cryptococcus neoformans selected for sensitivity to oxidants exhibit albinism (8); nonmelanized wildtype cells or mutants selected for albinism exhibit sensitivity to oxidants (9, 18); and melanin neutralizes oxidants and protects the melanized fungi C. neoformans, Wangiella dermatididis, and Alternaria alternata from killing by hypochlorite and by permanganate (8, 9). Thus, melanin may function as chemical armor in invasive disease. We reasoned that maintenance of the reducing capacity of extracellular melanin may be an important aggressive f...

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.