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A Wor1-Like Transcription Factor Is Essential for Virulence of Cryptococcus neoformans.

ABSTRACT: Gti1/Pac2 transcription factors occur exclusively in fungi and their roles vary according to species, including regulating morphological transition and virulence, mating and secondary metabolism. Many of these functions are important for fungal pathogenesis. We therefore hypothesized that one of the two proteins of this family in Cryptococcus neoformans, a major pathogen of humans, would also control virulence-associated cellular processes. Elimination of this protein in C. neoformans results in reduced polysaccharide capsule expression and defective cytokinesis and growth at 37°C. The mutant loses virulence in a mouse model of cryptococcal infection and retains only partial virulence in the Galleria mellonella alternative model at 30°C. We performed RNA-Seq experiments on the mutant and found abolished transcription of genes that, in combination, are known to account for all the observed phenotypes. The protein has been named Required for cytokinesis and virulence 1 (Rcv1).

SUBMITTER: Paes HC

PROVIDER: S-EPMC6243373 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A Wor1-Like Transcription Factor Is Essential for Virulence of Cryptococcus neoformans.

Paes Hugo Costa HC Derengowski Lorena da Silveira LDS Peconick Luisa Defranco Ferreira LDF Albuquerque Patrícia P Pappas Georgios Joannis GJ Nicola André Moraes AM Silva Fabiana Brandão Alves FBA Vallim Marcelo Afonso MA Alspaugh J Andrew JA Felipe Maria Sueli Soares MSS Fernandes Larissa L

Frontiers in cellular and infection microbiology 20181113

Gti1/Pac2 transcription factors occur exclusively in fungi and their roles vary according to species, including regulating morphological transition and virulence, mating and secondary metabolism. Many of these functions are important for fungal pathogenesis. We therefore hypothesized that one of the two proteins of this family in Cryptococcus neoformans, a major pathogen of humans, would also control virulence-associated cellular processes. Elimination of this protein in C. neoformans< ...[more]

PMID: 30483479

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Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Project description:Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.

Project description:Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity.IMPORTANCE The pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here, we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings significantly advance our understanding of a serious disease.

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A Wor1-Like Transcription Factor Is Essential for Virulence of Cryptococcus neoformans.

Publications

A Wor1-Like Transcription Factor Is Essential for Virulence of <i>Cryptococcus neoformans</i>.

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