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Microevolution during serial mouse passage demonstrates FRE3 as a virulence adaptation gene in Cryptococcus neoformans.

ABSTRACT: Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a "virulence acquisition gene" during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts. IMPORTANCE Cryptococcus neoformans is a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene, FRE3, in two strains that adapted in mice to become highly virulent, and overexpression of FRE3 recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity of C. neoformans to show reproducible changes in the expression levels of small numbers of genes termed "virulence adaptation genes" to effectively increase pathogenicity during the environment-to-mammal transition.

SUBMITTER: Hu G

PROVIDER: S-EPMC3977352 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Microevolution during serial mouse passage demonstrates FRE3 as a virulence adaptation gene in Cryptococcus neoformans.

Hu Guowu G Chen Shu Hui SH Qiu Jin J Bennett John E JE Myers Timothy G TG Williamson Peter R PR

mBio 20140401 2

Passage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in ...[more]

PMID: 24692633

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Project description:The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

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: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.

Dataset Information

Microevolution during serial mouse passage demonstrates FRE3 as a virulence adaptation gene in Cryptococcus neoformans.

Publications

Microevolution during serial mouse passage demonstrates FRE3 as a virulence adaptation gene in Cryptococcus neoformans.

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