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Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.

ABSTRACT: In 1999, heteroresistance to triazoles was reported in Cryptococcus neoformans strains isolated from an azole therapy failure case of cryptococcosis in an AIDS patient and in a diagnostic strain from a non-AIDS patient. In this study, we analyzed 130 strains of C. neoformans isolated from clinical and environmental sources before 1979, prior to the advent of triazoles, and 16 fluconazole (FLC)-resistant strains isolated from AIDS patients undergoing FLC maintenance therapy during 1990 to 2000. All strains isolated prior to 1979 manifested heteroresistance (subset of a population that grows in the presence of FLC) at concentrations between 4 and 64 microg/ml, and all 16 FLC-resistant AIDS isolates manifested heteroresistance at concentrations between 16 and 128 microg/ml. Upon exposure to stepwise increases in the concentration of FLC, subpopulations that could grow at higher concentrations emerged. Repeated transfer on drug-free media caused the highly resistant subpopulations to revert to the original level of heteroresistance. The reversion pattern fell into four categories based on the number of transfers required. The strains heteroresistant at > or =32 microg/ml were significantly more resistant to other xenobiotics and were also more virulent in mice than were those heteroresistant at < or =8 microg/ml. During FLC treatment of mice infected by strains with low levels of heteroresistance, subpopulations exhibiting higher levels of heteroresistance emerged after a certain period of time. The ABC transporter AFR1, known to efflux FLC, was unrelated to the heteroresistance mechanism. Our study showed that heteroresistance to azole is universal and suggests that heteroresistance contributes to relapse of cryptococcosis during azole maintenance therapy.

SUBMITTER: Sionov E

PROVIDER: S-EPMC2704677 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.

Sionov Edward E Chang Yun C YC Garraffo H Martin HM Kwon-Chung Kyung J KJ

Antimicrobial agents and chemotherapy 20090504 7

In 1999, heteroresistance to triazoles was reported in Cryptococcus neoformans strains isolated from an azole therapy failure case of cryptococcosis in an AIDS patient and in a diagnostic strain from a non-AIDS patient. In this study, we analyzed 130 strains of C. neoformans isolated from clinical and environmental sources before 1979, prior to the advent of triazoles, and 16 fluconazole (FLC)-resistant strains isolated from AIDS patients undergoing FLC maintenance therapy during 1990 to 2000. A ...[more]

PMID: 19414582

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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:Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Dataset Information

Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.

Publications

Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence.

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