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PLoS One
2016 Oct 03;1110:e0163919. doi: 10.1371/journal.pone.0163919.
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Amino Acid Permeases and Virulence in Cryptococcus neoformans.
Martho KF
,
de Melo AT
,
Takahashi JP
,
Guerra JM
,
Santos DC
,
Purisco SU
,
Melhem MS
,
Fazioli RD
,
Phanord C
,
Sartorelli P
,
Vallim MA
,
Pascon RC
.
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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.
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27695080
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Fig 3. Growth phenotype of wild type, single (aap4Δ and aap5Δ) and the double mutant aap4Δ/aap5Δ (two independent transformants of each) in rich medium YEPD, synthetic dextrose supplemented with ammonium sulfate and amino acids and synthetic dextrose supplemented with amino acids only at 30 and 37°C.Serial dilutions represent 104, 103, 102, 101 and 1 cell.
Fig 5. Growth pattern of H99 and double mutant (aap4Δ/aap5Δ) in YEPD at pH 5.2 without supplements or supplemented with NaCl (0.75 M), in YEPD at pHs 6 and 7 (without salt supplements).Serial dilutions represent 104, 103, 102, 101 and 1 cell. aap4Δ and aap5Δ single mutants under the same conditions as reference are presented in S2 Fig.
Fig 6. Growth rate in wild type and mutants under oxidative stress.Serial dilution (104, 103, 102, 101 and 1 cell) were spotted on YEPD not supplemented and supplemented with 5 mM H2O2 (A) and SD supplemented with ammonium sulfate and SD supplemented with amino acids only with 1 mM H2O2 (B). Growth was carried out at 30°C up to 72 hours.
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