<i>Cryptococcus neoformans</i> (VNIV) can colonize mature soil grown <i>Arabidopsis thaliana</i>.

<p>(A) Mating mixtures can induce chlorosis, (B) Colony forming units (CFUs + SEM) indicate that both individual and mating strains of <i>Cryptococcus</i> can colonize <i>A</i>. <i>thaliana</i> plants. Chlorosis was only associated with mated mixtures of <i>C</i>. <i>neoformans</i> (VNI).</p

Enhanced mating of <i>Cryptococcus</i> is observed on plant material-based agars.

<p><i>C</i>. <i>deneoformans</i> and <i>C</i>. <i>neoformans</i> display filamentation (arrows) around the periphery of the colony on many types of plant-based agars at four weeks post inoculation. Mating and the production of basidia were confirmed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s008" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s009" target="_blank">S9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s010" target="_blank">S10</a> Figs). Mating of <i>C</i>. <i>bacillisporus</i> x <i>C</i>. <i>gattii</i> was less prolific and was confirmed based on microscopic observation of hyphae, basidia, and spores. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s008" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s009" target="_blank">S9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171695#pone.0171695.s010" target="_blank">S10</a> Figs). For <i>C</i>. <i>bacillisporus</i> x <i>C</i>. <i>gattii</i> arrows highlight detectable areas of filamentation with confirmation by light microscopy.</p

Strain dependent changes in the virulence of <i>Cryptococcus</i> as a result of passage on plant-based agars are depicted.

<p>Kaplan-Meier survival curves of nine <i>Cryptococcus</i> strains grown on <i>Arabidopsis</i> agar are shown. <i>C</i>. <i>bacillisporus</i> NIH312 (E) demonstrated reduced virulence and <i>C</i>. <i>neoformans</i> A1-22 (H) displayed increased virulence following one week of growth on <i>Arabidopsis</i> plant agar. No significant differences were observed between YPD agar and <i>Arabidopsis</i> agar for any other strain.</p

Plants promote mating and dispersal of the human pathogenic fungus <i>Cryptococcus</i>

<div><p>Infections due to <i>Cryptococcus</i> are a leading cause of fungal infections worldwide and are acquired as a result of environmental exposure to desiccated yeast or spores. The ability of <i>Cryptococcus</i> to grow, mate, and produce infectious propagules in association with plants is important for the maintenance of the genetic diversity and virulence factors important for infection of animals and humans. In the Western United States and Canada, <i>Cryptococcus</i> has been associated with conifers and tree species other than <i>Eucalyptus</i>; however, to date <i>Cryptococcus</i> has only been studied on live <i>Arabidopsis thaliana</i>, <i>Eucalyptus sp</i>., and <i>Terminalia catappa</i> (almond) seedlings. Previous research has demonstrated the ability of <i>Cryptococcus</i> to colonize live plants, leaves, and vasculature. We investigated the ability of <i>Cryptococcus</i> to grow on live seedlings of the angiosperms, <i>A</i>. <i>thaliana</i>, <i>Eucalyptus camaldulensis</i>, <i>Colophospermum mopane</i>, and the gymnosperms, <i>Pseudotsuga menziesii</i> (Douglas fir), and <i>Tsuga heterophylla</i> (Western hemlock). We observed a broad-range ability of <i>Cryptococcus</i> to colonize both traditional infection models as well as newly tested conifer species. Furthermore, <i>C</i>. <i>neoformans</i>, C. <i>deneoformans</i>, <i>C</i>. <i>gattii</i> (VGI), <i>C</i>. <i>deuterogattii</i> (VGII) and <i>C</i>. <i>bacillisporus</i> (VGIII) were able to colonize live plant leaves and needles but also undergo filamentation and mating on agar seeded with plant materials or in saprobic association with dead plant materials. The ability of <i>Cryptococcus</i> to grow and undergo filamentation and reproduction in saprobic association with both angiosperms and gymnosperms highlights an important role of plant debris in the sexual cycle and exposure to infectious propagules. This study highlights the broad importance of plants (and plant debris) as the ecological niche and reservoirs of infectious propagules of <i>Cryptococcus</i> in the environment.</p></div

Douglas fir and Eastern hemlock infection model at one week post infection.

<p>(A) Top row displays Douglas fir inoculated with (A) <i>C</i>. <i>deneoformans</i> JEC21α, JEC20<b>a</b>, and JEC21α + JEC20<b>a</b> mixed; Middle row shows, <i>C</i>. <i>neoformans</i> H99α, KN99<b>a</b>, and H99α + KN99<b>a</b> mixture; Bottom row shows, C. <i>deuterogattii</i> (NIH312α), <i>C</i>. <i>gattii</i> (NIH194<b>a)</b>, and NIH312α + NIH194<b>a</b> mixture. (B) Inoculated buds and needles predominantly appear green and healthy but some browning or loss of needles are observed for both individually inoculated strains and mated mixtures. (B) Left column shows, <i>C</i>. <i>deneoformans</i> JEC21α, JEC20<b>a</b>, and JEC21α + JEC20<b>a</b> mixed; Middle column under labeled control shows, <i>C</i>. <i>neoformans</i> H99α, KN99<b>a</b>, and H99α + KN99<b>a</b> mixture; and right column shows, <i>C</i>. <i>deuterogattii</i> (NIH312α), <i>C</i>. <i>gattii</i> (NIH194<b>a)</b>, and NIH312α + NIH194<b>a</b> mixture.</p

<i>Arabidopsis thaliana</i> mutants display increased susceptibility to <i>Cryptococcus</i> colonization.

<p><i>A</i>. <i>thaliana jar1-1</i> mutants display increased colonization by <i>C</i>. <i>neoformans</i> (VNI) at four and twelve days post infection (dpi). <i>A</i>. <i>thaliana npr1-1</i> mutants display increased colonization to mixed <i>C</i>. <i>neoformans</i> infection at 4 and 12 days post-infection. Error bars represent CFUs + SEM.</p

<i>Cryptococcus</i> can colonize live Douglas fir and Western hemlock trees.

<p>Scanning electron micrographs are shown of mixed mating strains of (A) <i>Cryptococcus deneoformans</i> (VNIV) producing filaments on Douglas fir trees, and (B) colonizing Eastern hemlock trees. Colonization of <i>Cryptococcus neoformans</i> (VNIV) on Douglas fir (C) or Western hemlock (D), and of <i>C</i>. <i>deuterogattii</i> (VGII) <i>x C</i>. <i>gattii</i> (VGI) on Douglas fir (E) or Hemlock (F) are shown. Scale bar = 5 μm.</p

Conifer trees are susceptible to colonization by <i>Cryptococcus</i>.

<p>Colony forming units (CFUs + SEM) at three weeks post-inoculation are shown. (A) <i>Cryptococcus</i> cells were recovered from Douglas fir and Eastern hemlock seedlings. <i>C</i>. <i>neoformans</i> was recovered from both Douglas fir and Eastern hemlock; however, recovery of <i>C</i>. <i>deneoformans</i>, <i>C</i>. <i>gattii</i>, <i>C</i>. <i>bacillisporus</i>, <i>C</i>. <i>deuterogattii</i> strains was inconsistent. In a plant infection trial, Douglas fir, <i>Eucalyptus</i>, and Mopane infection models were compared utilizing engineered strains containing various drug-resistant cassettes. <i>Cryptococcus</i> cells were recovered from <i>Eucalyptus</i>, Mopane, and Douglas fir infected with individual and mated strains. Error bars represent +SEM.</p

Aerosolization at high humidity increases spray factor and viable aerosols for <i>Cryptococcus</i> during aerosolization in the Madison Chamber.

<p>(A and C ) Log spray factor plotted against relative humidity for broth-grown (A) <i>C. neoformans</i> var. <i>grubii</i> H99 (p = 0.0005, R² = 0.802) and (C) <i>C. gattii</i> EJB18 (p = 0.06, R² = 0.534). Spray factor increases with relative humidity. (B and D) Log aerosol concentration plotted against relative humidity for broth-grown (B) <i>C. neoformans</i> var. <i>grubii</i> H99 (p = 0.011, R² = 0.57) and (D) <i>C. gattii</i> EJB18 (p = 0.12, R² = 0.402). Log aerosol concentration increases with relative humidity. Filled circles and squares represent <i>C. neoformans</i> var. <i>grubii</i> (H99) and open circles and squares represent <i>C. gattii</i> EJB18.</p

Aerosol delivery of <i>C. neoformans</i> var. <i>grubii</i> yeast and yeast-spore mixtures.

<p>(A) <i>C. n.</i>var. <i>grubii</i>, H99 (MATα), KN99<b>a</b>, and a mated mixture containing spores was effectively delivered to mice as assayed by dose retained in lungs (95% RH and 1 hour exposure). (A) Increased tissue burden, dissemination to the brain, and (B) decreased weight was observed at 24 days post-exposure and demonstrates the developmental sequelae of cryptococcosis. No <i>Cryptococcus</i> colonization was obtained from brain or spleen tissues 1 hour post exposure. No significant differences in CFUs were observed between H99 (MATα), KN99<b>a</b>, or mated mixtures (with spores) at any 1 or 25 days post exposure. Mean value (n = 3–4, +/− SEM) are plotted.</p