Plants promote mating and dispersal of the human pathogenic fungus Cryptococcus

Abstract

Infections due to Cryptococcus 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 Cryptococcus 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, Cryptococcus has been associated with conifers and tree species other than Eucalyptus; however, to date Cryptococcus has only been studied on live Arabidopsis thaliana, Eucalyptus sp., and Terminalia catappa (almond) seedlings. Previous research has demonstrated the ability of Cryptococcus to colonize live plants, leaves, and vasculature. We investigated the ability of Cryptococcus to grow on live seedlings of the angiosperms, A. thaliana, Eucalyptus camaldulensis, Colophospermum mopane, and the gymnosperms, Pseudotsuga menziesii (Douglas fir), and Tsuga heterophylla (Western hemlock). We observed a broad-range ability of Cryptococcus to colonize both traditional infection models as well as newly tested conifer species. Furthermore, C. neoformans, C. deneoformans, C. gattii (VGI), C. deuterogattii (VGII) and C. bacillisporus (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 Cryptococcus 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 Cryptococcus in the environment.

Fig 1. Cryptococcus neoformans (VNIV) can colonize mature soil grown Arabidopsis thaliana.

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

Fig 2. Arabidopsis thaliana mutants display increased susceptibility to Cryptococcus colonization.

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

Fig 3. Cryptococcus can colonize live Douglas fir and Western hemlock trees.

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

Fig 4. Douglas fir and Eastern hemlock infection model at one week post infection.

(A) Top row displays Douglas fir inoculated with (A) C. deneoformans JEC21α, JEC20a, and JEC21α + JEC20a mixed; Middle row shows, C. neoformans H99α, KN99a, and H99α + KN99a mixture; Bottom row shows, C. deuterogattii (NIH312α), C. gattii (NIH194a), and NIH312α + NIH194a 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, C. deneoformans JEC21α, JEC20a, and JEC21α + JEC20a mixed; Middle column under labeled control shows, C. neoformans H99α, KN99a, and H99α + KN99a mixture; and right column shows, C. deuterogattii (NIH312α), C. gattii (NIH194a), and NIH312α + NIH194a mixture.

Fig 5. Cryptococcus can infect conifers and shows progressive disease associated symptoms.

Conifer infection model at four weeks post-infection displays exacerbated needle browning, bud drooping, and needle dropping consistent with infection. Douglas fir (top row) and Eastern hemlock (bottom row) four weeks post inoculation with, (left column) C. deneoformans JEC21α, JEC20a, and JEC21α + JEC20a mixed; (middle column) C. neoformans H99α, KN99a, and H99α + KN99a mixture; (right column) C. bacillisporus (NIH312α), C. gattii (NIH194a), and NIH312α + NIH194a mixture. Douglas fir displays more plant infection symptoms in contrast to Eastern hemlock.

Fig 6. Conifer trees are susceptible to colonization by Cryptococcus.

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

Fig 7. Cryptococcus can grow saprobically and mate on dead plant materials.

Scanning electron micrographs depict C. neoformans (VNI) displaying robust filamentation on sugar maple leaves (top row), northern white cedar needles (middle row) and long leaf pine needles (bottom row). Fused clamp connections are indicative of productive matings (arrow).

Fig 8. Enhanced mating of Cryptococcus is observed on plant material-based agars.

C. deneoformans and C. neoformans 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 (S7, S8, S9 and S10 Figs). Mating of C. bacillisporus x C. gattii was less prolific and was confirmed based on microscopic observation of hyphae, basidia, and spores. (S7, S8, S9 and S10 Figs). For C. bacillisporus x C. gattii arrows highlight detectable areas of filamentation with confirmation by light microscopy.

Fig 9. Strain dependent changes in the virulence of Cryptococcus as a result of passage on plant-based agars are depicted.

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