Melanin protects Cryptococcus neoformans from spaceflight effects

Abstract

As human activity in space continues to increase, understanding how biological assets respond to spaceflight conditions is becoming more important. Spaceflight conditions include exposure to ionizing radiation, microgravity, spacecraft vibrations and hypervelocity; all of which can affect the viability of biological organisms. Previous studies have shown that melanin-producing fungi are capable of surviving the vacuum of space and Mars-simulated conditions in Low Earth Orbit. This survival has been associated in part with the protective effects of melanin, but a comparison of fungal viability in the presence or absence of melanin following spaceflight has never been tested. In this study, we evaluated the protective effects of melanin by comparing the viability of melanized and non-melanized clones of Cryptococcus neoformans yeasts following a roundtrip to the International Space Station. Yeast colonies were placed inside two MixStix silicone tubes; one stayed on Earth and the other was transported inside for 29 days before returning to Earth. Post-flight analysis based on colony-forming unit numbers shows that melanized yeast viability was 50% higher than non-melanized yeasts, while no difference was observed between the Earth-bound control samples. The results suggest that fungal melanin could increase the lifespan of biological assets in space.

Fig. 1

Preparation of flight samples. A. Examples of non‐melanized and melanized clones of C. neoformans grown in solid minimal media. Scale bar, 1 cm. B. The MixStix is a 10 ml parylene‐coated silicon tube used to carry the fungal samples. It was compartmentalized into three volumes using two Clamps (A and B). Volume 1 contained two rectangular pieces of agar containing living melanized and non‐melanized C. neoformans lawns (approximately ~10⁸ cells). Volume 2 and 3 contained an uncoated and melanin‐coated dosimeter (RADTriage50 radiation sensor card) respectively. The sensor card was cut to fit the diameter of the MixStix tube. However, no useful data could be obtained from the sensor cards once cut. C. Shows a rectangular piece of melanized fungal lawn being placed inside the MixStix‐Volume 1.

Fig. 2

Mean daily radiation dose detected at the Japanese Pressurized Module by the International Space Station Radiation Environment Monitor 2 (REM2). The dashed line corresponds to the South Atlantic Anomaly (SAA) and the solid line is Galactic Cosmic Rays (GCR) in milli Gray per day. Data were provided by the Space Radiation Analysis Group (SRAG).

Fig. 3

Boxplots showing viability differences between melanized and non‐melanized yeasts following roundtrip to the International Space Station (ISS) and Earth‐bound control. Cell viability was evaluated by enumerating the CFUs of Earth‐bound and ISS‐flown yeast samples relative to the total number of cells inoculated; 500 and 250 cells per plate in quadruplicates (eight plates per sample condition). The viability of samples was analysed four independent times, but we had contamination issues with the Earth‐bound sample on two occasions. Hence, the percent viability of the ISS‐bound results corresponds to four biological replicas and the Earth‐bound results correspond to two replicas. The bars on the boxplots are 95% confident intervals. The black dot represents data values outside the confidence interval. Asterisks denote P < 0.05 comparing melanized and non‐melanized samples via ANOVA with Tukey tests.

Fig. 4

Light microscopy images of melanized and non‐melanized yeast cells following spaceflight and the Earth‐bound control sample. Light microscopy of Earth and International Space Station (ISS) C. neoformans specimens. Objective: 100× oil immersion. Scale bar, 5 μm. All images were captured using the same exposure time. Differences in the background darkness behind cells are not due to differences in exposure time but to the inherent heterogeneity of the counterstain (India Ink) on mounted glass slides. White arrows show examples of capsule fragments that were more prevalent in the non‐melanized sample, especially in the ISS‐bound.

Fig. 5

Boxplots showing cellular dimensions measured from light microscopy images. A. Capsule radius (μm). B. Cell body radius (μm). C. Total cell radius (μm). D. The capsule to cell body ratio. Measurements were done manually using the ImageJ software as previously described (Cordero et al., 2011). The bars on the boxplots are 95% confident intervals. Dots represent data values outside the confidence interval. Middle asterisks denote comparisons between International Space Station (ISS) and Earth‐bound using ANOVA with Tukey tests, while left and right asterisks denote comparisons between melanized and non‐melanized samples (n = 120 cells per group; one biological replicate). NS = not significant; and *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001 respectively.