Effects of Simulated Microgravity on the Proteome and Secretome of the Polyextremotolerant Black Fungus Knufia chersonesos.

Donatella Tesei,Katja Sterflinger,Ganesh Babu Malli Mohan,Jason E Stajich,Markus Kalkum,Kasthuri Venkateswaran,Abby J Chiang

doi:10.3389/fgene.2021.638708

Abstract

Black fungi are a group of melanotic microfungi characterized by remarkable polyextremotolerance. Due to a broad ecological plasticity and adaptations at the cellular level, it is predicted that they may survive in a variety of extreme environments, including harsh niches on Earth and Mars, and in outer space. However, the molecular mechanisms aiding survival, especially in space, are yet to be fully elucidated. Based on these premises, the rock-inhabiting black fungus Knufia chersonesos (Wt) and its non-melanized mutant (Mut) were exposed to simulated microgravity—one of the prevalent features characterizing space conditions—by growing the cultures in high-aspect-ratio vessels (HARVs). Qualitative and quantitative proteomic analyses were performed on the mycelia and supernatant of culture medium (secretome) to assess alterations in cell physiology in response to low-shear simulated microgravity (LSSMG) and to ultimately evaluate the role of cell-wall melanization in stress survival. Differential expression was observed for proteins involved in carbohydrate and lipid metabolic processes, transport, and ribosome biogenesis and translation via ribosomal translational machinery. However, no evidence of significant activation of stress components or starvation response was detected, except for the scytalone dehydratase, enzyme involved in the synthesis of dihydroxynaphthalene (DNH) melanin, which was found to be upregulated in the secretome of the wild type and downregulated in the mutant. Differences in protein modulation were observed between K. chersonesos Wt and Mut, with several proteins being downregulated under LSSMG in the Mut when compared to the Wt. Lastly, no major morphological alterations were observed following exposure to LSSMG. Similarly, the strains’ survivability was not negatively affected. This study is the first to characterize the response to simulated microgravity in black fungi, which might have implications on future astrobiological missions.

Highlights

Based on their nature as settlers in extreme environments, microbial extremophiles are of great interest to studies aiming to elucidate stress adaptation and survival mechanisms
Cell survivability in simulated microgravity was measured by colony forming units (CFU) using ImageJ software (Schneider et al, 2012) according to established protocol (Choudhry, 2016) and were: K. chersonesos WtLSSMG = 2.8 × 106 cell/mL, K. chersonesos MutLSSMG 2.5 × 106 cell/mL, K. chersonesos Wt1G 2.6 × 106 cell/mL, K. chersonesos Mut1G 2.3 × 106 cell/mL (Supplementary Table 1)
Investigations into the responses of microorganisms to space and Mars-like conditions have been performed with few black fungi species; some of them involved stress simulation in ground-based facilities (Pacelli et al, 2017), while others carried out the exposure of fungal strains inside or outside the International Space Station (Onofri et al, 2012, 2019; Pacelli et al, 2016)

Summary

Introduction

Based on their nature as settlers in extreme environments, microbial extremophiles are of great interest to studies aiming to elucidate stress adaptation and survival mechanisms. Successful examples of extremophiles can be found in the fungal domain, alongside bacteria, and archaea, which for a long time were considered to be the sole colonizers of habitats previously considered uninhabitable. Some of these fungal extremophiles show even higher resistance than that of prokaryotes (Shtarkman et al, 2013; Aguilera and González-Toril, 2019). Black fungi in particular represent a group of highly melanized microfungi, whose ability to survive in a variety of extreme environments has in recent decades attracted increasing attention (Selbmann et al, 2005; Sterflinger, 2006; Gorbushina, 2007; Onofri et al, 2007). Rock represents a harsh habitat and a quite ancient niche for life, believed to reflect early Earth conditions, and considered to be a model for extraterrestrial life (Selbmann et al, 2014)

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