Return of Mars samples is a high priority in the planetary science community and has remained an enduring goal of planetary exploration programs. Development of sterilization techniques to prevent potential contamination of Earth’s biosphere with unknown life-forms that could exist on planetary bodies requires the use of the most robust biological indicators. We argue that self-seeding proteinaceous particles (prions) represent the most robust biological agents found on Earth. To evaluate the impact of various sterilization techniques on prion activity, we used derivatives of yeast prion proteins Sup35 and Ure2, which are not harmful to humans. Our study demonstrated that effective antimicrobial modalities, which include prolonged dry heat (up to 200°C), vapor hydrogen peroxide, gamma irradiation (up to 100 kGy), and ambient air or wet He/water plasma (deposited energy density of up to 6.3 kJ/cm2), did not eliminate the biological activity of yeast prions. However, ultraviolet C (UVC) irradiation at a wavelength of 260–270 nm for 16–24 days eliminated Ure2 prion detection and biological activity, and prolonged UVC irradiation eliminated detection of Sup35 prions and reduced, although did not eliminate, their biological activity. These data suggest that UVC could be an essential component of in-flight sterilization techniques for all future planetary missions.
AaronKM, MoussessianA, NewlinLE, et al.Planetary protection for Europa radar sounder antenna. Advances Space Res, 2016; 57(9):2013–2021.
2.
AllenKD, WegrzynRD, ChernovaTAet al.Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+]. Genetics, 2005; 169(3):1227–1242; doi: 10.1534/genetics.104.037168
3.
BalbirnieM, GrotheR, EisenbergDS. An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc Natl Acad Sci U S A, 2001; 98(5):2375–2380.
4.
BaxterH, CampbellG, WhittakerA, et al.Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radio-frequency gas-plasma treatment. J Gen Virol, 2005; 86(Pt 8):2393–2399.
5.
BeatyDW, GradyMM, McSweenHY, et al.The potential science and engineering value of samples delivered to Earth by Mars sample return: International MSR Objectives and Samples Team (iMOST). Meteorit & Planetary Scien, 2019; 54(S1):S3–S152.
6.
BeckSE, HullNM, PoeppingC, et al.Wavelength-dependent damage to adenoviral proteins across the germicidal UV spectrum. Environ Sci Technol, 2018; 52(1):223–229.
7.
Bellinger-KawaharaC, CleaverJ, DienerT, et al.Purified scrapie prions resist inactivation by UV irradiation. J Virol, 1987; 61(1):159–166.
8.
BenardiniJN, SeaslyE, SpryJA. Updates in NASA Policy and Practice in Planetary Protection. In: 2023 IEEE Aerospace Conference. IEEE; 2023; pp. 1–8.
9.
BiancalanaM, KoideS. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta, 2010; 1804(7):1405–1412.
10.
BosakT, ShusterDL, SchellerEL, et al.Astrobiological potential of rocks acquired by the Perseverance rover at a sedimentary fan front in Jezero Crater, Mars. AGU Advances, 2024; 5(4):e2024AV001241.
11.
CataldoG, AffentrangerL, ClementBG, et al.The planetary protection strategy of Mars Sample Return Earth Return Orbiter mission. J Space Saf Eng, 2024; 11(2):374–384.
12.
ChenF, LyC, MikellidesI, et al.Mars 2020 mission biological return sample contamination control approach and verification. Astrobiology, 2023; 23(8):862–879.
13.
ChernovaTA, ChernoffYO, WilkinsonKD. Yeast models for amyloids and prions: Environmental modulation and drug discovery. Molecules, 2019; 24(18):3388.
14.
ChoudhuryB, RevazishviliT, LozadaM, et al.Distributed compact plasma reactor decontamination for planetary protection in space missions. Sci Rep, 2023; 13(1):1928.
15.
ChungS, KernR, KoukolR, et al.Vapor hydrogen peroxide as alternative to dry heat microbial reduction. Adv Space Res, 2008; 42(6):1150–1160.
CravenE, WintersM, SmithAL, et al.Biological safety in the context of backward planetary protection and Mars sample return: Conclusions from the sterilization working group. Int J Astrobiol, 2021; 20(1):1–28.
18.
DalyMJ. The scientific revolution that unraveled the astonishing DNA repair capacity of the Deinococcaceae: 40 years on. Can J Microbiol, 2023; 69(10):369–386.
19.
DebusA, RunavotJ, RogovskiG, et al.Mars 96 small station biological decontamination. Adv Space Res, 1998; 22(3):401–409.
20.
Diaz-AvalosR, LongC, FontanoE, et al.Cross-beta order and diversity in nanocrystals of an amyloid-forming peptide. J Mol Biol, 2003; 330(5):1165–1175.
21.
DobryninD, FridmanG, FriedmanG, et al.Physical and biological mechanisms of direct plasma interaction with living tissue. New J Phys, 2009; 11(11):115020.
22.
EhrenshaftM, SilvaSO, PerdivaraI, et al.Immunological detection of N-formylkynurenine in oxidized proteins. Free Radic Biol Med, 2009; 46(9):1260–1266.
23.
FichetG, AntlogaK, ComoyE, et al.Prion inactivation using a new gaseous hydrogen peroxide sterilisation process. J Hosp Infect, 2007; 67(3):278–286.
24.
GrunertJ. Treaty on principles governing the activities of states in the exploration and use of outer space, including the moon and other celestial bodies. In: The United States space force and the future of American Space Policy. Brill Nijhoff; 2022; pp. 249–254.
25.
HoffmanGL, PutzRE. Host susceptibility and the effect of aging, freezing, heat, and chemicals on spores of Myxosoma cerebralis. The Progressive Fish-Culturist, 1969; 31(1):35–37.
26.
JulákJ, JanouskováO, ScholtzV, et al.Inactivation of prions using electrical DC discharges at atmospheric pressure and ambient temperature. Plasma Processes Polymers, 2011; 8(4):316–323.
27.
KopalV. Treaty on principles governing the activities of states in the exploration and use of outer space, including the Moon and other celestial bodies. YB Air & Space Law; 1966; p. 463.
28.
KuwanaR, YamazawaR, AsadaR, et al.Excessive ultraviolet C irradiation causes spore protein denaturation and prohibits the initiation of spore germination in Bacillus subtilis. J Microorg Control, 2023; 28(1):15–25.
29.
McDonnellG. Decontamination of prions. In: Decontamination in Hospitals and Healthcare, Woodhead Publishing; 2013; pp. 346–369.
30.
MurrellW, ScottW. The heat resistance of bacterial spores at various water activities. J Gen Microbiol, 1966; 43(3):411–425.
31.
NaikiH, HiguchiK, HosokawaM, et al.Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T. Anal Biochem, 1989; 177(2):244–249.
32.
NaitoK, SawadaishiK, KawasakiM. Photobiochemical mechanisms of biomolecules relevant to germicidal ultraviolet irradiation at 222 and 254 nm. Sci Rep, 2022; 12(1):18217.
NelsonM. Mars water discoveries-implications for finding ancient and current life. Life Sci Space Res (Amst), 2015; 7:A1–A5.
36.
OsmanS, PeetersZ, La DucMT, et al.Effect of shadowing on survival of bacteria under conditions simulating the Martian atmosphere and UV radiation. Appl Environ Microbiol, 2008; 74(4):959–970.
37.
PillingerJM, PillingerC, Sancisi-FreyS, et al.The microbiology of spacecraft hardware: Lessons learned from the planetary protection activities on the Beagle 2 spacecraft. Res Microbiol, 2006; 157(1):19–24.
38.
PottageT, MackenS, GiriK, et al.Low-temperature decontamination with hydrogen peroxide or chlorine dioxide for space applications. Appl Environ Microbiol, 2012; 78(12):4169–4174.
39.
PrinceHN. D-values of Bacillus pumilus spores on irradiated devices (inoculated product). Appl Environ Microbiol, 1978; 36(2):392–393.
40.
PrusinerSB. Novel proteinaceous infectious particles cause scrapie. Science, 1982; 216(4542):136–144.
41.
ReidLO, RomanEA, ThomasAH, et al.Photooxidation of tryptophan and tyrosine residues in human serum albumin sensitized by Pterin: A model for globular protein photodamage in skin. Biochemistry, 2016; 55(34):4777–4786.
42.
ReyesAL, CrawfordRG, WehbyAJ, et al.Heat resistance of Bacillus spores at various relative humidities. Appl Environ Microbiol, 1981; 42(4):692–697.
43.
RohatgiN, SchubertW, KoukolR, et al.Certification of Vapor Phase Hydrogen Peroxide Sterilization Process for Spacecraft Application. In: International Conference on Environmental Systems. SAE International: Vancouver, BC; 2002.
44.
RohatgiN, SchwartzL, StabekisP, et al.Development of hydrogen peroxide technique for bioburden reduction. In: 35th COSPAR Scientific Assembly. COSPAR; Paris, France; 2004; p. 541.
45.
RohwerRG. Virus-like sensitivity of the scrapie agent to heat inactivation. Science, 1984; 223(4636):600–602.
46.
RossED, WicknerRB. Prions of yeast fail to elicit a transcriptional response. Yeast, 2004; 21(11):963–972.
47.
RutalaWA, WeberDJ. Disinfection and sterilization: An overview. Am J Infect Control, 2013; 41(5 Suppl):S2–S5.
Sant’AnnaR, FernándezMR, BatlleC, et al.Characterization of amyloid cores in prion domains. Sci Rep, 2016; 6(1):34274.
50.
SarliB, BowmanE, CataldoG, et al.NASA’s capture, containment, and return system: Bringing Mars samples to Earth. Acta Astronaut, 2024; 223:270–303.
51.
SchildR, JosephR. Life on mars discovered by NASA’s 1976 viking landers: Lichens, algae, moss, microbial mats, vesicular trace fossils in Utopia Planitia and Chryse Planitia. J Cosmol, 2022; 32:44–115.
52.
SchuergerAC, RichardsJT, NewcombeDA, et al.Rapid inactivation of seven spp. under simulated Mars UV irradiation. Icarus, 2006; 181(1):52–62.
53.
Schulze-MakuchD, FairénAG, DavilaAF. The case for life on Mars. Int J Astrobiol, 2008; 7(2):117–141.
54.
SetoEP, HirschAL, SchubertWW, et al.Heat inactivation of stable proteinaceous particles for future sample return mission architecture. Front Microbiol, 2022; 13:911091.
55.
SharmaA, BehrensSH, ChernoffYO, et al.Modulation of the formation of Abeta- and Sup35NM-based amyloids by complex interplay of specific and nonspecific ion effects. J Phys Chem B, 2018; 122(19):4972–4981.
56.
TaniguchiM, LindseyJS. Database of absorption and fluorescence spectra of >300 common compounds for use in PhotochemCAD. Photochem Photobiol, 2018; 94(2):290–327.
57.
TaylorKL, ChengN, WilliamsRW, et al.Prion domain initiation of amyloid formation in vitro from native Ure2p. Science, 1999; 283(5406):1339–1343.
58.
Ter-AvanesyanMD, DagkesamanskayaAR, KushnirovVV, et al.The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae. Genetics, 1994; 137(3):671–676.
59.
Tokmina-RoszykD, SinghC, ChenF, et al.Heat inactivation of proteins: Implications for the Mars Sample Campaign and other extraterrestrial sample return missions. Int J Astrobiol, 2025; 24.
60.
Von KeudellA, AwakowiczP, BenediktJ, et al.Inactivation of bacteria and biomolecules by low‐pressure plasma discharges. Plasma Processes Polymers, 2010; 7(3–4):327–352.
61.
WeinsteinRA, RutalaWA, WeberDJ. Creutzfeldt-Jakob disease: Recommendations for disinfection and sterilization. Clin Infect Dis, 2001; 32(9):1348–1356.
62.
YangH, SharmaA, DalyMJ, et al.The ternary complex of Mn(2+), synthetic decapeptide DP1 (DEHGTAVMLK), and orthophosphate is a superb antioxidant. Proc Natl Acad Sci U S A, 2024; 121(51):e2417389121.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
