1. Introduction
The search for exo-biosignatures and the characterization of exoplanetary atmospheres and surfaces is an objective of the NASA Astrobiology Program [
1,
2]. Life as we know it requires the availability of liquid water and therefore those planetary bodies outside Earth that have or may have had aqueous environments are of particular interest in the search for life beyond Earth. The dry river valleys on Mars are remnants of an oceanic history [
3], and liquid water may still exist on Martian polar caps or below its surface [
4]. Large oceans of liquid water are also believed to exist under the ice sheets of Jupiter’s moon Europa [
5] and Saturn’s moon Enceladus [
6]. This is evidenced by the Cassini mission’s observations of frozen water plumes at the south pole of Enceladus and the presence of oxygen, nitrogen, carbon, phosphorous, and sulphur, and the Hubble telescope’s images of similar cryogeyser activity on Europa. Estimates of the number of Earth-like planets orbiting Sun-like stars in our galaxy exceeds six billion [
7,
8,
9] and spectroscopic signatures of water vapour has been reported in the atmosphere of exoplanets such as K2-18b [
10].
Interestingly, chondrite meteorites contain up to 5%, mostly inorganic, carbon but a high proportion of organic compounds [
11]. A number of amino acids, polyols, sugars, sugar alcohols, and sugar acids have been found on carbonaceous chondrite meteorites including the Murchison and Murray meteorites [
12] and Tagish Lake meteorite [
13]. The Murchison meteorite that fell in Australia in 1969 contains over 100 amino acids amongst many thousands of organic molecules ranging from two to nine carbons [
14,
15,
16]. Although meteoritic amino acids could be formed in the parent bodies by the Strecker reaction, in which aldehyde or ketone reacts with cyanide and ammonia followed by hydrolysis to produce α-amino acid [
17] but would produce only α-amino acids (amino and carboxyl group at the same carbon), and cannot explain the formation of β, γ and δ structures. Rigorous analytical methodologies excluded the possibility that this was due to contamination with organic molecules from Earth.
Of the organic molecules found in these meteorites, most are chiral, existing as both L- and D-stereoisomers (enantiomers). Terrestrial biology utilizes the L-enantiomer of amino acids preferentially but not exclusively [
18] while abiotic processes typically produce racemic mixtures. In the Murchison meteorite, several amino acids including alanine and isovaline exist in non-racemic (L-excess) mixtures [
19,
20]. Although non-biotic explanations have been suggested for this homochirality, these observations have supported speculations about exo-biological signatures within the solar system [
21].
Two reports have described polymers of amino acids in carbonaceous meteorites, the first being of di-glycine [
22], and second large polymers of mainly glycine in the CV3 class carbonaceous chondrite Allende [
23] before further characterization of amino acid polymers in Acfer 086 and Allende meteorites [
23,
24] identified the first protein in a meteorite hence in any extra-terrestrial source [
25]. The amino acid signatures in the meteorites like Murchison and Tagish Lake meteorite [
13] include the presence of high concentrations of non-protein amino acids that are exceedingly rare on Earth, such as isovaline and α-aminoisobutyric acid, and extremely low concentrations of amino acids that are common in terrestrial biota [
19,
26]. Those non-protein α-dialkyl-amino acids have been used as an indication of the indigeneity of meteoritic amino acids. These amino acids have also been detected in peptides produced by some filamentous fungi, suggesting the possibility of a terrestrial biotic source for some of the amino acids observed in meteorites. However, the relatively simple distribution of the C4 and C5 amino acids in fungal peptides (peptaibols or peptaibiotics) is distinct from the complex distribution observed in many carbonaceous chondrites, and extensive diagnostic analyses of stable isotope composition ruled out fungal contamination as a source of meteorite amino acids [
27].
If microbial life evolved outside Earth, it is conceivable that the composition of such organisms may include such unusual but available organic molecules. Space missions such as the Mars 2020 seek evidence of exo-life and may attempt the retrieval of samples from planets or moons where life could exist. This poses potential biosecurity risks and problems of contamination of the Earth ecosystem and even infection by exo-microorganisms [
28]. Here, we investigate the hypothetical risk of inefficient immune responses upon encountering organisms composed of antigens that could contain these rare exo-amino acids. We pose the question as to whether such exo-peptides have the capacity to be processed by cells of the mammalian immune system and induce adaptive immune responses.
To do this, we synthesised peptides in vitro based on a backbone of an immunodominant peptide epitope of ovalbumin that incorporated isovaline and α-aminoisobutyric acid residues at a range of positions. We then examined the efficiency of these peptides to activate and induce expansion of CD8+ T cells from ovalbumin-specific T cell receptor (TCR) transgenic OT-I mice.
The progression of T cells from a resting state to fully activated, proliferating cells is a crucial step in the initiation of an immune response. CD8
+ T cells recognize pathogen-derived peptides of 8–10 amino acids in length that are bound to the major histocompatibility complex (MHC) class I receptor presented by antigen-presenting cells (APC) [
29,
30,
31]. Naïve CD8
+ T cells then undergo a program that drives them to expand and differentiate into cytotoxic effector cells that can kill and eventually clear the pathogen [
32]. The capacity to proliferate following cognate antigen recognition is an important aspect of mammalian T cell immune response. In our work, we analyse CD8
+ T cell activation and subsequent proliferation upon stimulation with exo- amino acid containing peptides. The results indicate that antigen recognition and immune response of mammalian cells occurred in response to the exo-peptides but that this was less efficient than for the canonical control peptide.
4. Discussion
In this paper, we consider whether the immune system is able to recognise peptides containing amino acids that are not commonly found in terrestrial organisms but are known to be common in meteorites, and therefore may be represented in non-terrestrial life forms. It is likely that NASA or commercial space exploration by companies such as SpaceX, Virgin Galactic or Blue Origin will promote travel and exploration of other planets and the sending of long-range probes to retrieve samples. Such missions could contaminate exo-environments with terrestrial microorganisms that temporarily survived some of the harsh conditions of space-radiation, vacuum, extremely variable temperatures, etc., [
37,
38] but could also encounter non-terrestrial microorganisms that may have evolved in aqueous environments that have the potential to colonize or even infect humans and other animals. Liquid water is likely to exist on Jupiter’s moon Europa and on Saturn’s moon Enceladus and may exist below the surface of Mars [
5,
6]. Experiments performed in NASA’s Viking mission inoculated Martian soil with
14C-labeled nutrients in water, but failed to provide conclusive evidence of extant life, although the release of
14CO
2 from these samples was intriguing [
39].
On Earth, the boundary conditions under which life can exist has shown that microbial life is possible even at extremes of temperature, pH, pressure, radiation, salinity, energy, and nutrient limitation, as long as there is liquid water. Extremophiles, which span all three domains of life: bacteria, archaea and eukaryotes are important because of their enormous biotechnology potential but also because what they can teach us about the fundamentals of biochemical and structural biodiversity. Extremophile research reinforces the suggestion that microbial life may exist on other planetary and celestial bodies [
40] demonstrated by several studies that showed the growth of earth microorganisms under lab-simulated planetary conditions, including Mars-like [
41,
42,
43] and Enceladus-like [
44] conditions.
A recent review proposed two hypothetical scenarios of the human immune system interacting with alien microorganisms-terrestrial microbes that have grown and adapted to an alien environment or alien exo-microorganisms with different biochemistries and antigenicity profiles [
28]. Both settings anticipate the possible encounter of the terrestrial immune defense systems with distinct or novel organisms and biomolecules that may have radical departures from terrestrial canonical signatures that activate immune responses.
The induction of innate and adaptive immune responses towards invasive microorganisms is crucial for defense. We tested putative “exo-peptides” with antigenic potential that included amino acid substitutions with isovaline (Iva) and α-aminoisobutyric acid (Aib) that are present abundantly in carbonaceous meteorites [
14]. This anticipates a potential hypothetical encounter of the immune defense system with an antigen that contains those rare amino acids. We tested whether these non-canonical amino acids affected antigen processing, presentation, and stimulation of T cell responses of the model ovalbumin SIINFEKL epitope. We showed that the consequences of MHC class I antigen presentation in terms of T cell induction and proliferation was less efficient when presented with these exo-peptides than with canonical peptide controls.
This has implications for astronauts and space exploration missions designed to retrieve samples from potentially biotic exo-environments. The effects of exposure to a novel microbe could be exacerbated in astronauts where the human body and the immune system have already been exposed to sustained extreme conditions and environmental stress [
45]. Although astronauts have been shown to be able to survive in good health after many months in space, there is evidence that space flight can progressively weaken immune responses [
46]. Human neutrophils and monocytes post space flight exhibit reduced capacities of bacteria phagocytosis and an attenuated oxidative burst and degranulation [
47,
48]. Decreased responsiveness for host defense cells against potential invading pathogens could have contributed to the observation that nearly half of all Apollo crew members suffered from microbial infections shortly after they returned from space missions. Reduced T cell activation following proliferation after exo-peptide stimulation in our study suggests a risk that the potency of protective immune surveillance mechanisms could be attenuated when challenged by novel antigenic signatures.
It is not clear whether an extraterrestrial microorganism adapted to a non-terrestrial, extreme environment would be pathogenic in a human host. Similarly, it is likely that they would be poorly adapted to the conditions of the human body, and their capacity to colonize and infect us would be limited. However, a thermophilic bacterium
Mycobacterium xenopi was found in a hospital’s hot water system and three out of 87 patients exposed to this microorganism developed pulmonary mycobacteriosis [
49] suggesting that some terrestrial extremophiles have pathogenic potential for humans. Gene expression of
Enterobacter bugandensis isolated from the International Space Station (ISS) showed an increase in expression of genes involved in antimicrobial resistance (AMR), multiple drug resistance (MDR) and genes related to virulence and disease [
50]. Additionally, the competency of bacteria to acquire foreign genetic material was observed to increase in microgravity [
50]. This number of changes to the pathogenicity of microbes might become relevant during prolonged space travel. Even if the pathogenic potential for exo-microorganisms was inherently attenuated, it is also possible that they could induce allergic reactions or create novel toxigenic compounds [
28].
We show that exo-peptides were recognized by the mammalian immune system, but that the strength of the immune response was decreased. We can anticipate that the conditions of space travel impact on the immune system [
45]. Astronauts on extended spaceflights experienced already a general decay in T cell function, accompanied by persistent reductions in production of cytokines such as interleukin (IL)-5, IL-6, IL-10, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) [
51]. In addition, studies on animals had shown that space flight not only affect cytokine production [
52] and leukocyte subpopulation distribution of T lymphocytes (CD8
+ T lymphocytes and interleukin-2 receptor-bearing T lymphocytes) [
53], but also severe inhibition of bone marrow response to the colony-stimulating factors have been recorded in experimental rats and monkeys [
52,
53,
54]. Both the altered leukocyte distribution and impaired ability of precursor cells to differentiate into mature, immunologically competent cells contribute to alterations in cell-mediated immunity and impaired immune function observed after space flight.
Naïve CD8
+ T cells play a key role in protective immunity in response to foreign by differentiation into cytotoxic effector cells [
32]. At peak response, these effector T cells secrete high amounts of cytokines [interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα)] and cytolytic molecules (granzymes and perforin). Subsequently, after elimination of the antigenic source, most of these effector T cells undergo apoptosis, and a few survive and become central memory and effector memory T cells [
55,
56]. This differentiation process is tightly controlled and changes in the nature, context and duration of antigen exposure can alter the process leading to T cell dysfunction, unresponsiveness and/or death. The experiments outlined here suggest that multiple elements of the vital roles of T cell mediated protection could be attenuated when responding to non-canonical antigens.
In conclusion, the amino acids isovaline (Iva) and α-aminoisobutyric acid (Aib) have been identified as common organic molecules on chondrites. We show that the mammalian immune system recognized peptides containing these exo-amino acids but that T cell activation and proliferation was reduced. Future studies should extent our studies to encompass the immunomodulatory effects of a wider range of organic components including exo-sugars and other novel organics molecules that have been found on meteorites.