Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Microbial growth in actual martian regolith in the form of Mars meteorite EETA79001.

Studies to understand the growth of organisms on Mars are hampered by the use of simulants to duplicate martian mineralogy and chemistry. Even though such materials are improving, no terrestrial simulant can replace a real martian sample. Here we report the use of actual martian regolith, in the form of Mars meteorite EETA79001 sawdust, to demonstrate its ability to support the growth of four microorganisms, E. coli. Eucapsis sp., Chr20-20201027-1, and P. halocryophilus, for up to 23 days under terrestrial conditions using regolith:water ratios from 4:1 to 1:10. If the EETA79001 sawdust is widely representative of regolith on the martian surface, our results imply that microbial life under appropriate conditions could have been present on Mars in the past and/or today in the subsurface, and that the regolith does not contain any bactericidal agents. The results of our study have implications not only for putative martian microbial life but also for building bio-sustainable human habitats on Mars.

Microbial Growth in Martian Soil Simulants Under Terrestrial Conditions: Guiding the Search for Life on Mars.

The search for life elsewhere in the Universe goes together with the search for liquid water. Life as we know it requires water; however, it is possible for microbial life to exist under hyperarid conditions with a minimal amount of water. We report on the ability of two typical terrestrial bacteria (Escherichia coli B and Eucapsis sp) and two extremophiles (Gloeocapsa-20201027-1 sp and Planococcus halocryophilus) to grow and survive in three martian soil (regolith) simulants (Mohave Mars Simulant-1 [MMS-1] F, Mars Global Simulant-1 [MGS-1], and JSC Mars-1A [JSC]). Survival and growth were assessed over a 21-day period under terrestrial conditions and with water:soil (vol:wt) ratios that varied from 0.25:1 to 5:1. We found that Eucapsis and Gloeocapsa sp grew best in the simulants MMS and JSC, respectively, while P. halocryophilus growth rates were better in the JSC simulant. As expected, E. coli did not show significant growth. Our results indicate that these martian simulants and thus martian regolith, with minimal or no added nutrients or water, can support the growth of extremophiles such as P. halocryphilus and Gloeocapsa. Similar extremophiles on early Mars may have survived to the present in near-surface ecological niches analogous to those where these organisms exist on Earth.

Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Campylobacter jejuni lipooligosaccharide sialylation, phosphorylation, and amide/ester linkage modifications fine-tune human Toll-like receptor 4 activation.

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.