Exploring the human archaeome: its relevance for health and disease, and its complex interplay with the human immune system.

This Review aims to coalesce existing knowledge on the human archaeome, a less-studied yet critical non-bacterial component of the human microbiome, with a focus on its interaction with the immune system. Despite a largely bacteria-centric focus in microbiome research, archaea present unique challenges and opportunities for understanding human health. We examine the archaeal distribution across different human body sites, such as the lower gastrointestinal tract (LGT), upper aerodigestive tract (UAT), urogenital tract (UGT), and skin. Variability in archaeal composition exists between sites; methanogens dominate the LGT, while Nitrososphaeria are prevalent on the skin and UAT. Archaea have yet to be classified as pathogens but show associations with conditions such as refractory sinusitis and vaginosis. In the LGT, methanogenic archaea play critical metabolic roles by converting bacterial end-products into methane, correlating with various health conditions, including obesity and certain cancers. Finally, this work looks at the complex interactions between archaea and the human immune system at the molecular level. Recent research has illuminated the roles of specific archaeal molecules, such as RNA and glycerolipids, in stimulating immune responses via innate immune receptors like Toll-like receptor 8 (TLR8) and 'C-type lectin domain family 4 member E' (CLEC4E; also known as MINCLE). Additionally, metabolic by-products of archaea, specifically methane, have demonstrated immunomodulatory effects through anti-inflammatory and anti-oxidative pathways. Despite these advancements, the mechanistic underpinnings of how archaea influence immune activity remain a fertile area for further investigation.

Complete genome sequence of Methanosphaera sp. ISO3-F5, a rumen methylotrophic methanogen.

Methanosphaera spp. are methylotrophic methanogenic archaea and members of the order Methanobacteriales with few cultured representatives. Methanosphaera sp. ISO3-F5 was isolated from sheep rumen contents in New Zealand. Here, we report its complete genome, consisting of a large chromosome and a megaplasmid (GenBank accession numbers CP118753 and CP118754, respectively).

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

The Impact of Pre-Slaughter Fasting on the Ruminal Microbial Population of Commercial Angus Steers.

Diet impacts the composition of the ruminal microbiota; however, prior to slaughter, cattle are fasted, which may change the ruminal microbial ecosystem structure and lead to dysbiosis. The objective of this study was to determine changes occurring in the rumen after pre-slaughter fasting, which can allow harmful pathogens an opportunity to establish in the rumen. Ruminal samples were collected before and after pre-slaughter fasting from seventeen commercial Angus steers. DNA extraction and 16S rRNA gene sequencing were performed to determine the ruminal microbiota, as well as volatile fatty acid (VFA) concentrations. Microbial richness (Chao 1 index), evenness, and Shannon diversity index all increased after fasting (p ≤ 0.040). During fasting, the two predominant families Prevotellaceae and Ruminococcaceae decreased (p ≤ 0.029), whereas the remaining minor families increased (p < 0.001). Fasting increased Blautia and Methanosphaera (p ≤ 0.003), while Campylobacter and Treponema tended to increase (p ≤ 0.086). Butyrate concentration tended to decrease (p = 0.068) after fasting. The present findings support that fasting causes ruminal nutrient depletion resulting in dysbiosis, allowing opportunistic pathogens to exploit the void in the ruminal ecological niche.

The hydrogen threshold of obligately methyl-reducing methanogens.

Methanogenesis is the final step in the anaerobic degradation of organic matter. The most important substrates of methanogens are hydrogen plus carbon dioxide and acetate, but also the use of methanol, methylated amines, and aromatic methoxy groups appears to be more widespread than originally thought. Except for most members of the family Methanosarcinaceae, all methylotrophic methanogens require external hydrogen as reductant and therefore compete with hydrogenotrophic methanogens for this common substrate. Since methanogenesis from carbon dioxide consumes four molecules of hydrogen per molecule of methane, whereas methanogenesis from methanol requires only one, methyl-reducing methanogens should have an energetic advantage over hydrogenotrophic methanogens at low hydrogen partial pressures. However, experimental data on their hydrogen threshold is scarce and suffers from relatively high detection limits. Here, we show that the methyl-reducing methanogens Methanosphaera stadtmanae (Methanobacteriales), Methanimicrococcus blatticola (Methanosarcinales), and Methanomassiliicoccus luminyensis (Methanomassiliicoccales) consume hydrogen to partial pressures < 0.1 Pa, which is almost one order of magnitude lower than the thresholds for M. stadtmanae and M. blatticola reported in the only previous study on this topic. We conclude that methylotrophic methanogens should outcompete hydrogenotrophic methanogens for hydrogen and that their activity is limited by the availability of methyl groups.

Altered Gut Microbial Fermentation and Colonization with Methanobrevibacter smithii in Renal Transplant Recipients.

Renal transplant recipients (RTRs) often suffer from posttransplant diarrhea. The observed dysbiosis in RTR may influence the fermentation processes in the gut. In this study, we aimed to investigate whether fermentation differs between RTRs and healthy controls (HCs), by measuring breath H2 and CH4 concentrations. Additionally, we determined the fecal presence of the methanogen Methanobrevibacter smithii (M. smithii), which plays a main role in the process of methanogenesis. Data from the TransplantLines Biobank and Cohort Study (NCT03272841) was used. A total of 142 RTRs and 77 HCs were included. Breath H2 concentrations in RTRs were not significantly different from HCs. Breath CH4 concentrations in RTRs were significantly lower compared with HCs (median [interquartile range (IQR)] 7.5 [3.9-10.6] ppm vs. 16.0 [8.0-45.5] ppm, p < 0.001). M. smithii was less frequently present in the feces of RTRs compared to HCs (28.6% vs. 86.4% resp., p < 0.001). Our findings regarding the altered methanogenesis in the gut of RTRs show similarities with previous results in inflammatory bowel disease patients. These findings provide novel insight into the alterations of fermentation after renal transplantation, which may contribute to understanding the occurrence of posttransplant diarrhea.

The Human-Associated Archaeon Methanosphaera stadtmanae Is Recognized through Its RNA and Induces TLR8-Dependent NLRP3 Inflammasome Activation.

The archaeon Methanosphaera stadtmanae is a member of the gut microbiota; yet, the molecular cross-talk between archaea and the human immune system and its potential contribution to inflammatory diseases has not been evaluated. Although archaea are as bacteria prokaryotes, they form a distinct domain having unique features such as different cell wall structures and membrane lipids. So far, no microbe-associated molecular patterns of archaea which activate innate immune receptors have been identified. By stimulating human myeloid cells with M. stadtmanae and purified archaeal nucleic acids, we identified both the microorganism and its RNA as potent stimuli for the innate immune system. To dissect the recognition and activation pathways induced by M. stadtmanae, human monocytic BLaER1 knockout cells were generated using the CRISPR/Cas9 system targeting components of TLR and inflammasome signaling. While the recognition of M. stadtmanae is mediated by TLR7 and TLR8, activation of the NLRP3 inflammasome depends solely on TLR8 engagement. Notably, this process resembles hallmarks of both the canonical and the recently described alternative inflammasome activation. Thus, we have demonstrated for the first time the specific recognition of and response to an archaeon by human cells at the molecular level.