Corinna Bang

The Intestinal Archaea Methanosphaera stadtmanae and Methanobrevibacter smithii Activate Human Dendritic Cells

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in humans health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents. Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.

Effects of Antimicrobial Peptides on Methanogenic Archaea

ABSTRACT As members of the indigenous human microbiota found on several mucosal tissues, Methanobrevibacter smithii and Methanosphaera stadtmanae are exposed to the effects of antimicrobial peptides (AMPs) secreted by these epithelia. Although antimicrobial and molecular effects of AMPs on bacteria are well described, data for archaea are not available yet. Besides, it is not clear whether AMPs affect them as the archaeal cell envelope differs profoundly in terms of chemical composition and structure from that of bacteria. The effects of different synthetic AMPs on growth of M. smithii, M. stadtmanae, and Methanosarcina mazei were tested using a microtiter plate assay adapted to their anaerobic growth requirements. All three tested methanoarchaea were highly sensitive against derivatives of human cathelicidin, of porcine lysin, and a synthetic antilipopolysaccharide peptide (Lpep); however, sensitivities differed markedly among the methanoarchaeal strains. The potent AMP concentrations affecting growth were below 10 μM, whereas growth of Escherichia coli WBB01 was not affected at peptide concentrations up to 10 μM under the same anaerobic growth conditions. Atomic force microscopy and transmission electron microscopy revealed that the structural integrity of the methanoarchaeal cells is destroyed within 4 h after incubation with AMPs. The disruption of the cell envelope of M. smithii, M. stadtmanae, and M. mazei within a few minutes of exposure was verified by using LIVE/DEAD staining. Our results strongly suggest that the release of AMPs by eukaryotic epithelial cells is a potent defense mechanism targeting not only bacteria, but also methanoarchaea.

First Insights into the Diverse Human Archaeome: Specific Detection of Archaea in the Gastrointestinal Tract, Lung, and Nose and on Skin

ABSTRACT Human-associated archaea remain understudied in the field of microbiome research, although in particular methanogenic archaea were found to be regular commensals of the human gut, where they represent keystone species in metabolic processes. Knowledge on the abundance and diversity of human-associated archaea is extremely limited, and little is known about their function(s), their overall role in human health, or their association with parts of the human body other than the gastrointestinal tract and oral cavity. Currently, methodological issues impede the full assessment of the human archaeome, as bacteria-targeting protocols are unsuitable for characterization of the full spectrum of Archaea. The goal of this study was to establish conservative protocols based on specifically archaea-targeting, PCR-based methods to retrieve first insights into the archaeomes of the human gastrointestinal tract, lung, nose, and skin. Detection of Archaea was highly dependent on primer selection and the sequence processing pipeline used. Our results enabled us to retrieve a novel picture of the human archaeome, as we found for the first time Methanobacterium and Woesearchaeota (DPANN superphylum) to be associated with the human gastrointestinal tract and the human lung, respectively. Similar to bacteria, human-associated archaeal communities were found to group biogeographically, forming (i) the thaumarchaeal skin landscape, (ii) the (methano)euryarchaeal gastrointestinal tract, (iii) a mixed skin-gastrointestinal tract landscape for the nose, and (iv) a woesearchaeal lung landscape. On the basis of the protocols we used, we were able to detect unexpectedly high diversity of archaea associated with different body parts. IMPORTANCE In summary, our study highlights the importance of the primers and data processing pipeline used to study the human archaeome. We were able to establish protocols that revealed the presence of previously undetected Archaea in all of the tissue samples investigated and to detect biogeographic patterns of the human archaeome in the gastrointestinal tract and on the skin and for the first time in the respiratory tract, i.e., the nose and lungs. Our results are a solid basis for further investigation of the human archaeome and, in the long term, discovery of the potential role of archaea in human health and disease. IMPORTANCE In summary, our study highlights the importance of the primers and NGS data processing pipeline used to study the human archaeome. We were able to establish protocols that revealed the presence of previously undetected Archaea in all of the tissue samples investigated and to detect biogeographic patterns of the human archaeome in the gastrointestinal tract, on the skin, and for the first time in the respiratory tract, i.e., the nose and lungs. Our results are a solid basis for further investigation of the human archaeome and, in the long term, discovery of the potential role of archaea in human health and disease.

Biofilm formation of mucosa-associated methanoarchaeal strains

Although in nature most microorganisms are known to occur predominantly in consortia or biofilms, data on archaeal biofilm formation are in general scarce. Here, the ability of three methanoarchaeal strains, Methanobrevibacter smithii and Methanosphaera stadtmanae, which form part of the human gut microbiota, and the Methanosarcina mazei strain Gö1 to grow on different surfaces and form biofilms was investigated. All three strains adhered to the substrate mica and grew predominantly as bilayers on its surface as demonstrated by confocal laser scanning microscopy analyses, though the formation of multi-layered biofilms of Methanosphaera stadtmanae and Methanobrevibacter smithii was observed as well. Stable biofilm formation was further confirmed by scanning electron microscopy analysis. Methanosarcina mazei and Methanobrevibacter smithii also formed multi-layered biofilms in uncoated plastic μ-dishesTM, which were very similar in morphology and reached a height of up to 40 μm. In contrast, biofilms formed by Methanosphaera stadtmanae reached only a height of 2 μm. Staining with the two lectins ConA and IB4 indicated that all three strains produced relatively low amounts of extracellular polysaccharides most likely containing glucose, mannose, and galactose. Taken together, this study provides the first evidence that methanoarchaea can develop and form biofilms on different substrates and thus, will contribute to our knowledge on the appearance and physiological role of Methanobrevibacter smithii and Methanosphaera stadtmanae in the human intestine.

Health- and disease-associated species clusters in complex natural biofilms determine the innate immune response in oral epithelial cells during biofilm maturation

The aim of the present study was to verify our hypothesis concerning the differential induction of various antimicrobial and immunomodulatory responses in oral epithelial cells by diverse bacterial species clusters. For this purpose, oral biofilms between 1 and 14 days of maturation (36 volunteers) were co-incubated with gingival epithelial cells. Subsequently, human β-defensin (hBD)-2, hBD-3, LL-37, interleukin (IL)-1β, IL-6, IL-8 and IL-10 mRNA expression profiles were quantified by quantitative reverse transcription PCR. The correlation between bacterial species and the host innate immune response was determined by relating these results to existing 16S rRNA phylogenetic analysis by amplicon sequencing (Langfeldt et al. 2014. PLoS One 9: e87449). Data were analysed by multiple factor analysis. Transcription of hBD-2 and hBD-3 was significantly associated with the abundance of species of the Prevotella cluster and the absence of species of the Streptococcus cluster. IL-1β, -6, -8 and -10 mRNA syntheses were significant correlated with Leptotrichia species [Leptotrichia 302H02 (0.448, P < 0.0001), Leptotrichia nbw822e09c1 (0.214, P = 0.008) and Leptotrichia wadei (0.218, P = 0.007)] of the Prevotella cluster. In the third dimension IL-10 and members of the Prevotella cluster were negatively correlated, whereas hBD-3 and IL-1β, IL-6 and IL-8 were positive correlated to axis 3, like members of the Proteobacteria cluster. In conclusion, distinct species of health- and disease-associated bacterial clusters induce antibacterial or immunomodulatory reactions in oral epithelial cells during early stages of bacteria-host interactions.

Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides

The methanogenic archaeon Methanomassiliicoccus luminyensis strain B10T was isolated from human feces just a few years ago. Due to its remarkable metabolic properties, particularly the degradation of trimethylamines, this strain was supposed to be used as “Archaebiotic” during metabolic disorders of the human intestine. However, there is still no data published regarding adaptations to the natural habitat of M. luminyensis as it has been shown for the other two reported mucosa-associated methanoarchaea. This study aimed at unraveling susceptibility of M. luminyensis to antimicrobial peptides as well as its immunogenicity. By using the established microtiter plate assay adapted to the anaerobic growth requirements of methanogenic archaea, we demonstrated that M. luminyensis is highly sensitive against LL32, a derivative of human cathelicidin (MIC = 2 μM). However, the strain was highly resistant against the porcine lysin NK-2 (MIC = 10 μM) and the synthetic antilipopolysaccharide peptide (Lpep) (MIC>10 μM) and overall differed from the two other methanoarchaea, Methanobrevibacter smithii and Methanosphaera stadtmanae in respect to AMP sensitivity. Moreover, only weak immunogenic potential of M. luminyensis was demonstrated using peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (moDCs) by determining release of pro-inflammatory cytokines. Overall, our findings clearly demonstrate that the archaeal gut inhabitant M. luminyensis is susceptible to the release of human-derived antimicrobial peptides and exhibits low immunogenicity towards human immune cells in vitro–revealing characteristics of a typical commensal gut microbe.

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.

Metaorganisms in extreme environments: do microbes play a role in organismal adaptation?

From protists to humans, all animals and plants are inhabited by microbial organisms. There is an increasing appreciation that these resident microbes influence the fitness of their plant and animal hosts, ultimately forming a metaorganism consisting of a uni- or multicellular host and a community of associated microorganisms. Research on host-microbe interactions has become an emerging cross-disciplinary field. In both vertebrates and invertebrates a complex microbiome confers immunological, metabolic and behavioural benefits; conversely, its disturbance can contribute to the development of disease states. However, the molecular and cellular mechanisms controlling the interactions within a metaorganism are poorly understood and many key interactions between the associated organisms remain unknown. In this perspective article, we outline some of the issues in interspecies interactions and in particular address the question of how metaorganisms react and adapt to inputs from extreme environments such as deserts, the intertidal zone, oligothrophic seas, and hydrothermal vents.