Our research is focusing on the understanding of host-microbe interactions. In many animals epithelial tissues are colonized by complex communities of microbes. These microbes influence the fitness of their hosts, ultimately forming a metaorganism consisting of a multicellular host and a community of associated microorganisms. The basis of our approach is the hypothesis that host-to-microbe, microbe-to-host and microbe-to-microbe communications select for a core microbiota in a given host species which contributes to metaorganism fitness and adaptation. Microbial symbionts represent a specific form of genetic inheritance, being either vertically acquired through the egg or horizontally transmitted through the environment. Changes in the bacterial community or genetic variation in the symbionts can produce phenotypic variation of the metaorganism and may affect the ecological tolerance and distribution of the host. Using the two cnidarian model systems Hydra and Nematostella, we aim to answer the question why and how do individuals from different taxonomic groups and even kingdoms of life interact with each other within a metaorganism in an often beneficial way?
Cnidaria have the simplest epithelia in the animal kingdom consisting of only two cell layers, but they preserved much of the genetic complexity of the common metazoan ancestor. Thus, general principles of host-microbe interactions that operate in all metazoan life including humans can be investigated in these animals. Due to the transparent appearance of the animals and the ability of transgenic labeling of single cells, microscopic analysis can be archived in an in vivo context on single cell as well as on whole tissue level.
Hydra has adopted a life cycle in which proliferation and population growths occur asexually by budding. This asexual budding is maintained by the continuous self-renewal capacity of its three somatic stem cell lineages leading to a non-senescence phenotype. Hydra is consisting of a simple tube and has one of the simplest epithelia in the animal kingdom, with only two cell layers, and few cell types derived from three distinct stem cell lineages: the ectodermal and endodermal epithelial stem cells, and the interstitial stem cells.
Nematostella is a marine organism and individuals are found in a large range of environmental conditions concerning temperature and salinity. Nematostella is a well-established developmental model system and the animals are easy to culture and to crossbred in the lab with a generation time of 3-4 month. The genome of Nematostella is sequenced (360 Mb) and in addition a large set of transcriptomic data is available. For functional studies the animals can be modified by the use of morpholinos, transgenesis and CRSIPR/Cas9.
1. Host-microbe communication: host mechanisms controlling behavior of bacterial colonizers
Epithelial surfaces of most animals are colonized by specific bacterial communities. Therefore, it is important for the host to control both the composition and behavior of bacterial colonizers. Up to know, only few host mechanisms targeting the behavior of bacteria have been identified.
Many gram-negative bacteria are able to coordinate their behavior using fatty-acid-derived signaling molecules of the acyl homoserine lactone (AHL) family. This cell-density-based gene regulation is known as quorum sensing (QS). Degradation of QS signals results in quorum quenching (QQ). It was proposed that bacteria produce QQ enzymes as a self-modulatory system of their own QS system, but also eukaryotic tissues seem to be able to interfere with the bacterial QS signaling directly.
We were able to show, to our knowledge for the first time in vivo, that an animal can manipulate bacterial communication (quorum sensing) signals produced by colonizing bacteria. Using the early branching metazoan Hydra as a model host, we identified by LC-ESI MS (liquid chromatography coupled electrospray ionization mass spectrometry) a new eukaryotic quorum quenching mechanism which enables the host epithelium to specifically modify the quorum sensing signals of its bacterial colonizers. While the host-modified signal represses bacterial genes involved in the biosynthesis of the flagellum, the non-modified signal induces the flagellar machinery in vitro and in vivo. Investigating the impacts of the different quorum sensing signals on metaorganism assembly in vivo elucidated, that the host-modified signal promotes, while the non-modified signal represses symbiont colonization.
These results show that a host organism is able to regulate the behavior of its bacterial colonizers by modifying bacterial quorum sensing signals. The animal thereby not only maintains the symbiotic function of its bacterial colonizers, but also supports the homeostasis of the metaorganism.
Currently we are investigating the source of the QQ activity of Hydra by MS analysis (together with the group of Prof. Andreas Tholey, Kiel) in more details. In collaboration with Prof. Tal Dagan and Dr. Nils Hülter we are using a gene targeting system for the generation of chromosomal knockout mutations for QS in Curvibacter sp. to study the influence of QS of Curvibacter sp. on host colonization. To investigate the QS activity of Curvibacter sp. during host colonization by fluorescence microscopy, we are developing Curvibacter sp. reporter strains expressing GFP under the control of QS sensitive promotors. By the use of these bioreporter strains we will be able to determine the in vivo dynamcis of QS and spatial distribution of Curvibacter cells at the host epithelium.
- Wein T, Dagan T, Fraune S, Bosch TCG, Reusch TB, Hülter NF Priority rules in microbiota colonization dynamics (2018). Front Microbiol., 9
- Pietschke C, Treitz C, Forêt S, Schultze A, Künzel S, Tholey A, Bosch TCG, Fraune S (2017) Host modification of a bacterial quorum sensing signal induces a phenotypic switch in bacterial symbionts. Proc Natl Acad Sci USA, 114(40):E8488-E8497
2. Establishment of bacterial colonization during host ontogeny
Marine invertebrates are in constant contact with a vast number of highly diverse microbes in their environment. As such, they are ideal models for examining prokaryote-eukaryote interactions. The establishment of host-bacterial colonization during development is a fundamental process influencing the fitness of many organisms, but the factors controlling community membership and influencing the establishment of the microbial ecosystem during development are poorly understood.
We have started to investigate the host molecular mechanisms involved in bacterial recognition and establishment in the marine model system Nematostella vectensis. By investigating the bacterial colonization from the earliest developmental stages to one year old adult polyps under different environmental conditions, we could clearly demonstrate the robust colonization process in Nematostella. The clear separation of bacterial communities of early developmental stages, juvenile and adult polyps is correlated to major transitions within the development of Nematostella, mostly independent of environmental factors. Thus, we hypothesize that the age dependent bacterial colonization in Nematostella is reflecting development dependent changes in the innate immune system. Therefore, one of the aims of this project is to characterize the immune system of Nematostella depending on the developmental age. We will concentrate on the immune response to bacterial colonization and on bacterial recognition.
- Domin H, Zurita-Gutiérrez YH, Scotti M, Buttlar J, Hentschel Humeida U, Fraune S (2018). Predicted bacterial interactions affect in vivo microbial colonization dynamics in Nematostella. Front Microbiol., 9, 728, doi.org/10.3389/fmicb.2018.00728
- Mortzfeld BM, Urbanski S, Reitzel AM, Künzel S,Technau U, Fraune S (2015) Response of bacterial colonization in Nematostella vectensis to development, environment and biogeography, Environ. Microbiol. doi: 10.1111/1462-2920.12926
3. Beyond the genome: impact of microbial communities and epigenetic regulations for thermal adaptation
The phenotype of an animal cannot be explained entirely by its genes. It is now clear that factors other than the genome contribute to the development and the dynamic homeostasis of multicellular animals. Two fundamentally important factors are epigenetic regulations and the microbial communities associated with the animal. Unlike the genes and regulatory regions of the genome, epigenetics and microbial composition can be rapidly modified by environmental cues, and may thus represent mechanisms for rapid acclimation of individuals to a changing environment. At present, the individual functions of epigenetics, microbiomes, and genomic mutations are largely studied in isolation, particularly for species in marine ecosystems. This leaves significant questions open for how these mechanisms intersect in the acclimation and adaptation of organisms to environmental changes.
The aim of this research is to determine how epigenetic regulations and microbial communities participate in thermal acclimation of a coastal marine species residing in a dynamic temperature environment, and how these non-genetic factors interact with each other. The model species used for this study, the sea anemone Nematostella vectensis, enables us to carry out unprecedented functional experiments to dissect the interactions between microbes and epigenetic mechanisms in the acclimation of the holobiont.
We are currently monitoring the physiological, transcriptomic, epigenetic and microbial changes associated with thermal acclimation. We will then separate the effects of microbial and epigenetic effects in a series of bacterial transplantation experiments. Finally, we will carry out gene knockdown and over-expression experiments to elucidate the function of critical host genes in epigenetic regulations and in the plasticity of the microbiota. We hypothesize that changes in the microbial community improve the thermal tolerance of the host, and that the epigenetic landscape is responding both to the shifts in temperature and to the altered microbial composition.
The project is performed in collaboration with Adam Reitzel (University of North Carolina at Charlotte, USA) and Gavin Huttley (The Australian National University, Australia)
- Fraune S, Forét S, Reitzel AM (2016) Using Nematostella vectensis to study the interactions between genome, epigenome, and bacteria in a changing environment, Frontiers in Marine Science 3, 148, doi: 10.3389/fmars.2016.00148
4. Bacterial interference with host stem cell function
Animal development has traditionally been viewed as an autonomous process directed by the host genome. But in many animals biotic and abiotic cues, like temperature and bacterial colonizers, provide signals for multiple developmental steps. Hydra offers unique features to encode these complex interactions of developmental processes with biotic and abiotic factors. Here, we investigated the impact of bacterial colonizers on stem cell proliferation and differentiation. In Hydra, stem cell differentiation into head specific cells involves the canonical Wnt pathway. Treatment with alsterpaullone (ALP) results in stem cell differentiation in the whole body column, the main stem cell compartment. In this project, we focus on two aspects. First, how do bacterial colonizers affect stem cell behavior? Second, how are these interactions integrated into the well characterized network of transcription factors and signal transduction pathways in Hydra? By using Hydra polyps colonized with a single bacterial species, we have begun to evaluate each ones contribution to Hydra development and stem cell activity. Intriguingly, some bacteria-dependent host genes show a distinct spatial-temporal expression pattern suggesting a distinct role in Hydra´s developmental and patterning processes. In addition, chemical interference experiments for the wnt-signaling pathway in germ-free and temperature treated polyps elucidate a deep cross talk between these external cues and stem cell proliferation and differentiation. By using transgenic Hydra models interfering with essential host signaling pathways and already identified candidate genes, we aim to elucidate the molecular pathways involved in this crosstalk and the corresponding bacterial signaling molecules mediating the observed effects.