Interactions between Hydra and symbiotic bacteria

In addition to host-photobiont interactions in Hydra vi rid is si та, all Hydra species are stably associated with a species-specific microbiome. Even after over 30 years of culturing in the lab under artificial conditions. Hydra species maintain their distinct bacterial composition (Fraune and Bosch 2007; Franzenburg et al. 2013b) which resembled the microbiota of polyps freshly collected from the wild. This, together with the cellular and molecular accessibility of the Hydra polyp for functional approaches, allows investigating the interaction between microbes and Hydra polyps in an unprecedented manner and at the same time provide insight into the evolution of host-microbe interactions.

Spatial localization of the bacteria in the Hydra host

The Hydra ectodermal epithelium can be considered as an inside-out gut, with the mucosal layer facing towards the outside (Schroder and Bosch 2016). A carbohydrate and glycoprotein-rich layer, called glycocalyx, covers the outer surface of the polyp (Figure 5.2). The glycocalyx is a multi-layered structure composed of at least five layers with first four layers anchored to the ectoderm. Interestingly, the presence of bacteria was detected only in the most distal layer of the glycocalyx (Fraune et al. 2015), which acts as the first barrier between the environmental bacteria and the host tissue. In H. oligactis, a few of the bacterial symbionts are endosymbiotic in the ectodermal epithelial cells (Fraune and Bosch 2007). Most of the functional studies were done in H. vulgaris AEP or H. magnipapillata, which do not harbor any endosymbiotic bacteria (Fraune and Bosch 2007; Fraune et al. 2015).

Summary of interactions between Hydra and symbiotic bacteria

FIGURE 5.2 Summary of interactions between Hydra and symbiotic bacteria.

Epithelial cells produce anti-microbial peptides (AMPs) belonging to Arminin/Hydramacin/ Periculin families. Gland cells in the endoderm express Kazal family AMPs. I-cells, after being differentiated into female germ-line cells express AMP- Periculinla. Anti-bacterial neuropeptides NDA-1, Hym-370. Hym-357. and the RFamide III family are expressed in nerve cells. Stem-cell transcription factor FoxO plays a regulatory role in the expression of Hydramacin/Arminin/Kazal, without affecting Periculin and NDA-1. Mucin containing vesicles (in blue) in ectodermal epithelial cells contribute to the establishment of the glycocalyx.

Bacteria provide protection against fungal infection

Being able to separate the Hydra host and bacterial partners provides an efficient analytical framework to disentangle the contribution of each partner to the working of the holobiont. The germ-free H. vulgaris AEP hosts are highly susceptible to the infection by Fusarium sp. Fungus, suggesting a strong protective role of bacteria against a fungal pathogen. Colonization of hosts with single bacterial symbiont species failed to provide resistance against the fungi pointing to the importance of a complete and diverse bacterial community in maintaining the holobiont homeostasis.

The innate immune system shapes the host microbiome

Since the epithelial layer of Hydra is constantly in contact with a large variety of bacteria including pathogens, a wide array of competing forces seems to ensure the selection of specific bacterial species from the pool of bacterioplankton (Deines et al.

2017) . But do Hydra polyps actively select their microbial symbionts? On testing neutrality in the microbial composition for Hydra vulgaris AEP, it was recently observed that the microbiome composition deviates from the theoretically predicted neutral community, indicating that under the prevailing environmental conditions a major part of the microbiota is affected by host-derived factors (Sieber et al. 2019).

These theoretical considerations are supported by experimental work, which over the last decade has shown that Hydra’s innate immune system plays a major role in shaping the species-specific microbiota (Bosch 2014; Klimovich and Bosch

2018) . The recognition of the MAMPs occurs by Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) like receptors (NLRs) (Bosch et al. 2009). The structure of Hydra TLRs is unconventional: it is composed of leucine-rich repeat (LRR) domain containing protein (HyLRR) and a separate Toll/interleukin-1 receptor (TIR) domain containing protein (HyTRR). Both these proteins interact to form a functional TLR. The TIR domain of HyTRR interacts with the TIR domain of myeloid differentiation factor 88 (MyD88) protein. Upon stimulation by MAMPs. the signaling finally activates c-Jun N-terminal kinase (JNK) and Hydra orthologue of nuclear factor kappa-light-chain-enhancer of activated В cells (HyNF-кВ). These transcription factors induce the expression of various anti-microbial peptides (AMPs) that play a crucial role in shaping the host microbiome (Augustin et al. 2009; Bosch 2014).

Endodermal epithelial cells express the AMP peptide family ‘Hydramacin’ (Bosch et al. 2009; Jung et al. 2009). Hydramacin expression is induced by MAMPs via the TLR signaling pathway and has antimicrobial activity against various gram-positive and gram-negative bacteria. The endothelial gland cells in the body column express a considerably high amount of Kazal2, a kazal-type serine protease inhibitor that targets the bacterial serine proteases (Augustin et al. 2009) (Figure 5.2), and provides protection against pathogenic bacteria in the gastric cavity. Although Hydra lacks any migratory phagocytic cells, it does possess phagocytic activity in the endodermal cell. It is through this property, that the aposymbiotic H. viridissima is able to establish a symbiosis with Clilorella algae. The same phagocytic property can also help to keep the endoderm free of bacteria by engulfing any bacteria entering with the food (Bosch et al. 2009).

Maternal protection of the developing progeny against pathogens or bacterial overgrowth is provided by the AMP Periculinla which is expressed exclusively in maternal germ line cells (Fraune et al. 2010). This AMP persists during the initial phases of embryogenesis up to blastula stage, and controls the bacterial load as well as assists in selection of the colonizing bacteria. The function of maternal Periculinla is later taken over by zygotic Periculin2b after midblastula transition. In an adult H. vulgaris AEP polyp, the endodermal knockdown of another family of AMP, the Arminin peptides, results in a reduced selection by the host for the colonizing bacterial partners. Since in the absence of Arminin peptides the bacteria from other donor species, H. oligactis and H. viridissima, can colonize the host, these AMPs are involved in maintaining a specific-specific microbiota.

The colonization of a newly hatched polyp follows a robust temporal trajectory (Franzenburg et al. 2013a). There is a high variability in the microbiota initially, followed by a transient increase in the relative abundance of the main colonizers of the adult microbiota. Following this, there is a drastic decrease in the microbial diversity before it stabilizes to adult microbiota at the end of four weeks.

Crosstalk between innate immunity and stem cell factors

In Hydra, and most likely in other organisms as well, the innate immune system is tightly linked to pathways controlling epithelial cell homeostasis. Hydra's Forkhead box transcription factor (FoxO) is key to maintaining the stem cell identity of the ecto- and endodermal epithelial cells (Boehm et al. 2012) (Figure 5.2). The expression of FoxO is limited to the stem cell zone and absent in the terminally differentiated cells. Epithelial FoxO loss-of-function mutants revealed that a deficiency in FoxO signaling leads not only to malfunctions in cell cycle progression, but also to dysregulation of multiple families of genes encoding antimicrobial peptides (AMPs). FoxO loss-of-function polyps were more susceptible to colonization by foreign bacteria, and impaired in selection for bacteria resembling the native microbiome. FoxO-deficiency reduces the expression of AMPs, resulting in decreased selective pressure on colonizing microbial taxa and ultimately in reduced resilience of the microbiome (Boehm et al. 2012; Mortzfeld et al. 2018).

Crosstalk between the microbiota and the nervous system

In addition to the epithelial cells, the nervous system also plays an important role in shaping the microbial community of Hydra. First indication of crosstalk between both the systems came from the observation that loss of nerve-cell lineage resulted in an increased anti-bacterial activity of the Hydra polyps (Fraune et al. 2009). A recent study (Augustin et al. 2017) provided evidence that some sensory and ganglion neurons express a cationic neuropeptide called NDA-1, secrete it into the mucus layer and regulate the spatial distribution of the main colonizer, the gramnegative bacterium Curvibacter, along the Hydra trunk. The density of Curvibacter colonization is relatively low in the foot and tentacles of Hydra, where NDA-1 is strongly expressed, compared to the body column. Additionally, NDA-1 is highly potent in killing gram-positive bacteria. Strikingly, other neuropeptides, such as Hydra specific Hym-357 and Hym-370, and a member of the highly conserved

RFamide family, all previously characterized as classical neuromodulators eliciting motor activity, turned out to be also potent against gram-positive bacteria. Taken together, these findings indicate that distinct nerve cells contribute to the composition and spatial structure of Hydra’s microbial community by expressing a variety of neuropeptides with distinct antimicrobial activities.

Based on these observations, we proposed (Klimovich and Bosch 2018) that during evolution the nervous system, in addition to its role in sensory input/motor output, plays a primordial role as part of the innate immune system.

All these observations, taken together, portray a rather complex network of molecular and cellular interactions controlling the establishment and maintenance of a stable microbiota in Hydra (Figure 5.2).

The level of complexity of the interspecies crosstalk in the holobiont Hydra is even larger since many of the bacterial partners harbour temperate phages bearing the capacity to cross infect other bacterial species. One such example is observed as competition between the top two main colonizers of H. vulgaris AEP. Curvibacter sp. and Duganella sp. (Li et al. 2015, 2017). Associated with the host, Curvibacter out-competes Duganella, while in-vitro, in monocultures the effect is reversed. However, the co-culture of both the bacteria in-vitro results in a non-linear reduction of growth of Duganella depending on the initial frequency of Curvibacter. When modelled mathematically, the interaction between both the bacteria in co-culture could only be explained by the presence of a third partner, a bacteriophage. Indeed, Curvibacter inhabits a pro-phage that can be activated to enter the lytic cycle and have lytic activity against Duganella. Thus, the in-vivo competition between the top two colonizers is likely to be mediated by a bacteriophage.

Effect of bacteria on host physiology

With the host factors have a profound effect on its microbiome, there also strong impacts of the bacterial partners on the host physiology and host behaviour. Hydra exhibits various behaviour like spontaneous contraction, feeding response to a stimulus and phototactic movement, to name a few. Spontaneous contractions are assumed to be regulated by the pacemaker activity of the neurons (Passano and Mccullough 1965). Interestingly, the frequency of the spontaneous contractions in germ-free H. vulgaris AEP animals is reduced to ~60% of the control animals (Murillo-Rincon et al. 2017). This effect can be restored, although not completely, by recolonization of these germ-free animals by normal microbiome. Moreover, the microbial extract from the in-vitro grown complete microbial community on a complex media, largely resulted in restoration of the contraction frequency. Bacteria, or bacteria-derived molecules, therefore, directly interfere with neuronal activity and function.

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