Author Profile--Bosch: from the Classics to Classical Music, amid a Steadfast Focus on Hydra

The freshwater polyp Hydra, which belongs to an ancient animal phylum, still must defend itself against pathogens, much like other more complex organisms. Further, microorganisms colonize the epithelial layers of Hydra, much as they colonize other organisms. How do hosts distinguish benign epithelial colonizers from threatening pathogens, and what role do those colonizers play for Hydra in its freshwater environment, which is teeming with microbes of all sorts, including potential pathogens?

Hydra, each about 0.5 cm in length, prove a particularly suitable organism for addressing questions of how hosts interact with epithelial-bound microorganisms. Hydra are cnidarians, which includes corals, jelly fishes, and sea anemones-an early branch on the animal side of the tree of life, whose closest sister group is the Bilateria (Fig. 1A). The last common ancestor of the Cnidaria and Bilateria, likely the first animal with a nervous system, branched off about 700 million years ago.

Hydra has a relatively simple three-dimensional tissue structure (Fig. 1B-D), and its malleable body consists of two cell layers, the ectoderm and endoderm. separated by a thin matrix called the mesoglea. The polar body has a head and tentacles at one end, and a foot at the other end of a hollow column (Fig. 2B). Epitheliomuscular cells cover the outside and line the gastric cavity, while interstitial cells between those two layers differentiate into nerve cells, cnidocytes, gland cells, and gametes. Although Hydra lacks migratory phagocytic cells, hemolymph, and permeability barriers, members of our lab determined that its epithelial cells activate in response to invading pathogens, increasing expression of genes encoding antimicrobial peptides and thus mediating this organism's innate immune responses.

Hydra, a Potential Source of Antimicrobial Drugs

Once induced, Hydra tissues produce highly selective antimicrobial peptides. For example, the endodermal epithelium produces the cationic 60-amino acid hydramacin-1, which contains eight cysteines, after being induced by microbial products. Once isolated from such tissues, hydramacin-1 can kill effectively several types of gram-negative pathogens that infect humans, including strains of Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae that produce extended spectrum beta-lactamases (ESBL). Additionally, this peptide is highly active against gram-positive strains such as Bacillus megaterium ATCC14581.

Such findings suggest that the structure of hydramacin-1 is a good starting point for designing antibiotics to combat infections in humans caused by pathogens such as methicillinresistant Staphylococcus aureus (MRSA), the vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis, and ESBL strains of Klebsiella pneumoniae, and Pseudomonas aeruginosa.

We screened Hydra tissues for other agents with antimicrobial activity. For example, in addition to peptides, a serine protease inhibitor that we designated kazal-2 is expressed in endodermal gland cells. It inhibits growth of S. aureus by blocking a specific serine protease of this gram-positive bacterium. Thus, kazal-2 and similar protease inhibitors might constitute a new class of antibiotics that, once optimized, could become highly effective against staphylococcal pathogens.

Finding this antimicrobial serine protease inhibitor in Hydra sheds new light on host defense mechanisms that developed early during metazoan evolution. Meanwhile, other findings from our lab suggest not only that antimicrobial peptides from Hydra can kill particular bacteria, but that they can help to shape the composition of the microbiota. For instance, overexpressing an endogenous antimicrobial peptide reduces overall bacterial levels while also drastically changing the resident microbial biodiversity. Thus, antimicrobial peptides apparently took part in the molecular conversation between bacteria and this host species during its evolution.

Hydra Epithelial Cells Select and Modulate Microbiota

The human intestine (Fig. 1E) is colonized by a complex and dynamic community of microorganisms that support a variety of functions. The stepwise microbial colonization of the intestine begins at birth and continues during the early phases of life, forming a microbiota that differs from one individual to another.

Similarly, a complex and dynamic community of microbes colonizes the Hydra epithelium and, here also, individuals from different Hydra species differ greatly in their microfauna (Fig. 2). Moreover, the microbiota of individual Hydra that were kept for years under controlled conditions are similar to the microbiota of individual polyps that are more recently isolated from lakes. This microbial continuity suggests that the colonizing bacteria become resident species and are not mere "tourists" passing through individual Hydra along with food, water, and other environmental components. The microbiota differences among Hydra species and their maintenance over long periods suggest that the Hydra epithelium imposes selective pressures on microorganisms, thus shaping this microbial community.

Meanwhile, environmental conditions can modulate bacterial communities that associate with particular types of Hydra. For example, when we cultured polyps of H. oligactis from the wild, that shift to laboratory conditions had drastic effects on the composition of its bacterial community. In particular, α-Proteobacteria remain the dominant species following long-term culture. However, other bacteria gradually disappear from the tissue collected in the wild following the shift to growth in the lab. Thus, Hydra not only is associated with specific symbiotic bacteria but that microbiota responds to changes in environment. Those environmentally responsive differences within microbiota fall within a narrow range, and, even under long-term culture, the bacterial communities from different Hydra species remain very different from one another. Thus, each Hydra species appears to select its own specific microbial community.

Associated Microbiota Influence and Benefit Hydra Hosts

Depleting microbiota damages the health and well-being of Hydra as well as mammals. Humans or other mammals lacking in intestinal microbiota, for example, tend to develop severe immune-response defects. Moreover, host genetic defects in immune responses along the epithelial layer can predispose individuals to inflammatory diseases. Although detailed explanations for these phenomena are lacking, there is little doubt that symbiotic bacteria help to maintain the balance between health and disease for their hosts.

Bacteria exert other profound effects on Hydra. For instance, microbiota-free Hydra cannot proliferate asexually by budding, according to findings reported in 1982 by Menachem Rahat and Chanan Dimentman at the University of Jerusalem in Israel. However, reinoculating such Hydra with medium containing bacteria from standard cultures of this freshwater animal restores its capacity to bud. Thus, resident microbiota strongly affect development in Hydra, albeit through mechanisms that we do not yet understand.

Although microbial imbalances underlie many human diseases, little is known about how epithelial homeostasis affects associated microbial community structures. Hydra provides an opportunity for experimentally approaching some of these issues. For example, the mutant strain sf-1 of Hydra magnipapillata produces temperature-sensitive interstitial stem cells. When this mutant is held for several hours at the restrictive temperature, it loses its entire interstitial cell lineage. However, both the ectodermal and endodermal epithelial cells remain undisturbed, and the morphology of the mutant and the integrity of its epithelium remain intact (Fig. 3B).

Meanwhile, these changes in cell composition lead to significant changes in the Hydra-associated microbial community. In particular, two bacterial phylotypes change drastically following the temperature shift. The ordinarily dominant bacterial phylotype of β-Proteobacteria decreases in the temperature-shifted polyps that lack the usual interstitial cells and their derivatives. Following the shift, a bacterial phylotype belonging to Bacteroidetes increases (Fig. 3). Thus, the epithelia and microbiota apparently interact directly.

What Hydra Offer to Our Understanding of Superorganisms

The numbers of symbiotic microorganisms and their aggregate genetic information far exceed the genetic information encoded by humans or other host animals. The human body thus belongs to its own diverse ecosystem, making it a superorganism. Because such communities formed long ago, microbes play important roles not only during the lives of individual hosts, but also while each of those host species evolved.

The holobiont, which consists of the host and its entire set of symbiotic microbiota, should be considered the entity that is subject to selective forces during evolution, according to Eugene Rosenberg at Tel Aviv University in Israel and his collaborators. Nonetheless, little is known about the part played by the microbiota and how microorganisms affect any species during evolution (Fig. 4). How do associated organisms coordinate their interactions at the molecular level? How do the genomes within a superorganism co-evolve? How do microorganisms affect their hosts and vice versa?

Although probing these complex interactions remains a challenging task, Hydra with its epithelial defense mechanisms provides a particularly suitable model for approaching some of these questions. These organisms carry many of the same gene families found in bilaterians and retain many genes that were lost from Drosophila and Caenorhabditis elegans.

Because Hydra-associated microbial communities are so complex, fulfilling this analytic challenge will not be easy. Indeed, such communities are distinct from those in the surrounding water, are specific for each Hydra species, are spatially and temporally stable, and also likely play multiple roles in their interactions with the Hydra host. Disturbing the balance between Hydra and its colonizing microbiota appears to foster disease. Characterizing these host-microbe interactions therefore will provide both fundamental and applied insights into the common ancestor of Hydra and other animals as well as how symbiotic bacteria contribute to the balance between health and disease in their hosts.

SUGGESTED READING

Augustin, R, S. Siebert, and T. C. G. Bosch. 2009. Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of hydra's innate immune system. Dev. Comp. Immunol. 33:830-837.

Bosch, T. C. G., R. Augustin, F. Anton-Erxleben, S. Fraune, G. Hemmrich, H. Zill, P. Rosenstiel, G. Jacobs, S. Schreiber, M. Leippe, M. Stanisak, J. Grötzinger, S. Jung, R. Podschun, J. Bartels, J. Harder, and J.-M. Schröder. 2009. Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence. Dev. Comp. Immunol. 33:559-569.

Fraune, S., Y. Abe, and T. C. G. Bosch. 2009. Disturbing epithelial homeostasis in the metazoan Hydra leads to drastic changes in associated microbiota. Environ Microbiol. 11:2361-2369.

Fraune, S., and T. C. G. Bosch. 2007. Long-term maintenance of species-specific bacterial microbiota in the basal metazoan Hydra. Proc. Natl. Acad. Sci. USA 104:13146-13151.

Jung, S., A. J. Dingley, R. Augustin, F. Anton-Erxleben, M. Stanisak, C. Gelhaus, T. Gutsmann, M. U. Hammer, R. Podschun, M. Leippe, T. C. G. Bosch, and J. Grötzinger. 2009. Hydramacin-1: structure and antibacterial activity of a protein from the basal metazoan hydra. J. Biol. Chem. 284:1896-1905.

Mazmanian, S. K., J. L. Round, and D. L. Kasper. 2008. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453:620-625.

Rahat, M., and C. Dimentman. 1982. Cultivation of bacteria-free Hydra viridis: missing budding factor in nonsymbiotic hydra. Science 216:67-68.

Rosenberg. E., O. Koren, L. Reshef, R. Efrony, and I. Zilber-Rosenberg. 2007. The role of microorganisms in coral health, disease and evolution. Nature Rev. Microbiol. 5:355-362.

Ruby, E. G. 2008. Symbiotic conversations are revealed under genetic interrogation. Nature Rev. Microbiol. 6:752-762.