Bacterial Colonization of Hydra Hatchlings Follows a Robust Temporal Pattern
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The ISME Journal (2013) 7, 781–790 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE Bacterial colonization of Hydra hatchlings follows a robust temporal pattern So¨ren Franzenburg1,4, Sebastian Fraune1,4, Philipp M Altrock2, Sven Ku¨ nzel2, John F Baines2,3, Arne Traulsen2 and Thomas CG Bosch1 1Zoological Institute, Christian-Albrechts University Kiel, Kiel, Germany; 2Max-Planck-Institute for Evolutionary Biology, August-Thienemann-Strae2,Plo¨n, Germany and 3Institute for Experimental Medicine, Christian-Albrechts-University of Kiel, Kiel, Germany Animals are colonized by complex bacterial communities. The processes controlling community membership and influencing the establishment of the microbial ecosystem during development are poorly understood. Here we aimed to explore the assembly of bacterial communities in Hydra with the broader goal of elucidating the general rules that determine the temporal progression of bacterial colonization of animal epithelia. We profiled the microbial communities in polyps at various time points after hatching in four replicates. The composition and temporal patterns of the bacterial communities were strikingly similar in all replicates. Distinct features included high diversity of community profiles in the first week, a remarkable but transient adult-like profile 2 weeks after hatching, followed by progressive emergence of a stable adult-like pattern characterized by low species diversity and the preponderance of the Betaproteobacterium Curvibacter. Intriguingly, this process displayed important parallels to the assembly of human fecal communities after birth. In addition, a mathematical modeling approach was used to uncover the organizational principles of this colonization process, suggesting that both, local environmental or host-derived factor(s) modulating the colonization rate, as well as frequency-dependent interactions of individual bacterial community members are important aspects in the emergence of a stable bacterial community at the end of development. The ISME Journal (2013) 7, 781–790; doi:10.1038/ismej.2012.156; published online 24 January 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: bacterial dynamics; cnidarians; host–microbe interaction; microbiota Introduction conventional larvae when reared on a nutrient-poor diet (Shin et al., 2011); and in anthozoan cnidarians All animals, ranging from simple invertebrates to shifts in the composition of the microbial commu- primates, are host to complex microbial commu- nity are associated with coral bleaching (Mao-Jones nities (Fraune and Bosch, 2007; Ley et al., 2008; et al., 2010). Thus, vertebrates and invertebrates Ochman et al., 2010). Microbial colonization appear not only to tolerate, but to require coloniza- appears to be an essential step in vertebrate ontogeny, contributing to the maturation of the tion by beneficial microorganisms for metabolism, immune system and gut development (Rawls et al., immune defense, development, behavior 2004; Mazmanian et al., 2005; Kelly et al., 2007). (Sandstrom et al., 2000; Xu et al., 2003; Nyholm Resident gut bacteria can cause disease if the and McFall-Ngai, 2004; Rawls et al., 2004; balance of the community is disrupted by antibiotics Mazmanian et al., 2005; Chow et al., 2010) and (Ayres et al., 2012). Neonatal recolonization of germ- most likely many other not yet identified functions. free mice with microbes prevents enhanced colitis Any animal with its associated microbes, therefore, and asthma sensitivity while exposure of adult mice can be considered as a metaorganism defined as ‘an to these conditions is not effective (Olszak et al., ensemble of individuals representing numerous 2012). In invertebrates, germ-free Drosophila larvae species that coexist and interact in an area or show drastically increased mortality compared with habitat’ (Drake, 1990). Membership in such a community is frequently influenced by interactions Correspondence: TCG Bosch, Zoological Institute, Christian- among species and properties, which emerge from Albrechts-University Kiel, Olshausenstrasse 40, 24098 Kiel, such interactions (Drake, 1990). In this sense, Germany. humans develop into ecological communities after E-mail: [email protected] being born with a sterile gastrointestinal tract that is 4These authors contributed equally to this work. Received 9 July 2012; revised 11 October 2012; accepted 28 successively colonized with microbial populations October 2012; published online 24 January 2013 until adult-like communities stabilize (Koenig et al., Bacterial colonization process of Hydra hatchlings S Franzenburg et al 782 2011; Walter and Ley, 2011). The processes, which inexpensive, easily handled, has a transcriptome control community membership in the neonatal gut and genome that more closely resembles humans and influence the colonization pattern during than those of flies and worms (Chapman et al., 2010; infancy and childhood are poorly understood. A Hemmrich et al., 2012), and is colonized by stable vitally important question to ask, therefore, is what and species-specific bacterial communities (Fraune are the factors and rules in a particular host that and Bosch, 2007). Unlike most animal systems, it is influence community assembly, composition and possible to examine temporal bacterial profiles of diversity. Because a dysregulation of host–microbe clonal lineages without being limited to fecal homeostasis can have severe impact on the host’s samples, as Hydra grows clonally under constant health, controlling and mediating ‘correct’ bacterial laboratory conditions. Hydra’s tube-like body colonization in early life would confer fitness (Figure 1a) resembles in several aspects the anatomy advantages to the host. This view has encouraged of the vertebrate intestine with the endodermal discussions as to what extent the microbiota is epithelium lining the gastric cavity and the ecto- controlled by the host through top–down mechan- dermal epithelium providing a permanent protec- isms involving the immune system, relative to tive barrier to the environment (Bosch, 2012). microbiota intrinsic bottom–up mechanisms (Ley Changes in Hydra’s epithelial homeostasis cause et al., 2006). Despite the importance of under- significant changes in the microbial community standing the factors that control bacterial coloniza- (Fraune et al., 2009), implying a direct interaction tion in man, however, the inaccessibility of the between epithelia and microbiota. Multifunctional microbial niches of the human gut, and the restric- epithelial cells recognize microbial-associated mole- tion of studies in infants to collection of fecal cular patterns with the help of the Toll-like receptor samples (Cilieborg et al., 2012) make it desirable to signaling pathway (Bosch et al., 2009; Franzenburg use animal models for understanding basic princi- et al., 2012). Intracellular recognition of bacteria in ples of colonization processes in detail. Here we Hydra is supposed to be mediated by a large number aimed to explore the assembly of bacterial commu- of nucleotide oligomerization domain-like receptors nities in the freshwater polyp Hydra. Our goal was (Lange et al., 2011). Prominent effector molecules to obtain mechanistic information about specific downstream of the conserved Toll-like receptor host–bacterial interactions and to elucidate the rules cascade are antimicrobial peptides. that determine how large and diverse bacterial Following fertilization, Hydra oocytes develop by populations colonize a newly born animal. a radial cleavage pattern outside the female polyp The cnidarian Hydra is an established animal (Figure 1a) (Martin et al., 1997). Gastrulation is model for studying host–microbe interactions. It is followed by a cuticle stage, which is characterized Figure 1 Hydra morphology and experimental design. (a) Female polyp of Hydra vulgaris (AEP) with a developing embryo. (b) Hydra hatchling eclosing from the cuticle. (c) Experimental design: First batch of hatchlings was used for immediate DNA extraction. Second batch of hatchlings was used for the establishment of clonal cultures, from which one polyp was removed for DNA extraction every week until week 15. Subsequently, the bacterial communities of these samples were determined by 454 sequencing. The ISME Journal Bacterial colonization process of Hydra hatchlings S Franzenburg et al 783 by a thick protective outer layer (Martin et al., 1997). universal 16S primer 27F and the barcoded reverse We have shown previously (Fraune et al., 2010) that primer V2_A_338R (50-CGTATCGCCTCCCTCGCGCCA early embryonic stages before the cuticle stage are TCAGNNNNNNNNNNCATGCTGCCTCCCGTAGGA colonized by a limited number of microbes, which GT-30) which contains the 454 FLX Amplicon primer are clearly distinct from later developmental stages. A, a sample-specific 10-mer barcode (N’s), a two Interestingly, the differential colonization is base linker and the universal 16S primer 338R. reflected in differences in antimicrobial activity Twenty-five microlitre PCR reactions were per- (Fraune et al., 2010). Young Hydra polyps directly formed using the Phusion Hot-Start II DNA poly- hatch from the cuticle stage (Figure 1b) (Martin merase (Finnzymes, Espoo, Finland) following the et al., 1997). To elucidate how bacterial commu- manufacturer’s instructions. PCR conditions con- nities assemble in newly hatched Hydra polyps, we sisted of an initial denaturation step (98 1C, 30 s)