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AMER. ZOOL., 38:593-608 (1998) The Development of Cooperative Associations Between Animals and Bacteria: Establishing Detente Among Domains' MARGARET J. MCFALL-NGAI 2 University of Hawaii, Pacific Biomedical Research Center, Kewalo Basin Laboratories, 41 Ahui Street, Honolulu, Hawaii 96813 SYNOPSIS. detente = n.(fr.) the relaxation of tensions between two nations, usually through cooperation and negotiation. [The Random House Dictionary, 2nd Edition, 1987] Despite the ubiquitous occurrence of cooperative associations between animals and bacteria, there is little understanding of how these interactions arose, how they evolved, and how they persist. Thus, an extensive database concerning the influence of bacteria on developmental pathways is not yet available. However, in much the same way that mutually beneficial liaisons are created between nations with vastly different histories and cultures, it is likely that highly refined developmental mechanisms exist in which a type of detente is created to retain the integrity of the associations between the partners, both within and between generations. These developmental pathways would be responsible for insuring that a balance of cell growth is established and maintained among the community members, comprised of animal and microbial cells, such that neither form of pathogenesis, i.e., overgrowth (war) or aposymbiosis (isolation), ensues. This contribution examines aspects of how alliances with prokaryotes may have been integrated into the mechanisms and patterns of host animal developmental programs. (Margulis, 1970). It is against this backdrop of cooperating cells that the Domain EuBiologists have long known that animal karya evolved. Thus, when animals radiatevolution, including the evolution of their ed, two possibilities presented themselves: developmental pathways, occurred in envianimals could develop mechanisms to inronments ecologically dominated by bactehibit interactions with bacterial cells; or, ria. The members of the Domain Bacteria they could take advantage of the already expreceded the animals with a long evolutionisting trend of interaction and foster coloary history in which almost all known metabolic pathways were derived (Gottschalk, nization by specific subsets of bacteria, 1986); prokaryotes evolved mechanisms for availing themselves of bacterial metabolic the use of a wide variety of organic and diversity. Most likely it was a combination of these inorganic energy sources available in their environments, forming interacting commu- two pressures that resulted in the formation nities of mutually dependent individuals of sets of complex prokaryotic communities (Madigan et al, 1997). The formation of in association with animals, which were difthe eukaryotic cell gave rise to the predict- ferent in composition from the bacterial able persistence of subsets of interacting communities in the surrounding environcommunities, but did not divorce these ment. Until recently, however, biologists units from specific interactions with less in- have focused primarily on the evolution of tegrated prokaryotes in the environment mechanisms by which animals would inhibit interactions with prokaryotes. This viewpoint has been largely driven by: INTRODUCTION 1 From the Symposium The Evolution of Development: Patterns and Process presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 26-30 December 1996, at Albuquerque, New Mexico. 2 E-mail: [email protected] 1) our concepts of the nature of the immune system; 2) the emphasis on bacteria as animal pathogens; and, 593 594 MARGARET J. MCFALL-NGAI 3) the technical difficulties associated with exploring the establishment and maintenance of cooperative animal-bacteria relationships. Nonetheless, both recent changes in our conceptual framework and advances in technology have brought about reevaluations in all three of these areas. First, over the past five years, there has been growing support for a paradigm shift in immunology in which the animal immune system rather than being considered a non self recognition system (i.e., "self" vs. "non-self"), is viewed as a system by which threats (i.e., "danger" vs. "non-danger") are detected (Matzinger, 1994; Duncan and Edberg, 1995; Matzinger and Fuchs, 1996). This change in viewpoint was primarily derived as an attempt to understand autoimmune disease, where a subset of "self" is treated by the immune system as "danger." However, it also presents a very important change in perspective for biologists who study animal-bacterial mutualisms. This shift in viewpoint allows for incorporation of the idea that, under certain circumstances, animal tissues tolerate, and even promote, the presence of biotic "non-self." In so doing, this new view reconciles the longstanding problem in immunology of whether to classify the bacterial symbionts as "self" or "non-self." It has been recognized since the 1970s that indigenous microorganisms induce the production of host antibodies poorly or not at all, especially when the microbes are restricted to the enteric tract (Foo and Lee, 1974; Morishita and Mitsuoka, 1973); and, in addition, intestinal bacteria can even share antigens with the mucosa (Foo and Lee, 1974). These observations suggested that indigenous microbiota evolved in a close immunological relationship with their host (Savage, 1977). Thus, a healthy animal can be denned as a community comprised of interacting, yet widely divergent, genomes. Taken in this context, the immune system is seen to function to provide defense against danger presented by cells both within and outside of the community. Any cell within that community can become a threat, such as miscommunicating cells of the host (e.g., autoimmune disease or cancer) or opportunistic pathogens (e.g., overgrowing intestinal Escherichia coli); or, by the more traditional view, the community itself can be threatened by interlopers from the outside, which would span the range from viruses to grafts of cells from conspecifics. Secondly, in addition to our historical view of the immune system, the traditional emphasis in microbiology on human bacterial pathogenesis has skewed our perspective (Garrett, 1994). The fact that human history has been so tremendously affected by outbreaks of bacterial disease (McNeill, 1977; McKeown, 1988) has promoted the study of this type of animal-bacterial interaction, and incidentally overshadowed investigations of the normal, essential interactions of the healthy individual (Lee, 1985). An eventual outcome of this restricted view was the "antibiotic" solution to microbial disease (Levy, 1992). Unfortunately, during this early phase of antibiotic development, the plasticity of the bacterial genome was not appreciated, and now bacterial pathogens with multiple antibiotic resistances have arisen and present a threat as never before (Cohen, 1992; Neu, 1992). Yet we continue to focus primarily on how these pathogens affect animal tissues, rather than how they interrupt the complex functions of the normal animal-bacterial community. It is clear that efforts must be directed toward exploring the nature of these "normal" interactions. Finally, there have been few experimental systems by which to approach the study of the complex interactions between prokaryotes and animals. Advances in the application of molecular genetics to diverse bacteria, the examination of nonculturable bacteria within complex communities using molecular probes (Hugenholtz and Pace, 1996), and the development of model systems, such as the Euprymna/Vibrio association described in the last section of this contribution, are offering avenues by which biologists can study cooperative interactions between animals and microbes. These shifts in emphasis in the fields of immunology and microbiology, and the development of new technology, have paved the way for a changing perspective on an- DEVELOPMENT OF COOPERATIVE SYMBIOSES imal-bacterial relationships. Once one entertains the idea that evolution fostered the creation of complex communities (=host + microbes), the door is opened for exploration of how the interactions among the partners affect all levels of the biology of these communities, including the evolution of the developmental mechanisms of the animal hosts. Thus, the development of an animal must not only be concerned with the fidelity of cell divisions and differentiations within the single genome of that animal, but must also incorporate mechanisms by which to foster with a similar fidelity the cell divisions and differentiations of microbial cells of a number of different genomes. The field of biology that is concerned with the influence of bacteria on animal development, as well as the development of animal-bacterial community structure that comprises the unit, has really only been considered in any detail by the bacteriologists. To incorporate this new avenue of research, animal biologists must not only define experimental programs to examine these interactions, but also create a strong theoretical framework within which to formulate questions. In this paper I address some of the issues that are relevant to a consideration of the influence of bacteria on animal development. Specifically, I consider key aspects of the relationships between animals and bacteria that have bearing on host development, discuss the state of our current database in this field, and finish by summarizing what is known for one model, the Euprymna-Vibrio symbiosis. THE NATURE OF COOPERATIVE ASSOCIATIONS Specific characteristics of cooperative animal-bacterial associations, sometimes termed "mutualisms," are likely to affect animal host developmental processes. Some of the more conspicuous influences are discussed below. Community structure In most cooperative associations between animals and bacteria, the animal host provides a nutrient-rich environment that promotes bacterial growth in exchange for the products of specific bacterial metabolic 595 pathways (such as those mediating chitin or cellulose breakdown, or vitamin synthesis; Madigan et al., 1997). As such, the most conspicuous and widespread type of animal-bacterial interaction is between hosts and their specific consortia of enteric tract microbiota, which usually are the primary cell type; e.g., in a typical omnivorous mammal, there is a 10:1 ratio of bacterial to animal cells (Savage, 1977, 1986; Duncan and Edberg, 1995). The enteric consortium usually consists of two principal components: the resident, indigenous microbiota, which are believed to have coevolved with the host, and nonspecific "tourist" microbes, which have been introduced from the environment and do not form permanent associations (Savage, 1977). While most animals have, and may require, specific interactions with microorganisms in their enteric tracts, occasionally in evolutionary history, specializations have occurred that drive the structure of the community, or a portion of the community, in one of two ways: toward a highly distinctive consortium driven by unusual diets, such as in the rumen (Hungate, 1966) or termite hindgut (Breznak, 1982); or, toward organs that involve a two-partner symbiosis, i.e., one species of bacterium in a specialized host organ, such as a light organ (McFall-Ngai and Toller, 1991; Haygood, 1993). Both of these types of derived conditions provide convincing illustrations of how interactions with microorganisms can profoundly influence both the evolution of specific animal groups and the ecology of their associated biosphere (Margulis and Fester, 1991). For example, the radiation of the ruminant mammals must have been dependent on their association with the rumen microbiota (McFarland et al., 1979; Stahl, 1985); and, the evolution and ecology of coral reef (Stoddard, 1969; Newell, 1972) and deep sea hydrothermal vent communities (Grassle, 1986; Tunnicliffe, 1988; Childress and Fisher, 1992) appear to have been driven by associations of animals with autotrophic microorganisms. Maintenance of community integrity over time—transmission of microbes between host generations Whether the community defining the symbiosis is consortial or two-partner, there 596 MARGARET J. MCFALL-NGAI is a growing body of evidence indicating Lee and Ruby, 1994). In both direct and that bacteria influence of the development indirect cyclic transmission, the bacteria are of the animal tissues with which they as- not actually present during embryogenesis. sociate (Gordon and Pesti, 1971; McFall- Nonetheless, during this period, the animal Ngai and Ruby, 1991; Schwemmler and must develop tissues to promote establishGassner, 1989; Saffo, 1992). How and ment of the association, and often rudiwhen these influences express themselves is ments of the more mature organ. For exdependent on mechanisms by which the as- ample, over evolutionary time selection sociations are maintained between genera- must have occurred to incorporate into the tions. host's developmental program the differThe principal modes of transmission entiation of cells with surface properties inhave been termed transovarian (or vertical) volved in recognition and adherence of the and cyclic (or horizontal) (Douglas, 1994). appropriate environmental bacteria. The In transovarian transmission, the bacterial first interactions of the host with such cysymbionts are translocated to the ovary and clically transmitted bacterial partners occur are transmitted in or on the egg. As such, at either hatching or birth. These interacthey participate directly in the process of tions of the animal and bacteria during postembryogenesis of the host, as well as in embryonic development can have profound postembryonic development. For example, conspicuous effects on the maturation of at least 11 % of all insect species examined the tissues of the host (Gordon and Pesti, vertically transmit specific symbionts that 1971; McFall-Ngai and Ruby, 1991). In addition to transmission between genreside in a tissue called the bacteriome (formerly, mycetome). These symbionts pro- erations, the dynamics of bacterial comvide vitamins and/or other nutrients that al- munities may be crucial to the normal prolow these insects to survive on nutritionally grams that occur within and between depoor diets (Douglas, 1989). In cyclic trans- velopmental stages in the life history of an mission, the bacterial partners are acquired individual, particularly in species with comfrom the environment anew each genera- plex larval life histories. In more simple life tion. While vertical transmission is restrict- histories, such as that of mammals, changes ed to a limited number of taxa, cyclic in- in gut microbiota are well documented in oculation with specific microbiota that be- the early postnatal development (Savage, come associated with the enteric tract and/ 1977). Colonization by pioneer species is or integument probably occurs in all ani- followed by an ecological succession, a progression that eventually results in a stamals (Savage, 1977, 1986). Cyclic transmission between generations ble, climax community of interacting mican be further divided into direct and indi- crobes. The community structure is then alrect. In direct transmission, the transfer of tered as a result of a dramatic dietary bacterial symbionts between generations is change, such as that occurring at weaning facilitated by the adult host, and is often a (Gordon and Pesti, 1971; Savage, 1977). hallmark of animal/bacterial communities While these processes have been studied with special metabolic capabilities. For ex- extensively in mammals, very little inforample, termite juveniles are fed symbiont- mation is available on the nature of the baccontaining feces of the adults (Breznak, terial communities associated with inverte1982), and cows inoculate their calves with brate larvae, and the extent to which these the rumen microbiota during grooming bacteria influence the often complex devel(Dehority and Orpin, 1988). With indirect opmental programs of their hosts. However, cyclic transmission, the most prevalent several examples have been reported in form of transmission between generations, which host species have been demonstrated the young acquire their inoculum from the to require bacteria for proper settlement and broader environment without the interces- metamorphosis (Sheltema, 1974; Hadfield sion of conspecifics, although the adult et al., 1994). For example, the larvae of population may be more or less responsible many sessile benthic species assess the nafor seeding the environment (Drasar, 1974; ture of substrate biofilms, and only settle DEVELOPMENT OF COOPERATIVE SYMBIOSES 597 association, the bacterial partner may be superficial (e.g., on the skin), or invested in Superficial the microvilli of a simple tissue (e.g., the enteric epithelium) or of a complex organ (e.g., squid and fish photophores). Alterdevelopmental/ evolutionary progression natively, the bacterial symbionts can be within the host cell, either surrounded by a vacuolar membrane or free in the cytoplasm. For the intracellular symbioses that Intracytoplasmic are cyclically transmitted, not only does this progression of intimacy between the bacterial and animal cells obviously occur during establishment of the mature relationship, but also it is believed to represent an [cslt/vacuolB membrane] evolutionary progression; i.e., intracellular symbioses are presumed to be more derived surface contact than extracellular symbioses, although there is little evidence to support this idea (Smith, exchange of gene products 1979). The location of the bacterial symbiont in relation to the animal cell does not neceshorizontal gene transfer •'>i\ sarily correlate with the degree to which the partners can and do exercise control over FIG. 1. Intimacy of host and microbial cells as defined by position and mechanisms of reciprocal sig- one another's metabolism. Instead, the innaling. A. From top to bottom, progression of micro- tensity of the dialogue, as reflected in bial cells from an extracellular, superficial location on changes in gene expression of the partners, the host cell, to free within the host cytoplasm, i.e., should be more meaningful in defining the without a vacuolar membrane. In intracellular, cyclic transmission this progression occurs developmentally, impact of the symbiosis on their biology and may also represent an evolutionary trend toward (Fig. IB). Such communication can be meincreasing interdependence of the symbiotic partners. diated remotely through surface compoB. Modes of signal exchange between host and micro- nents of the plasma or vacuolar membranes, bial symbiont [see text for details], a = host genes; b and second messenger pathways may be re= microbial genes. sponsible for transmitting the information to the partner's genomes. Similarly, the and metamorphose when bacteria with pre- cells can exchange gene products or metabscribed characteristics are present (e.g., olites, which either directly or indirectly inHofmann and Brand, 1987; Fitt et al., 1990; fluence gene expression within the recipient Maki et al., 1990). Yet how prevalent such cells. For example, during the interaction of a dependency might be is an area that re- Vibrio cholerae with mammalian intestinal mains to be explored. cells, the bacterium produces the protein cholera toxin (Spangler, 1992). The A subLocation of the cells of the partners and unit of this protein is introduced into the its relationship to mechanisms of host cell, where it perturbs the metabolism reciprocal signaling of that cell, which leads to changes in host Between species interactions.—Different cell gene expression (Lycke and Strober, bacteria colonize a variety of locations on 1989; Spangler, 1992). In contrast to animal or in the cells of their host. (Fig. 1A) The cells, bacteria do not typically import protopography of these associations may be teins, so animal cells usually influence bacexpected to influence the extent to which terial gene expression through smaller sigthe bacteria participate in the differentiation nal molecules. For example, leguminous of host cells as well as the overall mor- plants release flavenoids into the soil that phology of the tissues and organs that house are taken up by specific bacteria termed the microbiota. Thus, in a given cooperative "rhizobia" (Long, 1989; Hirsch, 1992). — - ». K--— v 598 MARGARET J. MCFALL-NGAI These flavenoids induce expression of bacterial genes that promote the development of the symbiosis between the legume and its specific species of rhizobia, which ultimately leads to formation of the mature, nitrogen-fixing root nodule (Appleby, 1984; Gussin et al., 1986). While these more indirect mechanisms of host/microbe exchange have been recognized for decades, a grasp of the implications associated with direct introduction of a genetic element into the cell of a symbiotic partner, either historically or during the ontogeny of a given symbiotic association, represents a frontier area in biology. The genetic makeup of an individual can be thought to consist of four types of elements: (i) the principal components of the nuclear genome that share a single, continuous evolutionary history and that obey the laws of Mendelian genetics; (ii) genes in organelles, such as the mitochondria and plastids, which coevolved with the nuclear genome of the eukaryotic cell; (iii) those associated genetic elements that may be derived from that genome, but have become independent, such as retroviruses in eukaryotes and phage or plasmids in prokaryotes; and, (iv) elements with a completely independent evolutionary origin that are, or have been, inserted into the genome—a phenomenon referred to as horizontal gene transfer (Amabile-Cuevas and Chicurel, 1993). The best documented example of horizontal gene transfer and its influence on host development occurs in the Agrobacterium tumefac/ens-dicotyledonous plant pathogenesis called crown gall disease. The virulence genes of this bacterium are encoded on satellite DNA, the Ti plasmid, which is transferred into susceptible cells of the plant host (Raineri et al, 1993; Madigan et al, 1997). Genes on this plasmid are incorporated into the plant's nuclear genome, where they cause changes in host gene expression and protein synthesis that result in the formation of the gall, a hypertrophied tissue that houses the bacterial symbionts (Hooykaas, 1994). In animal-bacterial associations, few such striking examples have been described, although convincing molecular evidence suggests that such transfers have occurred in the past; i.e., chromosomal genes have been identified in both bacteria and animals that have sequences more closely related to the genes of a distantly related organisms than to those of the nuclear genome of that individual (Dyer, 1989; Amabile-Cuevas and Chicurel, 1992; Smith et al, 1992; Amabile-Cuevas and Chicurel, 1993). Further, recent studies on symbionts of mammalian intestines have shown that bacterial symbionts can donate stable, transcribable pieces of DNA into host eukaryotic cells (Courvalin et al., 1995). Although these latter genes are not directly associated with the germ line of the host, the degree of mobility of a particular genetic element and the susceptibility of the recipient cell are likely to be heritable traits that may influence developmental patterns in the partners of the symbiosis. Within species interactions.—The intimate interactions described above between symbiotic cells can be transmitted to the other cells comprising the populations both within the host and among the symbiotic bacteria themselves, a process which often determines whether a symbiosis will be established, the nature of the interaction (pathogenic or mutualistic), and the course of development of the association. As in the communication between partners, there exist examples of both indirect and direct influences on the genome. Indirect effects that have been extensively studied in the host include the induction of cortical cell division in the root by interactions with rhizobia at the plant root hair (Long, 1989; Hirsch, 1992) and remote changes in the animal immune system that result from antigen sampling in the intestine (Kagnoff and Kiyono, 1996). Among bacteria, one of the best described examples of within-species communication is a process called "quorum sensing" (Fuqua et al., 1994, 1996). In recent years this form of cell-cell interaction, first described in marine luminous bacteria (Nealson et al., 1970), has been discovered in a rapidly growing list of bacterial species that, while phylogenetically diverse, share the characteristic of being either pathogenic or cooperative symbionts of certain animals or plants. Briefly, each of these bacterial species secretes one of a class of structurally related Af-acyl-homoserine lactone mol- DEVELOPMENT OF COOPERATIVE SYMBIOSES ecules (autoinducers). If cells of that particular species are in abundance, their specific autoinducer will accumulate in the surrounding environment. These bacteria then sense this elevated concentration, which signals the presence of a "quorum" of conspecifics, and leads to the induction of certain genes whose products are required or useful only when the bacteria are colonizing a host tissue. Well studied examples of such regulated activities are luciferase synthesis in symbiotic luminous bacteria (Nealson et al., 1970), and the production of the enzyme elastase by pathogenic pseudomonads (Passador et al., 1993). The prevalence of this form of signaling between bacterial cells in host tissues, as well as the homology of the systems as revealed by biochemical and genetic comparisons, indicate its antiquity, ecological plasticity, and success in bacterial symbiosis, and encourage an examination of other host-microbe interactions for its presence. In addition to such molecular signaling between symbiotic cells, direct gene transfer within a bacterial species has been reported for certain bacteria during initiation of a pathogenesis. (Mel and Mekalanos, 1996). In these cases, a set of "virulence" genes are carried by only a small subset of the bacterial population present outside of the host. Interaction with the host induces the transfer of these genes, which are carried either on bacteriophages or on plasmids, and renders the recipient capable of expressing required virulence determinants. For example, only a small proportion of the free-living population of the plant pathogen Agrobacterium tumifaciens carries the virulence Ti plasmid when outside of an association with a plant. However, the plant provides a confined space for the accumulation of an autoinducer that promotes the transfer of the plasmid to other conspecifics rendering them virulent (Farrand, 1993). Another example has recently been demonstrated in the V. cholerae association with mammals. Several of the cholera virulence genes are carried on a bacteriophage that is transferred to conspecifics under conditions encountered in the host tissues (Waldor and Mekalanos, 1996). 599 THE EXISTING DATABASE Studies of cooperative associations of hosts with bacteria have produced many elegant studies on a wide array of associations (reviewed in Saffo, 1992; Douglas, 1994; Losick and Kaiser, 1997). However, there are three specific research programs that have provided extensive experimental bases and that together are of heuristic value for developmental biologists seeking to understand how bacteria may influence general developmental programs of animals. They are research on: (i) germfree and gnotobiotic animals, (ii) bacterial pathogens, and (iii) the plant/nitrogen-fixing bacteria (rhizobia) symbioses. Rather than reviewing in detail what is known about the developmental biology of each system, I discuss below what each system might contribute to our understanding of the effects of microorganisms on animal developmental processes, as well as some caveats. With the exception of research on the rumen, studies of germfree and gnotobiotic animals were until recently the principal source of information on how extracellular relationships with cooperative bacteria, the most prevalent form of symbiosis, affect the biology of their hosts (Heneghan, 1973; Coates and Gustafsson, 1984; Wostmann et al., 1984). The results of this area of research have revealed that not only do germfree animals fail to thrive without intervention (Gordon and Pesti, 1971), but the tissues with which they associate and the immune system do not mature in the absence of interactions with the natural complement of bacteria (Gordon and Pesti, 1971; Woolverton et al., 1992; Duncan and Edberg, 1995). However, using germfree and gnotobiotic animals to understand the interactions of hosts with their indigenous gut microbiota has met with certain technical problems. Firstly, although many experiments have compared these animals with their normal conspecifics, the nature of what actually constitutes the "normal" animal state is still poorly understood; i.e., even aspects as fundamental as the composition of the microbial community have not been determined for any animal species, including 600 MARGARET J. MCFALL-NGAI Homo sapiens, in which it has been extensively studied (Savage, 1977, 1986; Duncan and Edberg, 1995). Certain species within the community have been difficult to designate as either indigenous or nonspecific, because of sampling problems. Further, it is not well understood how either changes in the diet of an adult host or the maintenance of the host in captivity affect community composition. Finally, despite recent advances in the ability to culture specific types of bacteria, particularly anaerobic species, a significant portion of the gut microbiota consists of bacteria that have not yet been grown in the laboratory (Duncan and Edberg, 1995). Newly developed molecular methods for cataloguing unculturable microbes (Hugenholtz and Pace, 1996) may help to define more fully the community. However, even once the community is more precisely defined, extensive experimental studies with germfree and gnotobiotic systems will continue to be difficult, because the multispecies community of microbes certainly works as a unit, and the results of studies attempting to isolate and experimentally manipulate one component in the absence of the others would be difficult to interpret. In spite of these inherent limitations of using germfree and gnotobiotic animals to study the dialogue between normal microbiota and their hosts, genetic engineering of specific members of the intestinal bacterial community is providing insight into the "messages" that pass between the host and bacterial cells during the onset of the association. For example, by exposing germfree mice to either wild-type or a genetically altered strain of the indigenous enteric bacterium, Bacteroides taiotaomicron, Bry et al. (1996) showed that production of normal levels of glycoconjugates by intestinal epithelia requires specific interactions with bacteria. Continued studies in this area promise to provide further insight into how bacteria participate in specific aspects of enteric remodeling during development. Perhaps the most robust database on animal-bacterial interactions has resulted from research into the nature of pathogenic associations. Numerous studies of bacterial pathogens and their activities have revealed the diversity of mechanisms by which such microbes interact with host cells, as well as the diversity of host cellular processes that are involved in responses to pathogens (Iglewski and Clarke, 1990; Salyers and Whitt, 1994; Cossart et al, 1996). The degree to which fully defining these interactions might lead to a better understanding of all types of animal/bacterial interactions, intracellular to extracellular, cooperative to pathogenic, remains to be determined. However, one might suspect that using pathogens to understand a normal condition would be similar to trying to understand the cooperative workings of a complex, multicultural city by examining the behavior of wartime occupation forces. With this limitation recognized, studies of pathogens have revealed to developmental biologists the extent to which bacteria are capable of altering the morphology, biochemistry and molecular biology of the cells and tissues with which they associate (Salyers and Whitt, 1994). Studies of bacterial pathogenesis have revealed that often normal host cell processes are incorporated into responses to pathogens, and pathogens themselves have a complex set of genes and gene products that have been associated with the mediation of their interactions with the host (Cossart et al., 1996). However, whether host and bacteria are using the same cellular responses that would be used to mediate cooperative associations is not well defined. Further, an inherent anthropocentric view has labeled as pathogens bacterial species that cause disease in humans, although their primary ecological niche might be to occur as cooperative symbionts in other hosts. In such associations, the same molecular "currency" of exchange may be used, with modifications resulting in a benign association, rather than a pathogenesis. For example, recently it was reported (Weis et al., 1996) that a halide peroxidase, erstwhile associated only with tissues responding to pathogens, occurs in high concentrations in the symbiotic organ of Euprymna scolopes, a sepiolid squid that supports a cooperative association with the luminous bacterium Vibrio fischeri. Similarly, on the bacterial side, cholera-toxin-like molecules and their DEVELOPMENT OF COOPERATIVE SYMBIOSES 601 regulators have been found in a number of coordinate interplay between cells with two luminous bacteria that are either enteric or very different genomes. light organ symbionts (Reich and SchoolDespite the fact that the hosts in the lenik, 1994, 1996; Reich et al, 1997). These gume/rhizobia symbioses are plants, studies two examples suggest that certain mole- of root nodule development illustrate to ancules that have traditionally been associated imal developmental biologists just how with pathogenesis may have evolved in- complex a dialogue can be required that restead for a variety of interactions of bacteria sults in a stable, cooperative association bewith animals, and it is the identity of the tween a microbe and a multicellular organhost and its response that determines the na- ism. ture of the symbiosis. THE EUPRYMNA/VIBRIO COOPERATIVE An understanding of how cooperative ASSOCIATION microorganisms can influence the developDetailed reviews of the basic development of a multicellular organism has been best studied in plant/bacterial associations, mental program of the Euprymna scolopes most notably the symbiosis between legu- light organ have recently appeared in Amerminous plants and nitrogen-fixing bacteria ican Zoologist (McFall-Ngai, 1994; Mont(rhizobia) (Long, 1989; Nap and Bisseling, gomery and McFall-Ngai, 1995). Thus, for 1990; Brewin, 1991; Hirsch, 1992; van the purposes of this contribution, I will simRhijn and Vanderleyden, 1995). Because ply provide an overview of the symbiosis, these symbioses involve one host and one and then focus on how this symbiosis inmicrobial species, both of which can be terfaces with the existing, above-described grown independently in culture, they have databases and how it provides new opporoffered an unparalleled, manipulable exper- tunities to developmental biologists. Euprymna scolopes is a sepiolid (bobtail) imental system for the study of the establishment and maintenance of a symbiotic squid endemic to the Hawaiian archipelago association. Free-living rhizobia invade root (Berry, 1912). This nocturnal predator burhairs of the plant, and induce the formation ies in the sand of the shallow waters of the of an infection thread in the root (van Rhijn backreef during the day, and emerges just and Vanderleyden, 1995; Madigan, 1997). after dusk to forage in the water column This structure acts as a conduit through (Moynihan, 1983). The conspicuous light which the bacteria are transported from the organ in the center of the mantle cavity of root surface to its cortex, where the mature E. scolopes houses a culture of the bactenitrogen-fixing nodule will form. The de- rium Vibrio fischeri (McFall-Ngai and velopmental program of the root nodule, Montgomery, 1990). It is hypothesized that which spans a few weeks, requires that the the luminescence of these bacteria is used host and microbes be able to recognize their as a camouflaging mechanism by the squid, specific partner, as well as induce responses where ventral luminescence is emitted of that orchestrate the morphogenetic process the same quality (color and intensity) as (Long, 1989; Hirsch, 1992). Decades of downwelling moonlight or starlight so that study of these associations have revealed the squid casts no shadow. This behavior, that dozens of genes in each partner, ex- called counterillumination, could be used pressed in a specific, reciprocal order, are by the squid to avoid being detected by eiessential for completion of development ther predators or prey (McFall-Ngai, 1990). The gross morphology of the symbiotic (van Rhijn and Vanderleyden, 1995; Denarie et al., 1996). The volley of signaling light organ can be observed by ventral disbetween the host and bacteria is similar to section of the squid, which reveals a bithe communication that occurs between tis- lobed organ associated with the ink sac sues within animal organs, such as the eye, (McFall-Ngai and Montgomery, 1990). during their proper development. However, Histological cross sections of the organ the salient difference lies in the fact that in show that it consists of a series of tissues the plant/bacteria interaction, there exists that together render it similar in architecture to the vertebrate eye (McFall-Ngai and 602 MARGARET J. MCFALL-NGAI Montgomery, 1990; Montgomery and McFall-Ngai, 1992). In the center of the organ is a network of epithelial cells with which the extracellular V. fischeri are directly associated. Surrounding this photogenic tissue is a thick reflector, which serves to direct luminescence ventrally out of the body of the squid. The reflector is in turn surrounded by diverticula of the ink sac, which act as both choroid and iris analogues, i.e., to prevent stray light from passing dorsally out of the animal, and to rotate dynamically around the ventral portion of the light organ to modulate the intensity of light emission. The entire ventral surface of the light organ is covered with a thick, transparent lens, which diffuses the point-source luminescence over the ventral surface of the animal (Montgomery and McFall-Ngai, 1992; Weis et al, 1993). Sepiolids with light organ symbioses exhibit cyclic transmission of their symbionts. Adult females of E. scolopes lay clutches of eggs (ranging in number from 50 to 450) on hard substrates in their environment (M. McFall-Ngai, personal observation). At around 20 days post-fertilization, following no parental care by the adults, the juvenile squid hatch from their egg cases (Montgomery and McFall-Ngai, 1993). The juveniles emerge just after dusk {i.e., light inhibits hatching; M. McFall-Ngai, personal observation) with light organs that are devoid of bacteria, and if these juveniles are not exposed to V. fischeri, a common constituent of the free-living bacterioplankton, the light organ remains sterile (McFall-Ngai and Ruby, 1991). However, under normal conditions, V. fischeri is abundant (approx. 200 cells/ml) in the seawater where adults are found (Lee and Ruby, 1994), most likely because of a diel behavior characteristic of the adults in which they release 90% of their bacterial culture into the water column each day at dawn (Boettcher et al., 1996). The juvenile E. scolopes ventilate symbiont-rich water into their mantle cavity and inoculation of the juvenile light organ occurs. In laboratory experiments using a similar density of bacteria as that occurring in the field, inoculation of the organ occurs in minutes to hours after hatching, a process that can be monitored by measuring light production of the growing symbiont culture (Ruby and Asato, 1993). During embryogenesis, when no bacterial symbionts are present, the squid host develops an organ anatomy and morphology that appears to promote efficient colonization of the light organ (Montgomery and McFall-Ngai, 1993). Superficially, each lateral face of the organ is covered with a complex, ciliated, microvillous epithelial field, composed of an anterior and posterior appendage and an area surrounding three pores. Observations with high speed cinematography showed that the cilia surrounding the pores and on the appendages, which form a ring, entrain bacteria-ladened water toward the pores (M. McFall-Ngai and R. Emlet, personal observation). The pores lead by long ducts to epithelia-lined crypts that form sequentially through the embryonic period, presumably through invagination (Montgomery and McFall-Ngai, 1993). In normal development, once the bacterial culture has become established, the superficial ciliated, microvillous field regresses within days (Fig. 2 and Fig. 3), and the light organ goes on to elaborate those tissues associated with function of the mature light organ, i.e., light modulation (McFallNgai, 1994). This process, which occurs from about Day 4 to Day 21 post-hatching, involves a dramatic thickening of the reflector, development of the ink sac diverticula, elaboration of the lens, and an overall remodeling of the rounded hatchling organ into the bilobed morphology characteristic of the adult (McFall-Ngai, 1994). In addition to these superficial changes in the organ, during normal symbiotic development the crypt cells associated with the bacteria swell to a volume four times that of the hatchling crypt cells, and the microvillar density increases four fold (Fig. 2). This developmental program strikingly resembles that of the legume/rhizobia symbioses both in pattern and timing. One principal difference is the presence in E. scolopes of a group of cells in the hatchling, i.e., the superficial ciliated, microvillous field, whose sole purpose appears to be the mediation of the inoculation process. Cells of rhizobia infect the already existing, functioning root hair, and induce creation of the DEVELOPMENT OF COOPERATIVE SYMBIOSES Initiation • Colonization 603 • Persistence FIG. 2. Early development of the squid/vibrio symbiosis. The diagrams represent a ventral view of the right side of the juvenile light organ. At the time of initiation (upper, left-most diagram), the ciliated field functions to promote infection by flagellated, V. fischeri in the environment. The bacteria enter the hatchling organ through pores on its surface, travel down ciliated ducts and come to reside in crypts lined with columnar epithelia (lower, left-most diagram). Bacterial colonization of the light organ (middle diagram) leads to massive cell death of the superficial, ciliated field, beginning at about 12 hr post-initiation at the tips of the appendages and at the edge of the field (depicted as small circles on the organ). This cell death program results in the eventual loss of the entire superficial, ciliated field (upper, right-most diagram), which characterizes the persistence stage of the symbiosis, i.e., that period when a stable association has been established. In addition, the host cells swell and the microvillar density of the brush border increases 4-fold over the first 4 days of the symbiosis. Bacterial cells lose flagellation and reduce in size in response to the symbiotic state. infection thread, which mediates the inoculation of the root nodule. As in the squid/ vibrio symbiosis (McFall-Ngai, 1994), the maturation of the functional, i.e., nitrogenfixing, organ is an elaborate process that follows a transformation from the inoculation morphology and requires a few weeks after infection to complete (Long, 1989). In addition to being similar to the legume/rhizobia symbioses in overall developmental pattern, the squid/vibrio symbiosis offers developmental biologists similar opportunities to dissect the process of symbiotic organ morphogenesis. Each of these types of associations, in contrast with consortial symbioses, has one host and one microbial species, both of which can be maintained in culture independent of the other partner. Further, both of these symbioses involve a prokaryotic partner, so that molecular genetics are readily applied to their study (Long, 1989; Ruby, 1996). In contrast with the legume/rhizobia symbioses no genetics is as yet available in the squid host. However, development of a genetic system may be possible; maintenance of a colony of only 12 female squid yields between 50,000 and 100,000 eggs per year (M. McFall-Ngai, personal observation), and techniques for raising the squid through their life cycle have been recently refined at the Marine Biological Laboratories in Woods Hole, Mass. (R. Hanlon, personal communication). However, manipulation of the host genome, using antisense probes or viral vectors may be more promising than conventional breeding approaches to genetics. Experimental manipulation of the squid/ vibrio symbiosis have shown that the partners have a profound influence on each other's development. Thus far, we have focused on the early developmental events, i.e., those that occur within the first four days of the symbiosis, a period which can be divided into stages of initiation, colonization and persistence (Fig. 2). By maintaining hatchlings in the presence (symbi- 604 MARGARET J. MCFALL-NGAI BACTERIAL DEVELOPMENTAL PATHWAY SYMBIOSIS DEVELOPMENTAL PATHWAY HOST DEVELOPMENTAL PATHWAY (embryogenesis) Hatching I • expulsion behavior - cell death signalling - brush border remodeling - /uxgenes induction - full colonization - flagella loss - size decrease - catalase induction (?) 1 day - CMS regression - crypt cell swelling - oxidatlve stress - competitive exclusion - iron acquisition - cyclic recolonization 4 days - lens formation (ALOH induction) - reflector elaboration - ink sac remodeling >10 days FIG. 3. Reciprocal signaling during the development of the squid/vibrio symbiosis. Host and bacterial development are characterized by changes in behavior, morphology, and biochemistry in response to interactions with one another. Molecular genetic studies of the bacteria, and responses of the host to mutant strains, indicate that a dynamic dialogue occurs between the two. The result of a successful dialog is the symbiosis developmental pathway, which leads to the establishment of the stable, mature association. ALDH = aldehyde dehydrogenase, the principal protein of the light organ lens; CMS = ciliated, microvillous surface; LPS = lipopolysaccharide surface of bacterial symbionts; lux = descriptive of genes associated with the bacterial operon that encodes light production capability; ROS = reactive oxygen species; Sym = symbiosis. otic) and absence (aposymbiotic = exposed to other bacterioplankton, in the absence of V. fischeri), we have been able to determine: 1) what components of the developmental program require interaction with symbionts; and, 2) which of these components require persistent influence of symbionts, and which require only transient interaction with V. fischeri cells. In the absence of the symbionts, the su- perficial epithelial field of the hatchling light organ does not regress (Montgomery and McFall-Ngai, 1994), the crypt cells do not swell (Montgomery and McFall-Ngai, 1994) and the brush border does not change in microvillar density (L. Lamarcq and M. McFall-Ngai, personal observation). The loss of the superficial field occurs as a result of bacteria-induced cell death (Montgomery and McFall-Ngai, 1994), which pro- DEVELOPMENT OF COOPERATIVE SYMBIOSES 605 gresses over the first four days of the sym- fischeri has resulted in the description of biosis. Despite the fact that this process re- several classes of mutant strains that have quires four days to complete, the presence revealed bacterial genes that are essential of bacterial symbionts is only required for for proper colonization of the host organ. approximately 12 hr (Doino and McFall- Ruby (1996) has divided these mutants into Ngai, 1995); i.e., antibiotic treatment to re- three categories, initiation, accommodation move bacteria from the light organ after this and persistence phenotypes. Initiation muearly stage still results in the complete re- tants are incapable of any colonization of gression of the superficial field (Doino and the light organ; accommodation mutants McFall-Ngai, 1995). Thus, the bacteria never colonize to levels characteristic of must be delivering a signal that produces wild type; and, persistence mutants are able an irreversible cascade of developmental to enter the light organ, may or may not events in the host. In contrast, the changes colonize fully, but are not able to persist in in the brush border require a continued in- the light organ past the first day or two. The teraction with the symbionts; antibiotic cur- behavior of several of these mutants suging at any time results in a return to hatch- gests that the symbiont is responding to the ling or aposymbiotic levels of microvillar change in oxidative environment of the density (L. Lamarcq and M. McFall-Ngai, crypts. personal observation). The extent to which These experimental studies of the squid/ cell swelling requires persistent interaction vibrio symbiosis, while still in their infancy, remains to be determined. are beginning to reveal the elaborate diaDuring this early period in the initiation logue that goes on between E. scolopes and of the squid/vibrio symbiosis profound V. fischeri during the establishment and changes are also seen in the morphology of eventual maintenance of the symbiotic the bacterial symbiont (Fig. 3). Between 12 state. As in the legume/rhizobia symbioses, and 24 hr after initiation of colonization, there are indications that the dialogue is rethe bacteria have lost the tuft of sheathed, ciprocal; i.e., the progression through depolar flagella that is characteristic of this velopment requires a volley of gene exbacterial species in its free-living niche pression between the partners (Fig. 3). In addition to providing insight into the (Ruby and Asato, 1993). In addition, they have decreased approximately 8-fold in vol- mechanisms by which bacteria may influume to dimensions of approximately 1 mm ence the normal development of the epitheby 0.5 mm as differentiated symbionts lial tissues with which they associate, the (Ruby and Asato, 1993). In addition, the squid/vibrio system promises to help biolbacterial symbionts induce luminescence ogists understand how cooperative associ1,000-fold (Ruby and Asato, 1993), and ations differ from pathogenic ones. The their growth rate is curtailed from an initial finding of host halide peroxidases in the doubling time of 0.5 hr to one of between light organ (Tomarev et al., 1993; Weis et ah, 1996) and genes associated with vibrio 5 and 6 hr (Lee and Ruby, 1994). Several biochemical and molecular pathogenesis in the bacterial symbiont changes in one or the other of the partners (Reich and Schoolnik, 1994, 1996) suggest correlate with these morphological and an- that some of the language between the partatomical modifications. Most notably, a sig- ners is conserved between the two very difnificant modulation of indicators of oxida- ferent types of associations. In summary, the study of the bacterial tive stress in the host light organ occurs over the first few days of the association; influence on animal development is a field specifically, there is some evidence of a on the frontier. As both the abiotic and bitransient increase in toxic oxygen species, otic environments of animals become afperhaps the result of a respiratory burst of fected by human intervention, it becomes the crypt cells, followed by a decrease in increasing important to understand how anthe levels of these molecular species (Small imals form and maintain stable associations with environmental microbes, and the conand McFall-Ngai, 1993). Application of molecular genetics to V. ditions that determine whether such asso- 606 MARGARET J. MCFALL-NGAI ciations become cooperative or pathogenic. Just as certain nations, such as Great Britain and Germany, have gone through periods of history as either allies or enemies, it is likely that the delicate balance that is created in cooperative associations has been historically, and will continue to be, susceptible to forces that threaten to compromise detente. 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