Controlling Symbiotic Microbes with Antimicrobial Peptides - ACS

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Controlling Symbiotic Microbes with Antimicrobial Peptides Peter Mergaert*,1 and Eva Kondorosi1,2 1Institut

des Sciences du Végétal, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France 2Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary *E-mail: [email protected]

The production of antimicrobial peptides (AMPs) in response to invading pathogenic microbes is an effective and ancient innate immune strategy which is conserved in all analyzed present-day eukaryotes. However, organisms are not only threatened by microbes but on the contrary, they often form beneficial symbiotic associations with them. There is a growing number of reported cases, both in animals and plants, which demonstrate the critical involvement of AMPs also in these symbiotic interactions. Notably, AMPs can intervene in the selection and maintenance of symbiotic microbial communities.

Acquiring New Capabilities through Symbiotic Associations Multicellular organisms live not free of germs but are inhabited with a multitude of microbes. This microbiota includes symbionts which co-evolved with their hosts for millions to billions of years. They contribute significantly to the hosts normal development and growth in a wide range of means. In this way, the microbial symbionts endow the hosts with capacities that they did not need to evolve on their own. The animal gut is crowded with symbiotic microbes. For example, the human gut has an estimated load of 10 to 100 trillion bacteria representing from several hundreds to thousands of species (1). These gut symbionts have a profound impact on gut development. They direct the proliferation and maturation of gut epithelial cells for epithelium renewal in drosophila (2), zebrafish (3, 4) and © 2012 American Chemical Society In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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mammals (5). Gut bacteria in mice have been shown to stimulate intestinal blood vessel development (6). The mammalian gut symbionts also shape the intestinal and systemic immune system by coordinating the differentiation of pro-inflammatory and anti-inflammatory T cells (7–10). Other well documented symbioses involve organ development and often require an active participation of the microsymbiont in the organogenesis. The marine squid Euprymna scolopes for example has a light-emitting organ which is colonized by luminescent Vibrio fischeri bacteria. This light organ helps the animal in evading predators. The sequential steps in the colonization and tissue differentiation of the light organ are coordinated by bacterial signals (11). Aphids and other insects harbor intracellular symbionts in specialized cells called bacteriocytes which are organized in the bacteriome organ located on the abdomen of the insect (12). Another well described system for symbiotic organ development is the symbiosis of legume plants with soil bacteria belonging to the Rhizobiaceae (collectively called rhizobium bacteria or rhizobia). This interaction leads to the formation of a dedicated organ, the nodule. Nodules develop on the root system of the host, and house the intracellular symbiotic bacteria. The nodule formation entirely depends on the presence of rhizobia and is induced by a specific bacterial signal called the Nod factor (13). The raison d’être of many symbionts is to provide the ability to harvest otherwise inaccessible nutrients. Gut microbes form an anaerobic bioreactor which hydrolyze ingested polysaccharides and ferments the resulting monosaccharides. The by-products of fermentation are short chain fatty acids which are absorbed by the host and utilized as carbon and energy source (14, 15). Aphids feed exclusively on plant phloem sap which constitutes a diet that is deficient in amino acids. The pea aphid Acyrthosphion pisum harbors the obligate endosymbiont Buchnera aphidicola in its bacteriocytes which synthesizes the essential amino acids missing in the phloem and provides them to the host (16). Likewise, the endosymbiotic rhizobium bacteria in legume nodules fix air-borne nitrogen gas, an abundant but stable chemical form of nitrogen, unusable for most organisms except nitrogen-fixing microbes. The ammonium resulting from the nitrogen reduction by the nodule rhizobia is assimilated by the host plant and used for its growth (17).

Hosts Actively Select and Control Their Microbial Symbionts An important issue in symbiosis is to pick out the right bacterial partner, often among a myriad of environmental microbes. Hosts are able to select actively their microbiota. In addition, once the host is colonized by the right symbionts, mechanisms must be put in place to ensure that the association is stable, that the symbionts do not overgrow the host or on the contrary, that the host does not eliminate the symbionts. Effective symbiont selection can happen through the vertical transmission of the symbionts from parent to offspring through the female germ line as it happens for example in the aphid-Buchnera symbiosis (16). But even microbes that are transmitted horizontally are successfully selected from the environment. This may rely on complex mechanisms implying developmental 216 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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processes which are induced by bacterial signals. Such mechanisms in squid and legumes are exquisitely specific, capable to select a single bacterial species from the extremely complex microbiota of the environment, the sea water or the rhizoshere soil respectively (18). The specific colonization of the squid light organ by V. fischeri for example is ensured by a complex series of developmental responses, including gathering of bacteria from the seawater by moving cilia, production of mucus that specifically aggregates V. fischeri, recruitment of haemocytes, cell differentiation and apoptosis. Some of these events are induced by bacterial peptidoglycan and lipopolysaccharide signals and require specific V. fischeri genetic determinants such as the two-component regulator RscS/SypG which controls the production of the Syp (Symbiosis polysaccharide) exopolysaccharide mediator of colonization (11, 19–21). Established V. fischeri populations in the light organ are kept in check by the daily expulsion of most of the bacterial population each dawn, followed by the regrowth of the remaining bacteria (22). In the case of the legume root nodules, the rhizobium-produced Nod factors induce the formation of the nodules and also the formation of tubular infection structures called infection threads which guide the rhizobia inside the plant tissues but exclude penetration of any other bacteria into the nodule tissues (13). Rhizobia are released massively from infection threads in the target nodule cells and differentiate into nitrogen fixing “bacteroids”. In some legumes, the host cells control the intracellular bacterial population by the production of AMPs which induce an irreversible, terminally differentiated state of the bacteroids (23). Also in the seemingly “open” system of the animal gut where bacteria are acquired by oral uptake, the symbionts are actively selected by the host. The human intestine contains, compared to the 55 known bacterial divisions (deep evolutionary lineages), few divisions and is dominated by only two of them, the Firmicutes and the Bacteroidetes. This contrasts with soils where plant polysaccharides are also degraded, containing at least twenty bacterial divisions and suggests that the human gut microbiota are at least in part selected by the gut epithelium (24). This is corroborated by studies of the bacterial diversity in the guts of 60 mammalian species including humans (25) or of wild great apes (26), both indicating that the microbial communities co-diversified with the host phylogeny. When gut microbiotas from mouse and zebrafish were reciprocally transplanted in germ-free zebrafish and mouse hosts respectively, the transplanted microbial communities were transformed to resemble the normal microbiota composition of the recipient host, thus revealing again an active selection by the host (27). The complexity of the gut microbiota is highly different among animals and the bacterial diversity in the studied invertebrates is apparently one to two orders of magnitude lower than in the mammals. Nevertheless, in each case this bacterial population has a specific composition. For example, the Drosophila gut is dominated by just a few dominant bacterial species (28). Even in Cnidaria, the simplest animals positioned in the earliest branches of the animal tree of life, the epithelial cells actively shape their bacterial community (29). Moreover, the symbiotic bacteria in animal guts are numerous and thus they pose a threat of invasion. Therefore, the animal host has to build the specific 217 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

gut microbiota and maintain it in a homeostatic relationship with the epithelial cells that are in contact with it. Then, how is this achieved? In mammalians, specific host factors that determine the composition of the gut microbiota and keep them in equilibrium are the innate and adaptive immune systems (30, 31) with a primary role for AMPs (32). But also in Drosophila and Cnidaria, AMPs are key factors that influence the structure of the gut microbial community (28, 33). Thus, host AMPs are conserved metazoan key actors in the interaction with epithelial microbiota.

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AMPs: Fighting Pathogens and Controlling Symbionts AMPs are important players in plant and animal innate immune systems. AMPs are extremely diverse, differing in their amino acid composition and structure. They affect immunity in broadly two ways. They can kill microbes and thus act as antibiotics displaying usually fast and broad spectrum killing activities against Gram-negative and Gram-positive bacteria, fungi as well as viruses and parasites (34). In addition, AMPs may enhance immunity by functioning as immune-modulators (35). AMPs are best known for this role in innate immunity, in fending off microbes from the environment and in fighting pathogenic infections. However, as underlined above, recent findings in different organisms including invertebrates, vertebrates and plants have demonstrated the unexpected importance of AMPs in the selection of bacterial symbionts and the control of established symbiotic populations. These new insights in symbiotic mechanisms and the natural roles of AMPs in symbiosis will be reviewed here. The examples in the literature of AMPs controlling gut microbiota will be briefly highlighted and then the focus will be on specific AMPs that control the endosymbiotic rhizobium bacteria in legume nodules.

Control of the Animal Gut Microbiota by AMPs Colonization of Epithelia in the Early Branching Metazoan Hydra The cnidarian Hydra (polyps) maintain a specific bacterial microbiota as indicated by comparing laboratory populations of different species and specimens directly isolated from the wild. Different species grown under identical laboratorium conditions over more than thirty years differed greatly in their microbiota but specimens living in the wild were colonized with a similar microbiota as the laboratory polyps of the same species (29). Hydra have a simple tube-like body enclosing the digestive cavity with at one end a mouth and tentacles. This body is enclosed by an equally simple tissue organization consisting of two single-cell layers, the outside ectoderm and the inside endoderm. In between these layers are interstitial cells which are stem cells that can give rise to various cell types including the germ line, nerve cells, gland cells and nematocytes (the venomous nettle cells on the tentacles, the distinguishing feature of Cnidaria). Genetic ablation of the interstitial cell lineage in the temperature-sensitive mutant strain sf1 of the species Hydra magnipapillata changes the microbiota of the animals considerably. In particular, the dominant β-proteobacteria phylotype 218 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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in the control animals is reduced in the interstitial-cell-line-depleted animals, and on the contrary, the Bacteroidetes phylotype becomes more abundant (36). These results indicate that the ablated cells produce factors that interfere with the microbiota and control its structure. Purification of antimicrobial activities in Hydra protein extracts as well as screening for differentially expressed genes has led to the identification of several AMP families and other antimicrobial proteins produced by the Hydra epithelial and interstitial cells (33, 37–40). The impact on the microbiota of one of those AMPs named periculin 1a was analyzed in more detail (33). It was noticed that the composition of the microbiota in early embryos was very different from those at later embryonic stages and adult animals. Thus the early embryos produce specific factors controlling the bacterial colonizers. One of these factors was biochemically identified as the AMP periculin 1a. This peptide is specifically expressed in a subset of the interstitial cells of the female germ line, in the developing oocytes and the early embryos (33). When the periculin 1a peptide was ectopically expressed in the ectodermal cell layer of adult polyps, the bacterial load in the animals was strongly reduced and interestingly, also the specific composition of the microbiota was dramatically changed with a strong decrease of the dominant β-proteobacteria, an equally strong increase of the α-proteobacteria and the appearance of new phylotypes (33). Together, these findings make a strong case for a role of AMPs in the selection of particular bacterial symbionts during the animal development and adult life in Hydra. The Drosophila Gut Among the major immune reactions of Drosophila in response to microbial infections is the inducible production of AMPs (41). AMPs are induced during the so-called systemic response in the fat body and secreted in the hemolymph circulatory system of the insect. Moreover, they are also induced locally in epithelial cells in a tissue-specific manner. Induction of the AMPs in the gut is mediated by the immune deficiency (Imd) pathway. The pathway is activated by the peptidoglycan recognition proteins which bind the peptidoglycan component of the cell envelop of gram-negative bacteria. This subsequently results through an intracellular signalling pathway in the proteolytic activation and nuclear translocation of the NF-κB transcription factor Relish, followed by the transcription of AMP genes (41). Thus, oral infection with bacterial pathogens will lead to AMP activation in the gut and clearance of the pathogen. However, this leaves open the question how the homeostasis between this innate immune response and the symbiotic microbiota is achieved. Surprisingly, it was found that the resident bacteria in the Drosophila gut chronically activate the Imd pathway as revealed by the nuclear localization of Relish in conventionally reared flies but not in germ-free flies. Yet, this does not activate AMP gene transcription (28). The reason is the repression of AMP expression by the homeobox transcription factor Caudal (28), well known for its role in embryo formation and development of the gastrointestinal tract (42) and for constitutive AMP expression in certain epithelial cells (43). Inactivation of Caudal via RNA interference (RNAi) provoked a spontaneous activation of AMP gene expression in the gut which was however dependent on the presence of the 219 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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microbiata and did not take place in germ-free flies. Thus AMP expression in the gut epithelia is the result of a balance between activation by Relish and repression by Caudal (28). Inactivation of Caudal by RNAi provoked along with the high production of AMPs, also a dramatic shift in the microbiota community (28). In particular, the abundance of a dominant Acetobacter species in control flies was greatly reduced by Caudal RNAi while in contrast, a minor Gluconobacter species of the wild type gut emerged as a dominant one. Still other gut bacteria were not affected by the Caudal inactivation. Ectopic AMP expression in transgenic flies resulted in the same changes in the gut bacterial community. Moreover, the sensitivity of these bacteria to the in vitro applied, pure AMP correlated with the in vivo observations. Thus, the observed modifications in the microbiota are the direct consequence of deregulated AMP expression (28). Additionally, the dysbiosis (microbial imbalance) resulting from the dominance of the Gluconobacter species in the gut of the AMP overexpressing flies led eventually to the rupture of the gut homeostasis and induced apoptosis of intestinal cells and host mortality (28). Apoptosis and mortality were high in conventionally reared Caudal RNAi flies but not in germ free flies and simulation of the dysbiosis by introducing the Gluconobacter strain in germ free wild type flies induced the same. Thus the dysbiosis is the direct cause of the gut pathology. All together, this study shows that a controlled and balanced expression level of AMPs under healthy conditions is essential for the maintenance of the normal gut flora and the microbial composition is on its turn important for the homeostasis between the microbiota and the innate immunity in the gut of flies. The Mammalian Gut Maintaining intestinal homeostasis in mammalians and particularly in humans has another level of complexity in light of the astonishing quantity and species diversity of their gut microbiota. Thus, it is a challenging task for the intestinal epithelia to prevent that these microbiota penetrate in the underlying tissues. This is achieved by a multitude of immunological barriers which are designed to minimize the direct contact of the epithelia with the intestinal bacteria and to rapidly eliminate bacteria that penetrate anyhow the epithelia and the underlying lamina propria tissue (reviewed in (30)). The latter occurs through phagocytosis by macrophages and T cell-mediated responses. The barrier which confines the microbiota essentially to the gut lumen is composed of three key elements (30). First, a protective mucus layer composed of mucin glycoproteins is produced and secreted by the goblet cells, a specific epithelial cell type. This layer creates a nearly bacteria-free zone on the surface of the epithelia which is lost in mutant mice lacking the major mucin protein MUC2 (44, 45). Second, immunoglobulin A (IgA), specific for intestinal bacteria, are produced and secreted across the epithelia in enormous quantities by plasma cells in the lamina propria of the intestine (46–49). A third component of the barrier is the secretion of a diversity of AMPs (30, 31). AMPs are produced by most epithelial cells but the primary producers are the Paneth cells. These are secretory cells in the small intestine epithelium, located in clusters at the base of crypts. The key role 220 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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of the Paneth cells in the protection of the epithelia became clear when Paneth cells were specifically ablated in transgenic mice expressing a toxin under the control of a Paneth cell specific promoter. In these mice, both the microbiota and pathogens penetrate the mucosal barrier and the epithelial tissues (50). Another study used mutant mice lacking the Nod2 receptor, sensing the bacterial molecule muramyl dipeptide. The Paneth cells in this mutant produce less of the α-defensin AMP (51). The Nod2-deficient mice have an increased quantity of symbiotic bacteria in the small intestine (52). Thus the AMPs produced by Paneth cells keep the microbiota load under control and contribute to the limitation of the contact between the symbiotic gut bacteria and the epithelial tissues. Moreover, Paneth cell-produced AMPs also regulate the specific composition of the microbiota in the lumen of the small intestine (32). α-defensin production by the Paneth cells can be modulated to produce more or less than the wild type levels by the use of transgenic mice, homozygous or hemizygous for the transgene HD5 encoding a human α-defensin (53) and of mice, heterozygous or homozygous for a mutated Mmp-7 allele, required for processing of mouse α-defensin in an active form (54). The composition of the microbiota in these complementary models showed reciprocal shifts (32). The most notable differences were for bacteria of the Firmicute phylotype which decreased at higher α-defensin production by the Paneth cells while the Bacteroidetes phylotype followed the opposite trend. This is in agreement with an earlier study which found by microscopic observation a shift in the bacterial population in the gut of HD5-expressing mice (55). In addition to the shifts in the dominant phylotypes, an important species in the mouse microbiota, known as segmented filamentous bacteria or SFB (formally Candidatus arthromitis) is eliminated from the intestinal microbiota in HD5-expressing mice (32). Thus Paneth cells and α-defensins control the composition of the intestinal microbiota in a similar way as described above in flies and polyps. It is well known that the mammalian gut symbionts shape the intestinal and systemic immune system by coordinating the differentiation of pro-inflammatory and anti-inflammatory T cells in the lamina propria (56). Specific bacterial species have been identified which stimulate the differentiation of particular T cell types. For example, SFB stimulate the pro-inflammatory TH17 cells (8, 9) as well as regulatory T cells (9). The gut symbiont Bacteroides fragilis, through the action of its PSA polysaccharide signal, suppresses TH17 production and stimulates regulatory T cells (57). Faecalibacterium prausnitzii (58) and a consortium of Clostridium species (10) are all stimulating anti-inflammatory regulatory T cells. Thus not surprisingly, the mice models with altered α-defensin production by the Paneth cells and resulting dysbiosis, displayed also an alteration in the T cell production in their lamina propria. Particularly, the notable change in SFB entailed a corresponding change in the TH17 cell population (32). Thus the AMPs also influence, albeit indirectly, the differentiation of the adaptive immune system in the gut.

221 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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Conclusions The three studies in the very divergent animals Hydra, Drosophila and mouse, using similar strategies, namely the mis-expression of AMP genes in the gut epithelia, come to the same conclusion that the profile and the level of AMPs produced by the gut are crucial to create and maintain a specific and favourable composition of the microbiota. This type of studies has important implications for the understanding of disorders such as inflammatory bowel disease and in particular Crohn’s disease. An improper balance between pro- and anti-inflammatory T cells critically affects the onset and the progression of these diseases (56). Crohn’s disease is also associated with dysbiosis (59). Moreover, several risk alleles for Crohn’s disease in genetically susceptible people affect the proper functioning of Paneth cells and their ability to secrete AMPs, including mutations in the above mentioned Nod2 gene and other genes affecting the secretory pathway required for AMP secretion (31). This correlates with the observed lower production of AMPs in Crohn’s disease patients (55). Together, these findings provide a coherent picture of Crohn’s disease. The presence of risk alleles lead to a reduced Paneth cell functioning and secretion of AMPs which on its turn modifies the composition of the intestinal microbiota and an associated imbalance of pro- and anti-inflammatory responses resulting in or perpetuating the chronic intestinal inflammation associated with the disease (32).

AMPs Control Differentiation of Nitrogen Fixing Endosymbionts in Legume Plants Establishing a Symbiotic Bacterial Population in Legume Nodules The formation of nodules on the roots of legume plants is activated by the Nod factor signal molecules produced by the rhizobium bacteria in the rhizosphere. Nod factors, recognized by specific membrane-bound receptors in the root epidermal cells, activate proliferation of the root cortical cells and the formation of a so-called nodule primordium. Growth by continuing cell divisions and endoreduplication-driven cell differentiation in the emerging organ leads then ultimately to the formation of a full-grown root nodule (60). In parallel with the organogenesis process, Nod factor signalling also initiates the formation of rhizobium-containing infection threads in the root epidermal cells (13). The tissues of the growing nodules are invaded by the infection thread network which grows towards the differentiating nodule cells and releases bacteria into their cytoplasm by an endocytotic process (61). Single or a few internalized bacteria are confined in organelle-like structures called symbiosomes. The bacterial release, combined with symbiosome multiplication and maturation ultimately fills-up the host cell completely with symbiosomes. Differentiation of the Symbiotic Rhizobium Bacteria Within the developing symbiosomes, the rhizobium bacteria differentiate into nitrogen-fixing bacteroids. Differentiated bacteroids are highly specialized 222 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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bacterial cells, entirely different from bacteria in culture, in the rhizosphere or in infection threads. They are adapted for nitrogen fixation and the existence as an organelle-like entity. This bacterial differentiation is made possible by a massive transcriptome switch and is in part regulated by the FixLJ two-component regulator which senses the low concentration of free-oxygen prevalent in nodules and activates among others the genes required for nitrogen fixation per se and for microaerobic respiration (62). Moreover, rhizobium differentiation requires a number of bacterial household functions (reviewed in (63)) including transport of dicarboxylic acids, amino acids and other nutrients (17, 64, 65), the lipopolysaccharide component of the bacterial envelop (66, 67) and a performant oxidative stress resistance mechanism (68). In many but not all legume plants, bacteroid formation is also associated with drastic modifications of the bacterial morphology and cytosol organization. In plants of the Inverted Repeat Lacking Clade (IRLC) which includes the model legume Medicago truncatula, bacteroids are considerably elongated, reaching a length up to 10 µm, and can sometimes be branched. These bacteroids have altered membrane permeability and a high amplification of their genome which is condensed in multiple nucleoids of variable size (23, 69). The intensive DNA amplification in bacteroids requires an unusual oxidative-stress-resistant, cobalamine-dependent ribonucleotide reductase for the synthesis of the required deoxy nucleotides (70). The polyploidy of the genome suggests that the bacteroid differentiation process includes an interference with the bacterial cell cycle. This process of bacteroid differentiation is irreversible (or terminal) since these differentiated bacteria cannot produce offspring. On the other hand, in other legume groups the morphology, membrane integrity and genome content of bacteroids are similar to those of free-living bacteria. These bacteroids can produce offspring and are therefore reversibly differentiated (23). While terminal differentiation of bacteroids is thus not essential per se for symbiotic nitrogen fixation, it possibly improves the symbiotic efficiency of the bacteroids as suggested by a comparison of the symbiotic performance of terminal and reversible bacteroids (71). However, this single case study should be extended with a more extensive comparison between both bacteroid types before a general conclusion can be made. The AMP-like NCR Peptide Family The terminal differentiation of Sinorhizobium meliloti in M. truncatula nodules is independent of the FixLJ-controlled physiological bacteroid differentiation pathway because bacterial mutants in this signalling pathway still display features of terminal bacteroid differentiation (72). On the other hand, it was found by using nearly isogenic rhizobium strains nodulating both IRLC and non-IRLC legume species, that the host rather than the bacterial genetic repertoire determines the terminal bacterial differentiation. Therefore, it was concluded that the host cells of IRLC legume nodules produce factors that direct the terminal differentiation of rhizobium in the symbiosomes (23). A transcriptome search in M. truncatula designed to identify those factors was based on the assumptions that the encoding genes were induced during the nodule organogenesis and expressed 223 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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in the infected nodule cells. Moreover, homologous genes were expected to be similarly expressed in the nodules of other IRLC legumes but not in plants with reversible bacteroid differentiation. Nodule-specific cysteine-rich (NCR) peptides were identified by this approach as likely candidates. Remarkably, the NCR gene family in M. truncatula encodes more than 450 different peptides which are most similar to defensin-like AMPs. Homologs have been found in other IRLC legumes but not in species forming nodules with reversibly differentiated bacteroids (73, 74). Equally remarkable, the expression of all the M. truncatula NCR genes, except for two which are also expressed in roots, is strictly nodule-specific (73, 74). Expression was not found in any other plant organ or in other biotic interactions with mycorrhizal fungi, rhizosphere bacteria, pathogens, nematodes or insects. Transcriptome analysis of nodules obtained with a large collection of symbiotic mutants of M. truncatula and of its bacterial partner S. meliloti revealed that NCR gene expression was correlated with symbiotic cell formation (72). Moreover, in the case of examples tested with in situ hybridization or promoter-GUS fusion, the expression of NCR genes was found to be restricted to the rhizobium-infected plant cells. However, different subsets of NCR genes had distinct expression domains, certain being expressed in young symbiotic cells while others in older or mature symbiotic cells (74). Some NCR genes are expressed at very high levels and based on EST counts, it was estimated that the combined transcripts of the NCR family constitute close to 5% of the total mRNA pool in nodules. Although the NCRs have only limited sequence homology with other peptides, their protein structure, gene organization and family structure resembles AMPs. The possible involvement of AMP-like peptides in bacteroid differentiation is meaningful in light of certain bacteroid features such as membrane modifications, inhibition of cytokinesis and inability to reproduce which are known effects of different types of AMPs. In the NCR peptide sequences, an N-terminal signal peptide can be recognized, which is a cleavable tag for targeting of the peptide in the secretory pathway. By expressing protein fusions of NCRs or their signal peptides in a heterologous system, it was shown that these signal peptides are indeed functional and mediate entry of the peptides in the secretory pathway (74). The mature NCR peptides (the C-terminal part of the proteins obtained after the cleavage of the signal peptide during translocation in the endoplasmic reticulum) are around 40 amino acids long and are typified by a conserved pattern of cysteine residues while the rest of the sequence is highly variable amongst the family members in contrast to the preserved signal peptides (74). This reflects an evolutionary pattern with the signal peptides subjected to purifying selection in contrast to the mature peptides which were subjected to diversifying selection (73). Similar observations are frequently made for AMP gene families, including conservation of signal peptides and cysteine residues but strong divergence in the functional mature peptide (75, 76). Interaction of NCR Peptides with Endosymbiotic Rhizobium Peptides in the size range expected for NCRs accumulate in the nodules and co-purify with the bacteroids. Specific antibodies and peptide sequencing 224 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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confirmed that these peptides correspond to NCRs. Moreover, in situ localization of the NCR peptides confirmed this localization and demonstrated that at least some NCRs are transported in the bacteroid cytosol (77). Thus NCRs are targeted to and accumulate in high amounts in symbiosomes and bacteroids and this localization of the peptides indicates that the bacteroids are their targets. Since symbiosomes are vesicles with a plant-derived membrane, protein transport towards the symbiosomes and bacteroids depends on the secretory pathway (72, 77–79). Thus the localization of the NCRs and the presence of the characteristic signal peptide are in agreement with a transport mechanism of the peptides through the secretory pathway. This was confirmed by the analysis of the M. truncatula dnf1 mutant. This mutant is deficient in a nodule-specific subunit of the signal peptidase complex of the secretory pathway. This endoplasmic reticulum-located enzyme complex is responsible for the proteolitic cleavage of the signal peptide of secretory proteins which is critical for their correct targeting (80). The dnf1 mutant forms non-functional nodules which contain infected nodule cells. However, the symbiosome bacteria do not differentiate into elongated bacteroids (77, 79). In this mutant, the signal peptide of the NCRs is not properly processed and by consequence the peptides are blocked in the endoplasmic reticulum and are not targeted to the bacteroids. Thus, obstructing NCR transport is correlated with the absence of bacteroid differentiation in the symbiosomes, in agreement with a role of the NCRs in this bacterial differentiation process (77). The transcriptome analysis of nodules and infected cells in M. truncatula has revealed that the majority of the up-regulated genes in these cells are involved in protein secretion (72). These cells have a remarkably well developed and abundant endoplasmic reticulum (72). The genes encoding the four conserved subunits of the signal peptidase complex (including Dnf1) are duplicated in the M. truncatula genome and one copy maintains a ubiquitous expression while the other one acquired a nodule-specific expression (79). Thus the symbiosome-containing nodule cells are highly specialized for protein, and particularly for NCR transport to the symbiosomes (72, 79, 81). In an opposite, gain-of-function approach, NCR genes were ectopically expressed in the infected nodule cells of Lotus japonicus, a legume with reversible bacteroid differentiation and lacking NCR genes. Expression of certain NCR genes was sufficient to induce features of terminal bacteroid differentiation with symbiosomes containing single and remarkably elongated bacteroids (77). Even in vitro application of NCR peptides to S. meliloti free-living bacteria results in bacteroid-like features, notably in high permeability of the membrane, inhibition of bacterial proliferation, DNA accumulation and cell elongation (77, 82). All together, these findings are in agreement with a major role of the NCR peptides in the terminal differentiation of the symbiosome-located rhizobia. Bacterial Protection against the Antimicrobial Activity of NCRs NCRs are similar to AMPs such as defensins and the analysis of the in vitro NCR activity demonstrated that some NCR peptides indeed possess genuine antimicrobial properties and effectively kill not only S. meliloti (77) but also both gram-positive and gram-negative bacteria (E. Kondorosi, unpublished 225 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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data) at similar concentrations as most other antimicrobial peptides. The in vivo and in vitro effects of NCRs on S. meliloti are dramatically different because NCR-challenged bacteroids in nodule symbiosomes maintain an active metabolism for nitrogen fixation, despite their inhibition for growth. This could be explained by a concerted in vivo action of several tens or hundreds of peptides, each likely present at very low concentration. This is hardly comparable to the in vitro effect of the peptides externally applied at high concentrations. Furthermore, particular conditions prevalent in nodules such as the low free oxygen concentration which is needed for the activity of the oxygen-sensitive nitrogenase enzyme could modulate the bacterial responses to the NCRs in such a way that the bacteroids remain alive, although with a complete loss of their reproductive capacity. Moreover, the S. meliloti BacA protein was identified as a factor that helps the symbiosome bacteria to survive the NCR exposure (82). The S. meliloti bacA gene was originally identified as an essential gene for the establishment of an effective symbiosis (83). BacA mutants induce nodule formation on Medicago plants and the bacteria are released from infection threads into the symbiotic nodule cells but they fail to differentiate into elongated, nitrogen-fixing bacteroids (72, 83). The BacA protein is conserved in many bacteria, including the rhizobia and interestingly, the protein is critical for effective symbiosis and bacteroid development in those legume hosts that produce NCR peptides such as Pisum sativum (pea) and Astragalus sinicus (IRLC legumes) but it is dispensable for this process in Phaseolus vulgaris (bean), Vigna unguiculata (cowpea), and L. japonicus, which are legume hosts naturally devoid in these peptides (84–89). The BacA protein is an integral membrane protein in bacteria that belongs to the ATP binding cassette (ABC) superfamily of membrane transporters. The transported substrate remains unknown although the BacA protein and its Escherichia coli homolog SbmA facilitate the uptake of proline-rich peptides suggesting that those proteins can function as peptide transporters (90, 91). S. meliloti BacA also affects the incorporation of unusual, very long chain fatty acids in the lipo-polysaccharide membrane (92) and Rhizobium leguminosarum bv. viciae BacA affects the transcription of membrane proteins. Thus BacA has pleiotropic effects on the bacterial envelop. The BacA-deficient mutant of S. meliloti in vitro was still able to induce cell enlargement and DNA amplification in response to NCR peptides at low concentrations suggesting that the protein is not essential for the differentiation process per se despite the inability of the bacA mutant to differentiate in planta. However, the mutant was hypersensitive for the antimicrobial activity of the NCR peptides applied at higher concentrations indicating that the BacA protein provides resistance to S. meliloti against the antimicrobial activity of the NCRs (82). Within the symbiosomes of M. truncatula nodules, the bacA mutant bacteria are similarly challenged with NCRs as the wild type bacteria but the mutant, contrary to the wild type strain, cannot survive the symbiosome environment. However, in the dnf1 mutant of M. truncatula where NCR transport to the symbiosomes is blocked, BacA is not critical anymore for bacterial survival in the symbiosomes. Thus BacA is required in symbiosis by protecting S. meliloti against the bactericidal effects of NCRs in M. truncatula nodules and thereby 226 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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enabling proper bacteroid development (82). The mechanism for this protection remains to be discovered. It could be related to a peptide transport function of the BacA protein or alternatively, it could be related to bacterial envelop features affected by BacA which are critical for the amount of membrane damage induced by the NCR peptides. Interestingly, BacA function is also crucial for the pathogenicity of bacteria such as Brucella and Mycobacterium (85, 93). These pathogens establish chronic infections in animal hosts where they need to withstand cocktails of AMPs to survive. The E. coli and Brucella bacA genes can complement the symbiotic defect of the S. meliloti bacA mutant (94, 95) and the Brucella gene can protect the mutant against the antimicrobial activity of NCRs (82). Thus BacA function is conserved and BacA-mediated protection of bacteria against host AMPs may be general and a critical stage in the establishment of symbiotic as well as pathogenic chronic host infections. Future Directions: What Is the Biological Meaning of Bacterial Differentiation? The different lines of evidence described above demonstrate that the NCR peptides are the principal mediators of bacteroid differentiation in M. truncatula symbiosomes. However, many unsolved questions are remaining, notably with respect to the mode of action of the peptides and to the biological meaning of the NCR-induced effects. Some NCR peptides inhibit in vivo and in vitro bacterial division leading to cell elongation. Such NCRs were localized at the division site of S. meliloti cells (77) suggesting that these peptides may interfere with the bacterial cell division machinery. However, the high sequence variety of NCRs suggests diversity in their functions, mode of actions and bacterial targets. Perhaps some peptides interfere with bacteroid metabolism and thereby optimize the efficiency of the nitrogen fixation as suggested by the higher symbiotic efficiency of terminal bacteroids as compared to reversible bacteroids (71). For example, the accumulation of the storage compound polyhydroxybutirate (PHB), which takes the host-supplied carbon away from nitrogen fixation, is very frequently observed in bacteroids but not in the bacteroids of IRLC legumes. Inhibition of PHB accumulation in those bacteroids could be a direct or indirect consequence of the terminal differentiation. Another raison d’être for the high diversity of NCR peptides could be an adaptation to the high diversity of rhizobia in soils. The diversifying selection that has shaped the NCR family is compatible with such a hypothesis (73). Additional, non-exclusive hypotheses can be put forward as to explain a better performance of terminally differentiated bacteroids. Polyploidy of the bacteroids may support higher metabolic activity of the cells in a general way as it is in the case of eukaryotic cells (60). Terminally differentiated bacteroids are always present as a single bacterium per symbiosome which may permit a very efficient nutrient exchange with the host cell. On the contrary, multiple reversibly differentiated bacteroids are present in a single symbiosome and these bacteroids have therefore a more limited and less efficient contact with the symbiosome membrane. Moreover, terminally differentiated bacteroids are effectively digested 227 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

by the host during the senescence process, at the end of the nodule life. This is less so for nodules containing reversibly differentiated bacteroids which can efficiently survive nodule senescence. Thus, nutrient recycling during senescence might provide an advantage to the plant.

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Concluding Remarks The illustrated examples in Hydra, Drosophila and mouse strongly suggest that AMPs controlling gut microbiota is most likely conserved in all branches of the animal tree of life. AMPs also control endosymbiotic bacteria as illustrated by the legume bacteroids. It would thus not come as a surprise to find the implication of AMPs also in other symbiotic systems as for example in the formation of the light organ in the V. fischeri-squid symbiosis or in the insect bacteriomes such as the one formed during the aphid symbiosis with Buchnera. A specific transcriptome analysis of the bacteriome in the weevil Sitophilus zeamais indeed identified the specific transcription of an AMP gene in bacteriomes (96). Terminally differentiated bacteroids are not only observed in IRLC legumes but in other legume clades as well (97). For example, spherical bacteroids are described in Aeschynomene species (98). Also here, bacteroid differentiation is induced by plant factors whose nature however remains to be discovered. Analyzing more symbiotic systems and the interaction of host AMPs and more generally the innate and adaptive immune systems with the microbial symbionts may change our thinking on the evolutionary roots of these immune systems. Did they evolve to respond to pathogens or rather to select and maintain symbiotic partners?

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