Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis

All animals are associated with microorganisms; hence, host-microbe interactions are of fundamental importance for life on earth. However, we know little about the molecular basis of these interactions. Therefore, we studied the deep-sea Riftia pachyptila symbiosis, a model association in which the tubeworm host is associated with only one phylotype of endosymbiotic bacteria and completely depends on this sulfur-oxidizing symbiont for nutrition. Using a metaproteomics approach, we identified both metabolic interaction processes, such as substrate transfer between the two partners, and interactions that serve to maintain the symbiotic balance, e.g., host efforts to control the symbiont population or symbiont strategies to modulate these host efforts. We suggest that these interactions are essential principles of mutualistic animal-microbe associations.


Contents
Supp. Table S1a All Riftia host proteins identified in this study Supp. Table S1b Potential autophagy-related Riftia proteins Supp. Table S1c Potential Riftia antimicrobial peptides Supp. Table S1d Genomes and metagenomes used for SMART analysis of eukaryote-like protein structures Supp. Table S1e Sampling dates, cruise number and number of biological replicates of Riftia samples used in this study Supp. Table S1f Number of proteins with significant abundance differences in pairwise comparisons of Riftia tissues Supp. Table S1g All Riftia symbiont proteins identified in this study Supp. Table S1h Hemerythrin and myohemerythrin isoforms in Riftia and other invertebrates used for alignment in Supp. Figure S3 Supp. Table S1i Carbonic anhydrase isoforms in Riftia as detected in this study and described in the literature The following Supplementary Tables are provided as separate PDF files: Supp. than in the mantle were suggested to be a reaction to oxygen and sulfur radicals produced by the 49 symbionts (1). Higher HSP70 levels were furthermore shown to concur with a higher stress tolerance caused by symbiont digestion and subsequent host bacteriocyte death, and in interaction with the mechanism to avoid sulfide toxicity (17)(18)(19), this strategy thus seems to be of minor importance in 112 Riftia. For some thiotrophic symbionts, e.g. of Bathymodiolus mussels, thiosulfate appears to be the 113 preferred sulfur form (20), and mitochondrial sulfide oxidation to thiosulfate thus likely also 114 promotes symbiont energy generation. As Riftia symbionts prefer sulfide (even though they can 115 probably also use thiosulfate; (21-23)), they would presumably not benefit from mitochondrial 116 sulfide oxidation by the host. to have circulating and non-circulating globins in addition to extracellular globins (reviewed in (30)).

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Riftia might possess, in addition to its extracellular hemoglobins, also the other two globin types, 132 which would make it the first described member of the animals, e.g. iron storage, metal detoxification and functions in the immune system (37-39, 44).

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Potentially, they might even be involved in nitrate transport, as nitrate binding and concentrating by   Figure S4). Tissue-209 specific expression of Riftia CA has been noted before (46, 47). Three of the CAs detected in our study 210 align closely to the CAs analyzed in these previous studies (Supp. Figure S4). Previous biochemical 211 experiments hinted towards the existence of membrane-bound Riftia CA (47, 48). Indeed, predicted 212 transmembrane helices and/or signal peptides in three of the CA sequences in our study indicate that 213 these could be membrane-bound (or even secreted) rather than cytoplasmic. These three CAs also 214 align more closely to each other than to the other CA sequences (Supp. Figure S4). Interestingly, 215 putative membrane-bound CA isoforms seem not to be restricted to one tissue, but might increase Supp. Figure S4: A) Alignment of carbonic anhydrases (CAs) detected in this study (Riftia_CA1 -Riftia_CA9) and 223 CAs detected in previous studies ((46, 47), for accession numbers see Supp. Table S1i). Lower case letters  Table S1i. We detected proteins involved in transport of inorganic carbon by the Riftia host to its symbiont, i.e., addition, it has previously been suggested that the host also provides the symbionts with pre-fixed 237 CO2, i.e., with small organic compounds (49, 50). Phosphoenolpyruvate carboxykinase, which we 238 detected in all host tissues, and pyruvate carboxylase, which was only lacking in S-rich plumes (Supp.

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Table S1a), catalyze incorporation of CO2 into oxaloacetate, which would then be available for host containing symbiont waste products. 262 We found two possible NO3⁻ transporters (probable peptide/nitrate transporter At3g43790: Text Figure 4). Although NarGHIJ is mainly known to be involved in dissimilatory nitrate reduction,

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Both symbiotic partners appear to be capable of producing the polyamine putrescine (Main Text 283 Figure 4). This contradicts a previous, enzyme activity-based study, in which arginine decarboxylase 284 and ornithine decarboxylase activity were presumed to be exclusively due to symbiont enzymes (58).

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Given the ubiquitous importance of polyamines (see below), it is probably advantageous for the host 286 to be less dependent on the symbiont for polyamine synthesis, even more so during times of reduced 287 symbiont digestion (i.e. in S-rich specimens, Main Text).

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Our results suggest that Riftia degrades nitrogenous compounds via the urea cycle. We detected all 10) Host polyamines may be involved in host-symbiont interactions 299 We detected host spermine synthase and spermidine synthase, which synthesize the polyamines 300 spermine and spermidine, only in the trophosome metaproteomes, but not in other tissues.   12) The Riftia immune system might protect the symbiosis against phages 330 The Riftia symbionts appear not to elicit a general host immune response, as a number of host 331 immune proteins such as lysozyme, tyrosine-protein kinases and peptidoglycan recognition proteins 332 were detected, but overall, their abundance in the trophosome was not higher than in other tissues 333 (Main Text Figure 2). Galaxins, proteins associated with coral exoskeletons (68)  Tevnia symbiont (73). The similarity of ELP patterns between the tubeworm symbionts and 367 symbionts of shallow-water bivalves (Supp. Figure S5) suggests parallels in interaction strategies 368 with their respective hosts. 369 We also observed differences in ELP distribution patterns between distinct organism groups (Supp.  Table S1d. 404 For further information about the selected protein groups, see Supp.   (40)).

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The symbiont metaproteome profile also hints at more oxic conditions in S-depleted than S-rich