Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions

Abstract

Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host-microbe associations. However, how host-symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host-microbe interactions can be quite variable, even between closely related systems.

Fig. 1

Relative abundance of proteins in major metabolic categories in B. thermophilus (Bth) and B. azoricus (Baz). Bubble size corresponds to protein abundance in %OrgNSAF (average values, for replicate numbers see Supplementary Table S1a; see Supplementary Tables S2 and S3 for a complete list of all identified proteins). Sample types: we analyzed the soluble proteome of symbiont-containing whole gill tissue (Gill) and symbiont-free foot tissue (Foot). In addition, we selectively enriched symbiont fractions (symbiont cell pellet, Sym) and host proteins (host-enriched supernatant, Host, Baz only) from gill tissue using gradient centrifugation, and analyzed their soluble proteome. For enhanced identification of membrane-associated symbiont proteins, we additionally analyzed the membrane proteome of whole gill tissue samples (gill membrane fraction, GM) and enriched symbionts (symbiont membrane fraction, SM, Baz only). Baz Sym samples were analyzed in an LTQ-Orbitrap Velos (V) mass spectrometer and in an LTQ-Orbitrap Classic (O) mass spectrometer. The heat map in the center shows ratios of symbiont protein abundance in B. thermophilus and B. azoricus Gill and Sym samples (Velos measurements only). Ratios were calculated from CLR-transformed %OrgNSAF values (see Supplementary Methods). Negative ratios (red cells) indicate higher abundance in B. thermophilus, while positive ratios (blue cells) indicate higher abundance in B. azoricus. Gray cells (NA) indicate proteins that were either not compared, or that lacked the minimum number of valid values for reliable ratio calculations (see also Supplementary Table S4). Major metabolic categories are indicated on the right. H hydrogen oxidation, P phage defense

Fig. 2

Biomass contributions of symbionts in B. thermophilus and B. azoricus. Total symbiont biomass was substantially higher in B. thermophilus than in B. azoricus in whole gill tissue as well as in enriched symbiont fractions and in gill membrane fractions. Biomass contributions were calculated from the total number of spectra recorded for each organism during MS/MS analyses [16]. Error bars indicate standard deviations (all B. thermophilus samples: n = 3; B. azoricus enriched symbiont fraction and whole gill tissue: n = 2; B. azoricus gill membrane fraction: two biological replicates were pooled for MS analysis). SOX sulfur-oxidizing symbiont, MOX methane-oxidizing symbiont

Fig. 3

Metabolic interactions in Bathymodiolus mussels. a Thiosulfate generated by mitochondrial sulfide oxidation may accumulate in host tissues and could be used as an energy source by the thiotrophic symbiont. Purple: host mitochondrial membrane-associated enzymes. Green: host mitochondrial matrix enzymes. Gray: thiotrophic symbiont enzymes. Tst thiosulfate sulfurtransferase, Sdo sulfur dioxygenase, Sqr sulfide:quinone reductase, III coenzyme Q complex of respiratory chain, IV cytochrome c oxidase complex, Dsr dissimilatory sulfite reductase complex, Apr adenylylsulfate reductase complex, Sat ATP sulfurylase. Please note that sulfate and thiosulfate transport across host and symbiont membranes involves transporter proteins, which are not shown in this figure, because their identities and exact functions are yet unclear. b Proposed model of amino acid cycling between host and thiotrophic symbionts in Bathymodiolus. The symbiont’s general l-amino acid ABC transporter Aap imports host glutamate and exports aspartate (and presumably other amino acids) synthesized by the symbiont. Red arrows indicate amino acid biosynthetic routes that are shared between host and symbiont, whereas black indicates routes that are exclusive to the host or the symbiont. Arrows with flat ends suggest an inhibitory action. OatA: host ornithine aminotransferase, AgxT: host alanine aminotransferase, AspC: symbiont aspartate transaminase, GltBD: symbiont glutamate synthase, CitT: symbiont citrate transporter, Dct: symbiont tripartite ATP-independent periplasmic transporter. Lys, Thr, Arg, Gln, Asp: lysin, threonine, arginine, glutamine, aspartate; G5S: l-glutamate 5-semialdehyde