The Influence of Symbiosis on the Proteome of the Exaiptasia Endosymbiont Breviolum minutum

Abstract

The cellular mechanisms responsible for the regulation of nutrient exchange, immune response, and symbiont population growth in the cnidarian-dinoflagellate symbiosis are poorly resolved. Here, we employed liquid chromatography-mass spectrometry to elucidate proteomic changes associated with symbiosis in Breviolum minutum, a native symbiont of the sea anemone Exaiptasia diaphana ('Aiptasia'). We manipulated nutrients available to the algae in culture and to the holobiont in hospite (i.e., in symbiosis) and then monitored the impacts of our treatments on host-endosymbiont interactions. Both the symbiotic and nutritional states had significant impacts on the B. minutum proteome. B. minutum in hospite showed an increased abundance of proteins involved in phosphoinositol metabolism (e.g., glycerophosphoinositol permease 1 and phosphatidylinositol phosphatase) relative to the free-living alga, potentially reflecting inter-partner signalling that promotes the stability of the symbiosis. Proteins potentially involved in concentrating and fixing inorganic carbon (e.g., carbonic anhydrase, V-type ATPase) and in the assimilation of nitrogen (e.g., glutamine synthase) were more abundant in free-living B. minutum than in hospite, possibly due to host-facilitated access to inorganic carbon and nitrogen limitation by the host when in hospite. Photosystem proteins increased in abundance at high nutrient levels irrespective of the symbiotic state, as did proteins involved in antioxidant defences (e.g., superoxide dismutase, glutathione s-transferase). Proteins involved in iron metabolism were also affected by the nutritional state, with an increased iron demand and uptake under low nutrient treatments. These results detail the changes in symbiont physiology in response to the host microenvironment and nutrient availability and indicate potential symbiont-driven mechanisms that regulate the cnidarian-dinoflagellate symbiosis.

Keywords: Aiptasia; Breviolum minutum; free-living; proteomics; symbiosis.

Figure 1

Physiological effects of different nutritional regimes in Breviolum minutum and Aiptasia. (A) Comparison of in hospite symbiont cell densities between well-fed and starved Aiptasia. n = 13 per treatment. (B) Maximum quantum yield of photosystem II (F_v/F_m) of cultured algae (ASW vs. f/2 medium) and in hospite (well-fed vs. starved). n = 10 per treatment at each time-point. Asterisks indicate significant differences: * p < 0.05. *** p < 0.0001. Different letters next to asterisks indicate significant differences between treatments, a (ASW and f/2), b (well-fed and starved). Values are mean ± SE.

Figure 2

Principal component analysis of all detected Breviolum minutum proteins. (A) Contrasting algae in hospite with the sea anemone Aiptasia versus free-living cells; (B) high (i.e., well-fed in hospite or cultured in replete f/2 medium) versus low nutritional regimes, meaning starved in hospite or cultured in artificial seawater (ASW). Ellipses represent 99% confidence intervals.

Figure 3

Conceptual diagram of the cellular processes under varying degrees of B. minutum compatibility. Coloured proteins were significantly more abundant in the colour-coded treatments. Grey = metabolites/solutes; Purple = Starved; Red = Well-fed; Orange = ASW; Turquoise = f/2. (1) Proteins involved in phosphoinositol (PI) manipulation, including GIT1 and INP53 were more abundant only in hospite, potentially for preventing host autophagy, and aiding communication and cell recognition. (2) Cell wall modification proteins (e.g., GAS2) were increased in symbionts regardless of nutritional state. (3) B. minutum carbonic anhydrase (CA) and vacuolar proton pumps (V), which work in harmony to convert bicarbonate ions (HCO₃^-) to carbon dioxide (CO₂), were abundant in both free-living treatments. CO₂ can be further used for photosynthesis. Upregulation of algal CAs and V-Type ATPase in the free-living state might be a response to a lack of readily available CO₂ provided by the host CAs and proton pumps when in hospite. (4) Ammonium is assimilated by B. minutum via the GS/GOGAT pathway; however, proteins associated with this pathway increased only in the free-living state, likely because the host controls the flow of nitrogen to its symbionts when in hospite. Moreover, nitrilase (NIT), which is involved in hydrolysing nitrile compounds for ammonia, only increased in abundance when in hospite, possibly as a response to nitrogen limitation inside the host. (5) Photosystem (PS) I and II proteins involved in light harvesting, as well as chloroplastic electron transport proteins, were highly increased in abundance in the nutrient-enriched treatments (i.e., Well-fed and f/2). (6) Proteins involved in B. minutum antioxidant mechanisms such as ascorbate peroxidase (A), superoxide dismutase (S), and glutathione s-transferase (G) were more abundant in high nutrient treatments (i.e., Well-fed and f/2), potentially as a cellular response for maintaining high rates of photosynthesis/respiration. Moreover, metacaspase-1B (M), which is involved in oxidative stress and apoptosis, was only abundant in the well-fed treatment in hospite. (7) Nutrient usage, such as for iron, was affected by nutrient regime. Multicopper oxidase (MCO), which is involved in the iron uptake pathway, increased in abundance in the high nutrient regimes. Moreover, soma ferritin (SF), an important iron storage protein, was more abundant in the ASW treatment.