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Resolving the molecular mechanisms of intracellular coral-algal symbiosis

Periodic Reporting for period 2 - SYMCELLS (Resolving the molecular mechanisms of intracellular coral-algal symbiosis)

Reporting period: 2018-12-01 to 2020-05-31

Symbiotic associations occur in all domains of life and are key drivers of adaption and evolutionary diversification. A prime example is the endosymbiosis between corals and eukaryotic, photosynthetic dinoflagellates which transfer critical nutrients to their coral host. Here, two very distinct cells, an animal host and a dinoflagellate symbiont cell, coordinate their functions to drive the productivity and biodiversity of a whole ecosystem. This highlights the need for understanding the cell biology of endosymbiosis. This is challenging because corals are difficult experimental subjects. Therefore, we apply a unique model systems’ approach using the symbiosis anemone model Aiptasia, modern organismal biology, cell biology, biochemistry, and comparative work with phylogenetically relevant organisms at the bench and corals in the field. Dissecting the cell biology of this endosymbiotic life-style at the mechanistic level is critical to understand its evolution and response to the environment.

Research on coral symbiosis is also timely, since coral reefs are home to >25% of all marine species and provide food and income to millions of people. Thus coral reef ecosystems have major ecological and economic impacts for society. Yet, coral reefs are currently threatened by ‘coral bleaching’ due to climate change. Environmental stress such as the increase in sea water temperature leads to the breakdown of coral-algal symbiosis, which - if not reversed in a timely manner - leads to coral death. Understanding the molecular basis of the symbiotic interaction of corals and their symbionts provides the basis to understand the mechanisms of bleaching and thus the basis to develop effective means to mitigate coral reef loss.

Most corals acquire symbionts anew each generation during larval stages and the first objective of the project aims to uncover the fundamental mechanisms involved in symbiont acquisition and integration into host cells. Nutrient transfer from symbiont to host is vital to the survival of corals in nutrient poor environments and the second objective is to uncover key mechanisms of the metabolic transfer between the partners, as well as exploring the mechanisms of how symbionts can persist intracellularly inside host cells.
During the first half of the project, we investigated distinct aspects of our main objectives. First we uncovered the function of a highly-conserved protein, the cholesterol transporter Niemann-Pick Type C2 (NPC2). Corals and their anemone relatives are unable to create cholesterol themselves and thus need cholesterol and other sterols from their symbionts. In mammals, the NPC2 protein binds sterols within specific cellular organelles (lysosomes) and participates in the recycling cycle of sterols for reuse of this essential cellular building blocks. Taking advantage of our Aiptasia anemone model system and comparative work with field-collected corals, we find that the sterol composition of the host is dictated by the strain of symbiont housed. Depending on symbiont type, the host can use different sterol mixtures flexibly and even replace cholesterol, a vital component of all animal cells, with other types of steroles derived from the symbionts. To do so, anemones and corals have evolved atypical NPC2 proteins which accumulate within the 'symbiosome', the organelles in which the symbiont reside intracellularly and directly bind to cholesterol and various sterols. Finally, we provide evidence that the atypical NPC2 proteins may be specifically adapted to the acidic environment inside the 'symbiosome'. In summary, we propose that the atypical NPC2s are involved in allowing corals to survive in nutrient poor environments.

In addition, we investigated the mechanisms involved in symbiont persistence. The innate immune system of animals allows the detection and clearance of invading microorganisms to prevent infection. However, many intracellular parasites and symbionts have evolved mechanisms to escape immune detection and to establish an intracellular niche. To uncover the mechanisms used by the symbiont to avoid detection, we established a comparative approach with true symbionts and non-symbiotic algae. Using a combination of microscopy, life imaging, gene expression analysis and chemical perturbation experiments we find that uptake of microalgae is indiscriminate, but only true symbionts persist inside host cells. Symbiont uptake broadly suppresses host innate immunity, including the conserved TLR signalling pathway. Immune suppression allows symbionts to escape expulsion ('vomocytosis'), the canonical fate of non-symbiotic algae, and promote niche formation. This finding was unexpected because pathogens invading professional immune cells in mammals are mostly cleared by intracellular digestions and has implications for our understanding of the evolution of innate immunity and of an intracellular lifestyle.

Along the way we also developed a protocol and molecular tools for microinjection of Aiptasia eggs as a prerequisite to make the model system amenable for genetic manipulations.
To date we made significant progress beyond the state-of-the art. As planned. we concluded a first set of newly developed tools and resources for Aiptasia larvae, a major milestone in establishing a novel model system to dissect the mechanisms of coral-algal endosymbiosis. Taking advantages of our toolbox including molecular techniques, high-resolution microscopy, biochemistry and comparative analyses in field-collected corals, we directly tested a long-standing hypothesis about the role of NPC2 proteins in endosymbiosis and significantly extended our previous understanding of the mechanism underlying nutrient transfer from symbiont to host. This part of the project represents an important example of the power of our model systems’ approach. Moreover, combining RNA-Seq with live imaging and cell biology allowed us to address a key question in the field: how doe symbionts persist inside animal host cells? Our finding that immune suppression during symbiont uptake plays a role to avoid expulsion changes our view on the ancestral immunity function, and provides a stepping stone towards a better understanding of the prerequisites for symbiotic associations to become an intimate partnership.

Towards the end of the project, I expect to provide a first molecular framework of the individual steps involved in symbiosis establishment. The steps include symbiont recognition, uptake via phagocytosis, integration into host cell function, interaction with host defence mechanisms, metabolic transfer between the partners and spreading throughout the host organism. The mechanistic analysis of these steps will have broader implications within the fields of ecology and evolution, and specifically important ramifications on coral reef ecosystem health.
Aiptasia anemone
symbiotic Aiptasia larva