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Dinoflagellate symbionts escape vomocytosis by host cell immune suppression

Nature Microbiology volume 6, pages 769–782 (2021)Cite this article

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Abstract

Alveolata comprises diverse taxa of single-celled eukaryotes, many of which are renowned for their ability to live inside animal cells. Notable examples are apicomplexan parasites and dinoflagellate symbionts, the latter of which power coral reef ecosystems. Although functionally distinct, they evolved from a common, free-living ancestor and must evade their host’s immune response for persistence. Both the initial cellular events that gave rise to this intracellular lifestyle and the role of host immune modulation in coral–dinoflagellate endosymbiosis are poorly understood. Here, we use a comparative approach in the cnidarian endosymbiosis model Aiptasia, which re-establishes endosymbiosis with free-living dinoflagellates every generation. We find that uptake of microalgae is largely indiscriminate, but non-symbiotic microalgae are expelled by vomocytosis, while symbionts induce host cell innate immune suppression and form a lysosomal-associated membrane protein 1-positive niche. We demonstrate that exogenous immune stimulation results in symbiont expulsion and, conversely, inhibition of canonical Toll-like receptor signalling enhances infection of host animals. Our findings indicate that symbiosis establishment is dictated by local innate immune suppression, to circumvent expulsion and promote niche formation. This work provides insight into the evolution of the cellular immune response and key steps involved in mediating endosymbiotic interactions.

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Fig. 1: Aiptasia larvae as a comparative system to dissect symbiont maintenance.
Fig. 2: Non-symbiotic microalgae are removed by expulsion.
Fig. 3: Symbiosis establishment relies on vomocytosis inhibition.
Fig. 4: Local suppression of host innate immunity is a prerequisite for symbiosis establishment.
Fig. 5: Immune stimulation enhances the expulsion of symbionts during initial interaction.

Data availability

Raw reads of the RNA sequencing data can be accessed at the National Center for Biotechnology Information SRA with the following accession numbers: SRX7119772–7119776 (cells from aposymbiotic larvae), SRX7119782–7119787 (symbiotic cells) and SRX7119777–7119781 (aposymbiotic cells from symbiotic larvae) (combined in the SRA project SRP229372); and SRX7229078–7229080 (M. gaditana-containing cells) and SRX7229075–7229077 (microalgae-free cells from M. gaditana-containing larvae) (combined in the SRA project SRP233508). Source data are provided with this paper.

Code availability

The Knime92 workflow used for analysis, as well as other raw data and R scripts for analysis, can be found at https://doi.org/10.24433/CO.0872345.v1.

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Acknowledgements

We thank D. Pavlinic and V. Benes (GeneCore Facility, EMBL Heidelberg) for assistance with the Smart-Seq2 protocol and sequencing library preparation; D. Ibberson (Deepseqlab, Heidelberg University) for assistance with the Smart-Seq2 protocol; C. Rippe for access to the bioanalyzer; L. Hambleton for help with antibody purification; B. G. Bergheim for initiating live imaging of Aiptasia larvae; M. Mercker (Bionum) for advice on statistical analysis; F. Frischknecht, T. Gilmore, T. Holstein and S. Lemke for advice; and R. May for advice and comments on the manuscript. Funding was provided by the Deutsche Forschungsgemeinschaft (DFG) (Emmy Noether Program Grant GU 1128/3-1) and H2020 European Research Council (ERC Consolidator Grant 724715) to A.G., a scholarship from the CellNetworks Excellence Cluster (Heidelberg University) Postdoctoral Program to S.R. and a PhD scholarship within the graduate school Evolutionary Novelty and Adaptation by the Baden-Württemberg Landesgraduiertenförderung Program to P.A.V.

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  1. These authors contributed equally: Marie R. Jacobovitz, Sebastian Rupp.

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  1. Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany

    Marie R. Jacobovitz, Sebastian Rupp, Philipp A. Voss, Ira Maegele, Sebastian G. Gornik & Annika Guse

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Contributions

M.R.J., S.R. and A.G. conceived of the study. M.R.J., S.R., P.A.V., I.M. and A.G. developed the methodology. P.A.V. and S.G.G. developed the software. M.R.J. and S.R. performed the formal analyses. M.R.J., S.R. and I.M. performed the investigation. M.R.J., S.R., P.A.V., I.M. and A.G. interpreted the data. A.G. provided the resources. P.A.V. and S.G.G. curated the data. M.R.J., S.R. and A.G. wrote the original draft of the manuscript. M.R.J., S.R., S.G.G., P.A.V. and A.G. reviewed and edited the manuscript. M.R.J. and S.R. visualized the data. A.G. supervised the project. M.R.J., S.R. and A.G. administered the project. A.G. acquired the funding.

Corresponding author

Correspondence to Annika Guse.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Alejandro Sánchez Alvarado, Simon Davy and Christian Voolstra for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Uptake of microalgae is indiscriminate.

a, Additional microalgae screened: Isochrysis sp., Chlorella sp., D. salina, and C. parkeae. Images are DIC and red autofluorescence of microalgae photosynthetic pigments. Scale bar indicates 25 µm. b, Aiptasia larvae were infected at 4-6 days post fertilization (dpf) for 24 hours and were washed into fresh FASW. Error bars represent SEM. n=3 for all except C. parkeae with n= 1.

Source data

Extended Data Fig. 2 N-deglycosylation of LAMP1 in Aiptasia homogenate.

Verification of α-LAMP1 antibody used in Figs. 2a, 3d,e by Western blot. LAMP1 has been observed to run at a higher than predicted weight (38 kDa) because it is heavily glycosylated101. Deglycosylation of homogenates of symbiotic and aposymbiotic adult Aiptasia CC7 using PNGase F resulted in a shift to a lower molecular weight. As control, extracts were detected with LAMP1 antibody pre-adsorbed with the peptide used for raising the antibody.

Source data

Extended Data Fig. 3 Inhibition of actin polymerization does not affect expulsion of non-symbiotic microalgae.

a, Analysis of the effects of different concentrations of LatrunculinB (LatB) on Aiptasia larvae to determine a suitable concentration for live imaging analysis. Larvae were incubated for 6 hours in LatB, washed, fixed and the f-actin levels were assessed by fluorescence microscopy. 0.01 µM does not affect the overall levels or distribution of actin. In contrast, LatB concentrations >0.1 µM substantially decreased actin levels and impacted the integrity of Aiptasia larvae (see arrowheads pointing to ‘holes’ within the organisms). Accordingly, an intermediate concentration of 0.05 µM LatB which substantially reduced f-actin levels without compromising larval integrity was used for live imaging in Fig. 3a). b, Inhibition of actin polymerization with Latrunculin B did not affect the time to expulsion of M. gaditana from infected Aiptasia larvae.

Source data

Extended Data Fig. 4 ERK5 and MAP2K5 homologues in Aiptasia.

Phylogenetic analysis of ERK5 and MAP2K5 from Aiptasia. a + b are collapsed trees of Aiptasia MAPK (A) or MAP2K (B) in comparison to several other cnidarian and vertebrate species. Red arrowheads or writing indicate presence of an Aiptasia homolog. Both Aiptasia ERK5 and MEK5 cluster within ERK5 (MAPK7) or MAP2K5, respectively. Full tree can be accessed through Supplementary Files 1 and 2.

Extended Data Fig. 5 Cell-specific characterization for transcriptomic analysis.

a, Schematic of Aiptasia larvae used for cell-specific sequencing. Ectodermal cells were removed resulting in only endodermal cells that were dissociated and selected for based on contents: aposymbiotic cells from symbiotic larvae (Symbiont-Apo), symbiotic cells from symbiotic larvae (Symbiont (red)), aposymbiotic cells from aposymbiotic larvae (Apo), cells containing M. gaditana from larvae infected with M. gaditana (M. gaditana (yellow)), and aposymbiotic cells from larvae infected with M. gaditana (M. gaditana-Apo). b, Principal Component Analysis (PCA) plot of host gene expression in different conditions.

Extended Data Fig. 6 Amino acid sequence similarity between human and Aiptasia MyD88.

Human MyD88 homo-dimerizes to trigger a downstream signaling cascade leading to immune activation. It consists of three domains, the death domain (DD), the interdomain (ID) and the C-terminal TIR domain97. The human TIR domain is key for homo-dimerization with other TIR domains from MyD88 or other TIR domain containing proteins. Three distinct regions contributing to homo-dimerization have been identified by crystallography, NMR and mammalian two-hybrid analysis98. However, the so-called BB-loop within the TIR domain, a solvent-exposed stretch of 7 residues (RDLVPGT) is particularly critical for homodimerization in human MyD88. Accordingly, cell-permeable peptides mimicking the 7 residues of the BB-loop of human MyD88 interfere with homo-dimerization55,99,100. The TIR domains (black box/upper alignment) of mammals and Aiptasia are well conserved (∼50% sequence identity). Moreover, the BB-loop (red box) is almost identical and key residues (*) are conserved. Identical amino acids have black background, similar aa have gray background and aa with white background are not similar according to blosume62 scoring.

Supplementary information

Reporting Summary

Supplementary Data 1

Phylogenetic tree of MAPK with a focus on ERK5 (MAPK7).

Supplementary Data 2

Phylogenetic tree of MAP2K with a focus on MAP2K5.

Supplementary Video 1

Three-dimensional reconstruction of LAMP1 staining in Aiptasia larvae infected with a symbiont. LAMP1 is stained magenta, DNA is stained with Hoechst (cyan) and autofluorescence of the symbiont is shown in white.

Supplementary Video 2

Three-dimensional reconstruction of LAMP1 staining in Aiptasia larvae infected with N. oculata. LAMP1 is stained magenta, DNA is stained with Hoechst (cyan) and autofluorescence of the symbiont is shown in white.

Supplementary Video 3

Three-dimensional reconstruction of LAMP1 staining in Aiptasia larvae infected with C. velia. LAMP1 is stained magenta, DNA is stained with Hoechst (cyan) and autofluorescence of the symbiont is shown in white.

Supplementary Video 4

Intracellular/attached microalgae (symbionts) move in cohesion with the larva, whereas non-intracellular microalgae (asterisk) clearly move independently within the gastric cavity. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 5

Long-term imaging of larva infected with a symbiont. The symbiont can be seen dividing at ~8 h after the start of imaging. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 6

Z-stack of a symbiont within the gastric cavity for one time point during acquisition. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 7

Long-term imaging of larva infected with M. gaditana. M. gaditana can be seen being expelled and taken up again. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 8

Long-term imaging of larva infected with N. oculata. N. oculata can be seen being expelled and taken up again. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 9

Long-term imaging of larva infected with C. velia. C. velia can be seen being expelled and taken up again. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Video 10

Long-term imaging of larva infected with beads. Beads can be seen being expelled and taken up again. Autofluorescence of microalgae is shown in red. The timestamp is given in hours, minutes and seconds.

Supplementary Table 1

Live imaging statistics.

Supplementary Table 2

Statistics of transcriptional suppression of host cell immunity. Enumeration of the modulation of innate immunity genes over ten immune pathways, with some genes present in multiple pathways and multiple transcripts annotated as the same gene (for example, TRAF3).

Source data

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 2

Unprocessed western blot.

Source Data Extended Data Fig. 3

Statistical source data.

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Jacobovitz, M.R., Rupp, S., Voss, P.A. et al. Dinoflagellate symbionts escape vomocytosis by host cell immune suppression. Nat Microbiol 6, 769–782 (2021). https://doi.org/10.1038/s41564-021-00897-w

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