Deciphering the nature of the coral-Chromera association

Abstract

Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.

Fig. 1

a Uninfected A. digitifera larva showing the absence of red fluorescence. b Chromera-infected larva at 4 h post-infection. Chromera chloroplasts are responsible for the red fluorescence observed

Fig. 2

a Summary of the differential gene expression profile in Chromera-infected larvae compared to controls at 4, 12, and 48 h. An adjusted P ≤ 0.05 and E-value cut off ≤10⁻¹⁰ were used to filter differentially expressed genes and for BLASTX searches against the Swiss-Prot database. b, c Volcano plots showing the coral genes differentially expressed at 4 h b and 48 h c after Chromera infection compared to the uninfected control condition. The red dots represent the significantly differentially expressed transcripts at adjusted P ≤ 0.05

Fig. 3. Integrative model of the genes and pathways of larvae of Acropora digitifera involved in the interaction with Chromera.

Upregulated and downregulated genes/functions are in blue and red text, respectively. The initial contact phase (left panel) involves upregulation of ribosomal and mitochondrial functions (based on enrichment of GO terms), and suppression of host immune responses including the downregulation of the pattern recognition receptors (PRRs) that can recognize signature microbial compounds (i.e., microbe-associated molecular patterns or MAMPs). The PRRs that were detected included toll-like receptors (TLRs), a nucleotide-binding oligomerization domain (Nod) protein, scavenger receptors (SRs), lectins, and the complement protein C3. Moreover, the pancreatic secretory granule membrane major glycoprotein GP2, which serves as an uptake receptor for pathogenic bacteria in man, was strongly downregulated. Suppression of PRR–MAMP interactions leads to inactivation of nuclear factor kappa-B (NF-kappa-B), which is a master regulator of immunity. The phagosome formation phase (central panel) involves downregulation of genes involved in phagocytosis and actin remodeling as well as differential expression of genes implicated in endosomal trafficking that enhance the maturation of the phagosome and lysosome fusion (see Fig. 4 for more details about those genes). The Chromera tolerance phase (right panel) involves complex changes in the apoptotic network. During this phase, genes likely to have both anti-apoptotic and pro-apoptotic functions were differentially expressed

Fig. 4. Heat map of genes likely to be involved in immune, inflammatory, stress, and ROS responses in Chromera-infected (I1, I2, and I3) and uninfected control larvae (C1, C2, and C3) at 48 h post-infection.

The hierarchical clustering shown here was obtained by comparing the expression values (fragments per kilobase of transcript per million; FPKM) for Chromera-infected samples against the controls. Expression values were log₂-transformed and median centered by transcript. The blue-red scale represents the relative expression values

Fig. 5

Heat maps of a genes likely to be involved in phagosome maturation and lysosomal fusion, b genes likely to have pro-apoptotic functions, and c genes likely to have anti-apoptotic functions in Chromera-infected (I1, I2, I3) and uninfected control larvae (C1, C2, C3) at 48 h post-infection. The hierarchical clustering shown here was obtained by comparing the expression values (fragments per kilobase of transcript per million; FPKM) for Chromera-infected samples against the controls. Expression values were log₂-transformed and median centered by transcript. The blue-red scale represents the relative expression values

Fig. 6. The coral host modulates the endocytic pathway and enhances phagosome maturation and lysosome fusion in response to Chromera

Upon phagocytosis, the phagosome acquires the GTPase Rab5 via fusion with early endosomes. The Rab5 effector ALS2 was also upregulated. Rab5 acts to recruit phosphatidylinositol-3-kinase (PI3K), which generates phosphatidylinositol-3-phosphate (PI3P) and recruits early endosomal antigen (EEA1) from endosomes. EEA1 is a Rab5 effector protein and its upregulation triggers fusion of the phagosome with a late endosome. During the phagosome maturation process, Rab7 replaces Rab5 and the intermediate phagosome fuses with late endosomal vesicles, acquiring a suite of proteins that includes the proton-ATPase pump (V-ATPase), lysosome-associated membrane glycoprotein 1 (LAMP1), CD63, and lysosomal hydrolases. Vacuoles containing Chromera fuse with late endosomes/lysosomes as indicated by the upregulation of genes encoding Rab7, LAMP1, and CD63 (plus late endosomal/ lysosomal adapter, SNAP-associated protein and vesicle-associated membrane protein 7 (VAMP7) “not shown”). The observed upregulation of the lysosomal V-ATPase signals the formation of phagolysosomes, which are typically rich in hydrolytic enzymes and have an extremely low pH, presumably in order to eliminate Chromera. Genes in red text are downregulated, while those in blue are upregulated