Analysis of the immune-related transcriptome of a lophotrochozoan model, the marine annelid Platynereis dumerilii

Abstract

Background: The marine annelid Platynereis dumerilii (Polychaeta, Nereididae) has been recognized as a slow-evolving lophotrochozoan that attracts increasing attention as a valuable model for evolutionary and developmental research. Here, we analyzed its immune-related transcriptome. For targeted identification of immune-induced genes we injected bacterial lipopolysaccharide, a commonly used elicitor of innate immune responses, and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes.

Results: Sequence analysis of 288 cDNAs revealed induced expression of numerous genes whose potential homologues from other animals mediate recognition of infection (e.g. complement receptor CD35), signaling (e.g. myc and SOCS), or act as effector molecules like ferritins and the bactericidal permeability-increasing protein. Interestingly, phylogenetic analyses implicate that immune-related genes identified in P. dumerilii are more related to counterparts from Deuterostomia than are those from Ecdysozoa, similarly as recently described for opsin and intron-rich genes.

Conclusion: Obtained results may allow for a better understanding of Platynereis immunity and support the view that P. dumerilii represents a suitable model for analyzing immune responses of Lophotrochozoa.

Figure 1

Confirmation of the enrichment of immune-related transcripts in the subtracted cDNA library by quantitative real-time PCR analysis. The relative amount of cDNAs of 18 S rRNA, α-tubulin, BPI, and SOCS in the subtracted cDNA library (white bars) is shown relative to their amount in unsubtracted cDNA library (black bars). The cDNA amount of the house-keeping genes 18 S rRNA and α-tubulin were reduced about 2.5 fold by the subtraction procedure. In contrast, potentially immunity-related genes BPI and SOCS were found to be 1.3 and 5.1 fold enriched in the subtracted cDNA library, respectively. Results represent mean values of three independent determinations ± S.D.

Figure 2

Sequence alignment of potential Platynereis CD35 with human CD35. The partial sequence of the putative CD35 from Platynereis (AM697681), was aligned with the amino-terminal sequence from human CD35 (Hsap-CD35, NP_000642). Red color indicates 80% consensus and blue color 50% consensus.

Figure 3

Alignment and phylogenetic analysis of the myc oncogene. (A) The partial protein sequence of myc from P. dumerilii (AM697687) was aligned with corresponding sequences from S. purpuratus (NP_999744), D. melanogaster (Q9W4S7), C. elegans (NP_001022773), human c-myc (P01106), and human n-myc (1202343A). The human myc-like protein (NP_001028253) was used as out-group. Red color indicates 70% consensus and blue color 40% consensus. (B) A Bayesian protein tree generated using the aligned protein sequences revealed that Platynereis myc shows highest relation to human c-myc. Posterior probabilities are plotted at the nodes. The scale bar represents the substitutions per site according to the model of amino acid evolution applied.

Figure 4

Alignment and phylogenetic analysis of the ferritin isoforms. (A) The protein sequences of the identified ferritin subunits from P. dumerilii (Pdum-1, AM697674; Pdum-2, AM697675) were aligned with corresponding sequences from H. sapiens (Ferritin heavy chain, P02794; light chain, P02792; mitochondrial ferritin, Q8N4E7), S. purpuratus (Spur1, XP_001184706; Spur2, XP_796152), D. melanogaster (Dmel1, NP_524873; Dmel2, AAF07879; Dmel3, NP_572854), C. elegans (Cele1, NP_504944; Cele2, NP_491198), Suberites domuncula (Sdom1, CAC84556; Sdom2, CAC84555), Hydra vulgaris (Hydra, ABC25029), Arabidopsis thaliana (Athal, NP_195780), and Escherichia coli (Ecoli, NP_416418). Red color indicates 90% consensus and blue color 40% consensus. (B) A Bayesian protein was generated using aligned ferritin sequences including bacterial ferritin from E. coli as out-group. This analysis revealed that Platynereis ferritin 1 group near to ferritins from the sponge Suberites domuncula and the sea urchin S. purpuratus whereas the Platynereis ferritin 2 isoform groups near to the ferritins from Hydra and Drosophila. Posterior probabilities are plotted at the nodes. The scale bar represents the substitutions per site according to the model of amino acid evolution applied.

Figure 5

Sequence alignment of the potential Platynereis BPI with homologues from other organisms. (A) The partial sequence of the BPI from P. dumerilii (Platynereis, AM697673), was aligned with sequences from C. elegans (Caenorhab., NP_510689), from S. purpuratus (Strongylo., XP_001192950), and from Homo sapiens (Human_BPI, NP_001716; Human_BPI2, NP_777592; Human_BPI3, NP_079503; Human_LBP, NP_004130) As out-group we used human cholesteryl ester transfer protein (Human_CETP, NP_000069). Red color indicates 80% consensus and blue color 40% consensus. (B) A Bayesian protein tree was generated using the aligned region of proteins and we found that Platynereis BPI exhibit highest relation to the C. elegans BPI. However, the analysis reveals that BPI from P. dumerilii is more ancestral in sequence when compared to BPI from C. elegans. Posterior probabilities are plotted at the nodes. The scale bar represents the substitutions per site according to the model of amino acid evolution applied.

Figure 6

cDNA sequence and deduced amino acid sequence of a putative antimicrobial peptide from P. dumerilii. The identified cDNA of a putative antimicrobial peptide (AM697680) is shown including the deduced protein sequence. Start and stop codons are indicated by red shading, hydrophobic amino acids by gray shading and cysteine residues potentially involved in disulfide bridging by yellow shading.

Figure 7

Quantitative real-time RT-PCR analysis of transcriptional levels of selected Platynereis genes in response to immune challenge. The mRNA levels of selected genes in immune challenged animals (black bars) were determined and are shown relative to their expression levels in untreated animals (white bars). The transcription rates of myc, SOCS, and BPI genes were found to be increased over 1.6, 8, and 16 fold, respectively, in response to LPS injection. In contrast, the amount of 18 S rRNA transcripts were not significantly influenced. Results represent mean values of three independent determinations ± S.D.