MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers

Abstract

Toll-like receptor (TLR) signaling is one of the most important signaling cascades of the innate immune system of vertebrates. Studies in invertebrates have focused on the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, and there is little information regarding the evolutionary origin and ancestral function of TLR signaling. In Drosophila, members of the Toll-like receptor family are involved in both embryonic development and innate immunity. In C. elegans, a clear immune function of the TLR homolog TOL-1 is controversial and central components of vertebrate TLR signaling including the key adapter protein myeloid differentiation primary response gene 88 (MyD88) and the transcription factor NF-κB are not present. In basal metazoans such as the cnidarians Hydra magnipapillata and Nematostella vectensis, all components of the vertebrate TLR signaling cascade are present, but their role in immunity is unknown. Here, we use a MyD88 loss-of-function approach in Hydra to demonstrate that recognition of bacteria is an ancestral function of TLR signaling and that this process contributes to both host-mediated recolonization by commensal bacteria as well as to defense against bacterial pathogens.

Fig. 1.

Interference with the conserved Hydra TLR-signaling pathway. (A) Function of TLR signaling during metazoan evolution. Cnidaria are the sistergroup to all bilateria and they diverged from the common eumetazoan ancestor ∼600–700 million years ago (19). (B) Schematic representation of the conserved TLR-signaling pathway in Hydra. Note that the functional Hydra TLR is assembled by two proteins (HyLRR and HyTRR) (23) and the exogenous JNK inhibitor SP600125 is shown. (C) MyD88–Hairpin construct for generation of transgenic Hydra (p, promoter; T, terminator, as, antisense; s, sense). (D) Live image of a MyD88-control polyp (control). (E) Live image of a MyD88-knockdown polyp (MyD88⁻) showing EGFP expression in the endodermal and the ectodermal cell lineage. (F) RT-PCR amplifying myd88 shows down-regulation in MyD88⁻ polyps compared with control polyps and wild-type (WT) H. vulgaris (AEP). RT-PCR was normalized using the Hydra actin gene. (G) Absence of bacteria after antibiotic treatment was confirmed by PCR amplification of the bacterial 16S rRNA gene on genomic DNA normalized to Hydra actin.

Fig. 2.

Microarray analysis reveals differential gene expression due to MyD88 down-regulation and the absence of the associated microbiota. (A) Graphic representation of differentially regulated (≥1.5-fold change, P ≤ 0.05) contigs in MyD88⁻ and germ-free compared with control polyps. Note the overlap between both experiments. Down-regulated contigs are highlighted in red, up-regulated contigs in green. (B) Categorization of differential contigs. Pie charts were separated in MyD88- but not bacterial-regulated contigs (Left), MyD88- as well as bacterial-regulated contigs (Center), and MyD88-independent bacterial-regulated contigs (Right). Contigs were assigned into self-chosen categories.

Fig. 3.

JNK phosphorylation is mediating the expression of several MyD88-downstream genes. Relative expression level of the candidate genes upon administration of the JNK inhibitor SP600125 (34), determined by qRT-PCR. Note that the expression of 12837, 19777, 34924, and 7659 is influenced by SP600125 in a concentration-dependent manner. cDNA amounts were equilibrated by elongation factor 1 α. The graphic shows means + SD (n = 3).

Fig. 4.

454 sequencing of bacterial 16S rRNA reveals minor impact of MyD88 in bacterial colonization of Hydra polyps. (A) Bar charts representing bacterial communities of Hydra polyps on class level (means of five replicates). Rare bacterial taxa (<1% relative abundance) were grouped to the fraction “others.” (B) Experimental design. Germ-free MyD88⁻ and control polyps were inoculated with potential bacterial colonizers derived from pond water, Hydra-culture supernatant, and Hydra-tissue homogenates. Single polyps were removed from clonally growing cultures 2 wk and 19 wk postinoculation and subjected to 454 sequencing of the microbiota. (C and D) Bacterial communities clustered using principle coordinate analysis of the weighted UniFrac distance matrix. Percent variation explained by the principle coordinates is indicated in the axes. (E) Weighted UniFrac differences calculated by pairwise comparisons of the bacterial profile to control polyps. Statistical analysis was carried out using two-tailed t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig. 5.

MyD88⁻ and control polyps show differential susceptibility to infection by P. aeruginosa (P.a.) (A) Phenotypic scores of the Hydra infection model. Disease always starts with swelling of the tentacle tips (score 1), followed by subsequent shortening (score 2), and loss (score 3) of tentacles. Score 4 indicates the loss of body shape with maintenance of an intact epithelium. Score 5 is characterized by tissue lysis. (B) Temporal profile of P.a.14 infection in Hydra. Polyps were incubated in 1 mL Hydra medium containing 1.8 × 10⁸ cfu P.a.14. Values are plotted as mean + SEM (n = 24). (C) Detailed representation of the time point 96 h postinfection from B. Each dot represents one polyp; horizontal line shows the mean. Note that three polyps died in the MyD88-knockdown group, whereas the maximum score observed in the control group was 2 (tentacle shortening). Statistical significance was tested by two-tailed Mann–Whitney test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).