Expression and putative function of innate immunity genes under in situ conditions in the symbiotic hydrothermal vent tubeworm Ridgeia piscesae

Abstract

The relationships between hydrothermal vent tubeworms and sulfide-oxidizing bacteria have served as model associations for understanding chemoautotrophy and endosymbiosis. Numerous studies have focused on the physiological and biochemical adaptations that enable these symbioses to sustain some of the highest recorded carbon fixation rates ever measured. However, far fewer studies have explored the molecular mechanisms underlying the regulation of host and symbiont interactions, specifically those mediated by the innate immune system of the host. To that end, we conducted a series of studies where we maintained the tubeworm, Ridgeia piscesae, in high-pressure aquaria and examined global and quantitative changes in gene expression via high-throughput transcriptomics and quantitative real-time PCR (qPCR). We analyzed over 32,000 full-length expressed sequence tags as well as 26 Mb of transcript sequences from the trophosome (the organ that houses the endosymbiotic bacteria) and the plume (the gas exchange organ in contact with the free-living microbial community). R. piscesae maintained under conditions that promote chemoautotrophy expressed a number of putative cell signaling and innate immunity genes, including pattern recognition receptors (PRRs), often associated with recognizing microbe-associated molecular patterns (MAMPs). Eighteen genes involved with innate immunity, cell signaling, cell stress and metabolite exchange were further analyzed using qPCR. PRRs, including five peptidoglycan recognition proteins and a Toll-like receptor, were expressed significantly higher in the trophosome compared to the plume. Although PRRs are often associated with mediating host responses to infection by pathogens, the differences in expression between the plume and trophosome also implicate similar mechanisms of microbial recognition in interactions between the host and symbiont. We posit that regulation of this association involves a molecular "dialogue" between the partners that includes interactions between the host's innate immune system and the symbiont.

Figure 1. Box plot showing expression of target genes in the trophosome compared to the plume as determined by qPCR.

The fold expression level differences, of 18 target genes, in the trophosome were compared to those of the plume. The upper and lower ends of the boxes indicate the 25th and 75th percentiles, respectively. The length of the box depicts the interquartile range within which 50% of the values are located. The solid black lines denote the median. Capped error bars represent the minimum and maximum values, excluding outliers (Table 4). The dotted black line represents expression levels in the plume (y = 1), thus genes with medians >1 exhibit higher expression levels in the trophosome compared to the plume, and inversely genes with medians <1 show higher expression levels in the plume. All expression levels are normalized to the expression of actin. The gene abbreviations are as follows: hbB1, hemoglobin B1; HSP70, heat shock protein 70; LITAF, lipopolysaccharide-induced tumor necrosis factor-alpha; PGRPrpi1–5, peptidoglycan recognition protein 1–5; TLR2p, toll-like receptor 2 precursor; A2MRAP, alpha-2 macroglobulin receptor associated protein; CAbr, carbonic anhydrase (branchial plume); CAtr, carbonic anhydrase (trophosome); EF1α, elongation factor 1-alpha; LBPIP, LPS induced bactericidal permeability increasing protein; MMIF, macrophage migration inhibitory factor; MR, macrophage mannose receptor 1-like protein; NF-κBic, NF-κB inhibitor (cactus); ROSm, reactive oxygen species modulator.

Figure 2. Model of host-symbiont interactions between pattern recognition receptors (PRRs) and microbe-associated molecular patterns (MAMPs) in R. piscesae.

The branchial plume (p) of Ridgeia exchanges metabolites with the environment. The plume lacks symbionts but may still use PRRs to interact with microorganisms found in the surrounding vent fluid and seawater. The center of the worm is comprised mainly of one organ, the trophosome (t) made up of lobules that contain bacteriocytes housing the intracellular bacterial symbionts (endosymbionts) as well as blood vessels (bv) that transfer metabolites. Central bacteriocytes (c) harbor healthy and actively dividing endosymbionts. Towards the periphery of the lobules, bacteriocytes and symbionts appear to undergo terminal differentiation and apoptosis with many of the degenerative symbionts undergoing autophagy. We suggest a significantly greater response of the trophosome to MAMPs via PRRs that may trigger signal transduction cascades, ultimately helping to regulate symbiostasis. PRR expression in the trophosome may occur in the bacteriocytes and/or in the surrounding vasculature. Given the densities of endosymbionts, bacteriocytes encounter high concentrations of MAMPs. A constant turnover of bacteriocytes may also release extracellular MAMPs into the trophosome periphery.