Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

Abstract

Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

Transport of symbiont-encoded cellulases from the gill to the gut of shipworms via the enigmatic ducts of Deshayes: a 174-year mystery solved

“…Our results do not provide support for the recently proposed mechanism by which symbiotic bacteria are expelled from the surfaces of the gills and transported via ciliary action of the branchial grooves to the mouth [ 16 ]. Indeed, we saw little evidence of antibody labelling on the external surfaces of the gill or the branchial grooves, except at the most anterior end of the body where nearly all surfaces showed antibody labelling.…”

“…Nor did we see evidence of abundant bacterial cells on the external surfaces of the gills or branchial grooves using a bacteria-specific fluorescent oligonucleotide probe ( figure 3 b,d,e and electronic supplementary material, figure S9E). Additionally, in previously published proteomic [ 15 ] and transcriptomic [ 16 , 17 ] analyses, the symbiont-encoded proteins detected in the shipworm gut were primarily secreted cellulases. We would expect that other abundant bacterial proteins would be detected in the gut as well if intact bacterial cells rather than secreted cellulases were transported from gill to gut.…”

“…Shipworms differ in that they digest wood in the caecum and the diverticula of the digestive glands, gut compartments that are largely devoid of microbes [10]. Although the caecum lacks a conspicuous microbiome, the gills of shipworms harbour dense populations of endosymbiotic gammaproteobacteria capable of producing cellulolytic enzymes [11][12][13][14][15][16][17]. These symbionts are housed in specialized cells called bacteriocytes in a tissue within the gills referred to as the gland of Deshayes [18].…”

“…More recently, the existence of the ducts of Deshayes and their relevance to enzyme transport in shipworms was questioned [ 16 ]. In that study, both symbiont- and host-encoded cellulolytic enzymes were detected in the gut of a second shipworm species, Lyrodus pedicellatus [ 16 ], however no evidence for the presence of the ducts of Deshayes was found.…”

“…More recently, the existence of the ducts of Deshayes and their relevance to enzyme transport in shipworms was questioned [ 16 ]. In that study, both symbiont- and host-encoded cellulolytic enzymes were detected in the gut of a second shipworm species, Lyrodus pedicellatus [ 16 ], however no evidence for the presence of the ducts of Deshayes was found. Instead, it was proposed that symbiont-encoded enzymes are transported from gill to gut within the cells of symbiotic bacteria that are shed from the gills and transported via ciliary action of the branchial groove to the mouth and digestive system where they augment the activity of host-encoded cellulases [ 16 ].…”