A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ

Abstract

The lifelong relationship between the Hawaiian bobtail squid Euprymna scolopes and its microbial symbiont Vibrio fischeri represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. “Biofilm-up” and “biofilm-down” mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in V. fischeri and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors. IMPORTANCE The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner

“…(Table 1); such an advance will enable researchers to interrogate the function of candidate genes that have been identified by studying natural variation in the host. In addition, many emerging tools are available for the study of the light organ symbiont, V. fischeri (see Visick , as yet uncultivated Verrucomicrobia and some culturable Flavobacteriia and Gammaproteobacteria species 114,115,126 ". Approximately 50 core operational taxonomic units (OTUs) belonging to these taxa have been described from wild-caught populations off E. scoiopes 114 ", representing approximately 80% of the ANG community.…”

“…Approximately 50 core operational taxonomic units (OTUs) belonging to these taxa have been described from wild-caught populations off E. scoiopes 114 ", representing approximately 80% of the ANG community. Each tubule of the ANG is dominated by a specific bacterial taxon 115 , but the nature of this segregation is not understood. It may suggest that specific tubule microenvironments promote the growth and selection of d ifferent types of symbiont, or there are founder effects (that is, the microorganism that colonizes the space first proliferates and remains the dominant competitive species in that space).…”

“…It may suggest that specific tubule microenvironments promote the growth and selection of d ifferent types of symbiont, or there are founder effects (that is, the microorganism that colonizes the space first proliferates and remains the dominant competitive species in that space). Haemocytes also traffic into the ANG tubules, although whether they have a similar role as in the light organ has not been determined 115 . As with the light-organ symbiosis, the ANG symbionts are environmentally transmitted at each host generation 114,127 .…”

Acquisition of bioluminescent trait by non-luminous organisms from luminous organisms through various origins

“…3a). A recent study identified the diketopiperazine molecule required for a symbiotic relationship between the microbial symbiont A. fischeri and its host Euprymna scolopes [124]. But the same bacterial species failed to colonize other potential hosts, like monocentrid fish (Fig.…”

Diketopiperazine Gels: New Horizons from the Self-Assembly of Cyclic Dipeptides

“…CDPs are indeed attracting interest as drug carriers, thanks to their demonstrated ability to cross membranes and penetrate cells [ 29 ]. Their aggregates can display fluorescence [ 30 ], and they are widely studied for their diverse biological activities [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ], which make them promising for biomedical applications, as recently reviewed [ 39 ]. Further, they are being investigated also for optoelectronic [ 40 ] and catalytic [ 41 ] applications, as biodegradable and environmentally-friendly substitutes of more traditional options.…”