Although our understanding of the importance of the partnership of humans and their microbiome in health and disease is rapidly developing, its complexity is high and support for causality is scarce. Model organisms serve to tackle these limitations. Recent updates on the squid–vibrio symbiosis show mechanistic details that underlie the establishment and maintenance of a symbiotic association along the apical surfaces of an animal epithelium.
Refers to Visick K. L., Stabb, E. V. & Ruby, E. G. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00557-0 (2021) | Nyholm, S. V. & McFall-Ngai, M. J. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00567-y (2021).
It is hard to make a lasting relationship. Great partnerships are built on three core principles: identifying and attracting the potential partners that will complement one another, ongoing communications and relationship management. The laboratories of Edward Ruby and Margaret McFall-Ngai spent decades uncovering the mechanisms underlying the highly specific lasting partnership between the Gram-negative luminous bacterium Vibrio fisheri and the bobtail squid Euprymna scolopes. In two articles in Nature Reviews Microbiology, Ruby1 and McFall-Ngai2 build on their earlier work and review the recently discovered molecular, biophysical and biochemical features that work in concert to build this partnership (Fig. 1). Each of the two articles focuses on one of the two partners. Nyholm and McFall-Ngai2 review our current understanding of how the squid recruits the symbiont partner and keeps them happy. Visick et al.1 take a closer look at the Vibrio bacterium and explain the microbial characteristics that are important for the symbiosis.
A single squid maintains about a billion cells of V. fischeri in the two light organs. Here, at the underside of the squid, the bacteria produce bioluminescence that provides a sort of ‘counter illumination’ and camouflage against predators from below. Similar to our intestinal epithelia, the surfaces of the juvenile squid are covered with a chitin-rich mucus and cilia, which beat to direct the bacteria close to the pores. There, V. fischeri signals the host to ‘unlock the doors’ by activating a chitinase and also antimicrobials. Once inside, V. fischeri first ‘closes the door’ by inducing apoptosis of the structures that have allowed entry and immediately begins to influence a number of developmental pathways in the squid, including changes in the innate immune system, haemocyte infiltration and gene expression even in remote tissues such as the eyes and gills.
Three strategies help to stabilize and maintain the partnership between the squid and V. fischeri. First, the partners are complementing each other: the squid provides a nutrient-rich environment for the heterotrophic bacterium, and the host acquires luminescence. Second, there is ongoing communication that includes interactions at the symbiotic partner cell surfaces as well as uptake of symbiont biomolecules into host cells. Third, within 12 h after colonization, the resident symbionts lead to a profound daily rhythm in the host. Every dawn, the squid pumps out up to 95% of the bacteria, which leads to a disappearance of the bioluminescence. As the day goes by, the bacteria proliferate and then use their quorum sensing system to activate bioluminescence at night. Bacterial activity over the day–night cycle affects the cellular restructuring of the crypt epithelia, an acidification of the crypts in the hours before venting, and the migration of haemocytes from the host circulatory system into the crypts at night where these cells release chitin stores that the symbionts can metabolize. Over the day–night cycle, the expression of many squid genes cycles. So, it is not surprising that the whole squid transcriptome and also hemolymph biochemistry and metabolite signature oscillates on symbiont-driven daily rhythms3. Prominent examples include antimicrobial genes and components of the molecular clock of circadian rhythms (cry genes). It is this rhythm that seems to stabilize and maintain the partnership.
In the accompanying article, Visick et al.1 focus on the symbiont V. fisheri. This bacterium made history in the 1980s because it helped to discover the bacterial communication system, quorum sensing, that controls its bioluminescence. What still makes this bacterium so attractive are the novel functional genomics and mutant approaches developed in the Ruby laboratory to obtain mechanistic insights into this partnership. Their work has established that chemotaxis, a secreted exopolysaccharide (Syp-PS), and a swimming motility driven by flagella along a gradient of squid-generated chitin oligosaccharides have an important role in colonization (Fig. 1). Within hours after colonizing, V. fischeri begin to activate the lux operon to produce a high level of bioluminescence. ‘Dark mutants’ initially colonize, but within two days disappear. Focusing on the metabolic interaction, Ruby et al. discovered that the host provides certain amino acids such as glutamate to the symbiont. Later on in the symbiosis, the important primary carbon source seems to be chitin4. Mutations that increase the effectiveness of poorly colonizing strains of V. fischeri demonstrate the importance of a histidine kinase that negatively regulates biofilm formation at the host interface5. Similar mutations have helped to understand the evolution of this symbiosis6,7 emphasizing the importance of Syp-PS synthesis. Mutants have also revealed that a small RNA, SsrA, delivered to the squid cells via outer membrane vesicles (OMVs) (Fig. 1) is essential for the symbiotic partnership8. Although technically challenging, within the V. fischeri population different strains could be detected, which varied greatly in both aggregation behaviour and their effect on host ciliated surfaces9. The importance of strain variation is increasingly being recognized in the human microbiome as well.
“Great partnerships are built on three core principles: identifying and attracting the potential partners that will complement one another, ongoing communications and relationship management”
Defining the individual microorganism–host conversations, that is ‘the cellular dialogues in the holobiont’10, is a challenging but necessary step on the path to understanding the function of the associations as a whole. So what do we owe the squid–vibrio model? The two Reviews represent a major step in defining key regulators that drive the cellular and molecular changes essential for establishing and maintaining this partnership. Obviously, information from a single Vibrio bacterium can cause many changes in squid development, physiology and behaviour. What a difference to other symbiosis systems with their thousands of different symbiont species. This makes us wonder how much are we as humans being informed by the much more diverse and complex microbiota? The work fascinates by opening up remote space and raising exiting new questions. What are the mechanisms promoting symbiosis specificity, and which membrane receptors control the entry of the symbiont? How does the squid modulate bacterial functionality? How will the symbiotic relationship change depending on environmental context? The two Reviews make it quite clear how difficult it is to establish a lasting relationship. The key to success is effective communication. Communication within the growing community of squid–vibrio researchers was certainly also crucial in why this very special field of research has become so successful and of general importance also from a biomedical point of view.
Visick, K. L., Stabb, E. V. & Ruby, E. G. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00557-0 (2021).
Nyholm, S. V. & McFall-Ngai, M. J. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00567-y (2021).
Koch, E. J., Moriano-Gutierrez, S., Ruby, E. G., McFall-Ngai, M. & Liebeke, M. The impact of persistent colonization by Vibrio fischeri on the metabolome of the host squid Euprymna scolopes. J. Exp. Biol. 223, (2020).
Schwartzman, J. A. et al. The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation. Proc. Natl Acad. Sci. 112, 566–571 (2015).
Brooks, J. F. & Mandel, M. J. The histidine kinase BinK is a negative regulator of biofilm formation and squid colonization. J. Bacteriol. 198, 2596–2607 (2016).
Bultman, K. M., Cecere, A. G., Miyashiro, T., Septer, A. N. & Mandel, M. J. Draft genome sequences of type VI secretion system-encoding Vibrio fischeri strains FQ-A001 and ES401. Microbiol. Resour. Announc. 8, e00385-19 (2019).
Guckes, K. R. et al. Incompatibility of Vibrio fischeri strains during symbiosis establishment depends on two functionally redundant hcp genes. J. Bacteriol. 201, e00221-19 (2019).
Moriano-Gutierrez, S. et al. The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses. PLoS Biol. 18, e3000934 (2020).
Koehler, S. et al. The model squid–vibrio symbiosis provides a window into the impact of strain-and species-level differences during the initial stages of symbiont engagement. Environ. Microbiol. 21, 3269–3283 (2019).
Bosch, T. C. G. & Hadfield, M. G. Cellular Dialogues in the Holobiont (CRC Press, 2020).
The authors declare no competing interests.
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Giez, C., Bosch, T.C.G. Beating in on a stable partnership. Nat Rev Microbiol (2021). https://doi.org/10.1038/s41579-021-00575-y