A conserved chemical dialog of mutualism: lessons from squid and vibrio

Abstract

Microorganisms shape, and are shaped by, their environment. In host-microbe associations, this environment is defined by tissue chemistry, which reflects local and organism-wide physiology, as well as inflammatory status. We review how, in the squid-vibrio mutualism, both partners shape tissue chemistry, revealing common themes governing tissue homeostasis in animal-microbe associations.

Keywords: Development; Innate immunity; MAMPs; Nutrition; Stress; Symbiosis.

Figure 1. The anatomy of the juvenile squid-vibrio mutualism

A) The squid’s light organ is the anatomical structure that maintains the bioluminescent symbiont V. fischeri and modulates light output. The organ is located underneath the mantle of the squid, just atop the funnel: a structure used to move water into and out of the mantle cavity. The immature light organ (boxed in red, and enlarged at right) has bilateral ciliated fields and appendages. At the base of the appendages are pores, leading into the crypts of the light organ. B) A cross-section of the light organ, boxed in blue in (A), shows that the symbiont (teal) is maintained in extracellular crypts lined with a polarized epithelium that is photoreceptive. Structures surrounding the crypts, such as the reflector (indicated in green dashed lines), ink sac, and lens manipulate the light produced by the symbiont for host behaviors.

Figure 2. The winnowing of the symbiont

The events described in this figure take place in the external ciliated epithelium of the light organ (lower left, indicated by red box). A) The first contact of host and symbiont is accompanied by the presentation of microbial products. Along the surface of the light organ’s ciliated epithelium, peptidoglycan (PGN), shed by seawater bacteria, induces mucus production (purple). V. fischeri bind to the cilia. The binding event may also promote presentation to host tissues of lipopolysaccharide (LPS), which are released by V. fischeri during rotation of its sheathed flagella. B) The mucus is a site of chemical selection. In addition to sialylated mucins (pH 6.3), antimicrobial peptides (AMP) and nitric oxide (NO) shape the chemistry of the mucus matrix. The mucus is an environment that not only excludes Gram-positive microbes (G+), but also allows V. fischeri to out-compete any co-aggregating Gram-negative ones such as Vibrio parahaemolyticus. C) The mucus chemistry prepares V. fischeri for colonization. Contact with V. fischeri induces the expression of host enzymes that result in chitin sugars (CS) to accumulate in the mucus matrix. The exposure to CS, as well as to NO, primes V. fischeri for migration into, and survival within, the light organ crypts. Specifically, after CS priming, V. fischeri chemotaxes towards a gradient of the sugar that emanates from the pores (indicated at center of red box), which serve as the entrance to the crypts.

Figure 3. Accommodations of host and symbiont in the light-organ crypts

A) V. fischeri (Vf, green), attenuates host production of hypochlorous acid (HOCl, yellow), and nitric oxide (NO, purple), upon transit into the crypts. The process of initial colonization occurs within the first 6 h after contact between host and symbiont. Around 12 h post-colonization, the presentation of MAMPs (PGN & LPS) by the symbiont has delivered an irreversible morphogenic signal to light-organ tissues. During subsequent development of the persistent association, the ciliated fields of the light organ regress, and the crypt structure becomes more complex. By 2 weeks the ducts leading to the crypts have condensed from three to one, and continued colonization by the symbiont has induced a physiological state in host tissues that prevents secondary colonization. B) Symbiont-derived chemical cues orchestrate physiological and morphological changes in the light organ. Bioluminescence (teal) induces swelling of the crypt epithelium. Peptidoglycan (PGN) induces release of mucus, and the secretion of antimicrobial factors (AMP, red squares) into the crypts. Lipopolysaccharide (LPS), attenuates the local production of NO in the crypts, and induces the migration of hemocytes into the ciliated appendages, which is followed by apoptosis and regression of these structures. C) V. fischeri luciferase is a mixed-function oxidase that produces light upon the oxidation of FMNH₂ and an aliphatic aldehyde by oxygen. Oxygen is also a substrate of the dopaquinone-producing host enzyme phenoloxidase (PO). In the absence of sufficient aliphatic aldehyde, the oxidation of FMNH₂ and reduction of oxygen produce toxic hydrogen peroxide (H₂O₂), but no light. Hydrogen peroxide is a substrate of the toxic hypochlorous acid-producing host enzyme myeloperoxidase (MPO). Greater production of light by the symbiont may reduce the host’s production of antimicrobial compounds (red pathways), facilitating the persistence of the symbionts in the crypts.

Figure 4. Biological rhythms in the host and symbiont

The daily cycle of symbiosis is characterized by 4 stages (I – IV). The physiological and behavioral attributes of this rhythm are a combination of symbiont-independent (grey/black), and symbiont-dependent (colored) events, depicted as the temporal change in the interface between the crypt epithelium and the symbionts. (I) Just before dawn, the microvilli at the apical surfaces of the epithelial cells efface. (II) With the dawn light cue, the contents of the light-organ crypts are expelled. If an animal is colonized, this event clears the crypts of ~95% of the symbiont population. (III) The host microvilli reappear and the remaining symbionts re-populate during the day: a period of behavioral quiescence for the squid. Host-derived nutrients, including amino acids and phospholipids (P-lipids) support this bacterial proliferation. Just before dusk, several events take place: lipopolysaccharide (LPS) levels in the crypt are predicted to increase due to a decrease in LPS-degrading, host alkaline phosphatase production; in addition, chitin sugar (CS)-bearing hemocytes migrate into symbiont-colonized light organ tissues. (IV) At dusk, the symbiont population ferments CS, producing acid, and releasing oxygen from the host carrier-protein, hemocyanin. The oxygen released from hemocyanin fuels bioluminescence, while the acidification of the crypts may lead to an increase in PGN levels due to its ability to decrease peptidoglycan recognition-protein 2 activity. The host is active in the water column during this night phase, and uses symbiont bioluminescence in its behaviors.