A Single Host-Derived Glycan Impacts Key Regulatory Nodes of Symbiont Metabolism in a Coevolved Mutualism

Abstract

Most animal-microbe mutualistic associations are characterized by nutrient exchange between the partners. When the host provides the nutrients, it can gain the capacity to shape its microbial community, control the stability of the interaction, and promote its health and fitness. Using the bioluminescent squid-vibrio model, we demonstrate how a single host-derived glycan, chitin, regulates the metabolism of Vibrio fischeri at key points in the development and maintenance of the symbiosis. We first characterized the pathways for catabolism of chitin sugars by V. fischeri, demonstrating that the Ccr-dependent phosphoenolpyruvate-pyruvate phosphotransferase system (PTS) prioritizes transport of these sugars in V. fischeri by blocking the uptake of non-PTS carbohydrates, such as glycerol. Next, we found that PTS transport of chitin sugars into the bacterium shifted acetate homeostasis toward a net excretion of acetate and was sufficient to override an activation of the acetate switch by AinS-dependent quorum sensing. Finally, we showed that catabolism of chitin sugars decreases the rate of cell-specific oxygen consumption. Collectively, these three metabolic functions define a physiological shift that favors fermentative growth on chitin sugars and may support optimal symbiont luminescence, the functional basis of the squid-vibrio mutualism.

Importance: Host-derived glycans have recently emerged as a link between symbiont nutrition and innate immune function. Unfortunately, the locations at which microbes typically access host-derived glycans are inaccessible to experimentation and imaging, and they take place in the context of diverse microbe-microbe interactions, creating a complex symbiotic ecology. Here we describe the metabolic state of a single microbial symbiont in a natural association with its coevolved host and, by doing so, infer key points at which a host-controlled tissue environment might regulate the physiological state of its symbionts. We show that the presence of a regulatory glycan is sufficient to shift symbiont carbohydrate catabolism, acetate homeostasis, and oxygen consumption.

FIG 1

Prioritization of carbohydrate catabolism in V. fischeri. (A) The effects of deletions of genes encoding PTS proteins (NagE1, NagE2, and crr) on growth yield with chitin sugars, aminosugars, or non-PTS carbohydrates as substrates. The percent cell yield (based on the culture OD₆₀₀), relative to that for the wild type, was calculated following 24 h of growth in a minimal medium containing 30 mM of one of seven sole carbon sources (n = 2): (GlcNAc)₂, chitobiose; GlcNAc, N-acetylglucosamine; GlcN, glucosamine; NANA, N-acetylneuraminic acid. Sugars shown in the gray box are PTS-transported sugars that require EIIA^crr (Ccr). (B and C) Relative promoter activity for the glpFK locus during growth in a tryptone-based medium in the absence or presence of glycerol (B) or a glycerol- and tryptone-containing medium in the absence or presence of GlcNAc (C) (n = 2; results shown are representative of two independent experiments). (D) Effect of GlcNAc addition on the rate of glycerol consumption by V. fischeri wild-type and mutant strains (n = 3). a, b, and c indicate statistically different mean values, determined by a two-way analysis of variance with post hoc Bonferroni t tests. Error bars indicate standard errors.

FIG 2

Repression of the acs-dependent acetate switch by chitin sugars. (A) Detection of acetate in the culture supernatant after growth in LBS under anoxic and oxic growth conditions, either with or without 20 mM GlcNAc (n = 2; results shown are representative of two independent experiments). Activity of the acs promoter region during growth in a tryptone-based medium, either with or without 20 mM GlcNAc added, of either PTS mutants (n = 2; results are representative of two independent experiments) (B) or in the presence or absence of 20 mM GlcNAc and/or 100 nM C8-HSL (n = 3). a and b indicate groups of statistically similar means, assessed as described for Fig. 1.

FIG 3

Repression of aerobic respiration by chitin sugars. (A) Growth of wild-type V. fischeri (solid lines) and a cytochrome oxidase mutant strain (Δcco; dashed lines) under moderate aeration in LBS medium (left panel), LBS containing 20 mM GlcNAc (center panel), or LBS with 20 mM GlcNAc added at the arrow (right panel) (n = 3). (B) Average cell length of wild-type (open) and Δcco strain (gray) cells during growth in LBS medium at two stages of growth: early phase (OD₆₀₀, 0.1) and mid-log phase (OD₆₀₀, 0.8). (C) Oxygen consumption of wild-type (open), Δcco mutant (gray), and ΔnagE1 mutant (hatched) cells, expressed as the rate per OD₆₀₀ unit, that takes place between OD values of 0.3 and 0.8 during growth in a peptide-based medium, either with or without 20 mM GlcNAc added. Error bars indicate standard errors (n = 3). Statistical comparisons were performed as described for Fig. 1.

FIG 4

Chemical complementation of chitin sugar-modulated metabolic processes. (A) Cocolonization of squid for 48 h with wild-type V. fischeri and one of three isogenic strains carrying a mutation in either a PTS or an acetate switch protein (pathways are noted above plots). Cocolonization was performed for 48 h in the absence (open) or presence (hatched) of 20 mM GlcNAc. CS, chitin sugars. (B) Competition in cocultures of wild-type V. fischeri and the indicated mutants growing in SWT medium either without (open) or with (hatched) 20 mM GlcNAc. RCI, relative competitive index. The P value above the box-and-whisker plot represents the statistical similarity to a theoretical mean of 1.0, calculated by a one-sample t test (n = 20 squid per condition). Inner fences were determined by Tukey’s method. For comparisons in which either the wild type or the mutant performed significantly (>95%) better, P values are given in bold. Error bars indicate standard errors.

FIG 5

Impact of chitin sugars on symbiont physiology. Host-derived chitin is hydrolyzed to form chitin sugars. Chitin sugar catabolism by V. fischeri inhibits secondary (i.e., non-PTS) carbohydrate catabolism, acetate uptake, and respiratory oxygen consumption. The result of this physiological shift is an increased availability of host-provided oxygen to the symbiont’s luminescence-producing enzyme, luciferase. Luminescence, which is used in the squid’s behavior, is required to support a sustained colonization of the light organ by V. fischeri.