Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance

Abstract

Epithelial surfaces of most animals are colonized by diverse microbial communities. Although it is generally agreed that commensal bacteria can serve beneficial functions, the processes involved are poorly understood. Here we report that in the basal metazoan Hydra, ectodermal epithelial cells are covered with a multilayered glycocalyx that provides a habitat for a distinctive microbial community. Removing this epithelial microbiota results in lethal infection by the filamentous fungus Fusarium sp. Restoring the complex microbiota in gnotobiotic polyps prevents pathogen infection. Although mono-associations with distinct members of the microbiota fail to provide full protection, additive and synergistic interactions of commensal bacteria are contributing to full fungal resistance. Our results highlight the importance of resident microbiota diversity as a protective factor against pathogen infections. Besides revealing insights into the in vivo function of commensal microbes in Hydra, our findings indicate that interactions among commensal bacteria are essential to inhibit pathogen infection.

Figure 1

Hydra ectodermal glycocalyx is colonized by a complex bacterial community. (a) Schematic drawing of the freshwater polyp Hydra indicating the tissue areas in which the glycocalyx and the bacterial colonization was examined. The letters correspond to further panels in this figure. (b) Hydra ectodermal epithelial cells prepared by HPF/FS fixation provide excellent preservation of the glycocalyx layer revealing five distinct layers (c1–c5); pm, plasma membrane. (c) Total bacterial community colonizing the surface of the ectodermal epithelium in Hydra, stained with SYBR gold. (d, e) Raster electron micrograph (REM) of bacterial cells located on the surface of ectodermal cells. (f) Transmission electron micrograph (TEM) of a rod-shaped bacterium (red arrows) located within the outer layer (c5) of the glycocalyx covering ectodermal epithelial cells. (g–i) Fluorescence in situ hybridization (FISH) analysis of bacteria removed from the ectodermal epithelium. Bacteria cells were stained with the phylotype-specific probe for Curvibacter sp. (Curvi_442) (g) and with the eubacterial oligonucleotide probe EUB338 (h). Overlay images indicating the specifically labeled bacteria in yellow (i).

Figure 2

GF Hydra polyps are prone to fungal infection by Fusarium sp. (a) Raster electron micrograph (REM) of a control polyp showing no fungal infection. (b) GF Hydra polyp infected by Fusarium sp. (c) Fungal hyphae in association with Hydra producing a spore. (d) Fungal hyphae grown in liquid R2A medium. (e) Spores isolated from the supernatant of a liquid Fusarium sp. culture. (f) Phylogenetic position of Fusarium sp. (isolated from infected Hydra) within the Nectriaceae (based on ITS region, maximum likelihood using Kimura 2-parameter model+G). Bootstrap values are shown at the corresponding nodes. The branch-length indicator displays 0.05 substitutions per site.

Figure 3

In vitro activity of bacterial isolates against Fusarium sp. In vitro plate diffusion assay for fungal inhibition by bacteria isolated from Hydra tissue. Statistical analysis was conducted using analysis of variance (ANOVA; *P<0.05, **P<0.01, ***P>0.001; n=5. Acid., Acidovorax sp.; Curv., Curvibacter sp.; Duga., Duganella sp.; Pelo., Pelomonas sp.; Pseu., Pseudomonas sp.; Undi., Undibacterium sp.).

Figure 4

Examples of in vitro activity of co-cultured bacteria against Fusarium sp. (a) Example of an additive effect of two bacterial isolates in a plate diffusion assay. (b) Example of a synergistic effect of two bacterial isolates. (c) Example of an antagonistic effect of two bacterial isolates. Statistical analysis was conducted using two-way analysis of variance (ANOVA) to test the interaction effect (synergy or antagonism) of two bacterial isolates to fungal growth (*P<0.05, **P<0.01, ***P>0.001) (see also Table 2). Acid., Acidovorax sp.; Curv., Curvibacter sp.; Duga., Duganella sp.; Pelo., Pelomonas sp.; Pseu., Pseudomonas sp.; Undi., Undibacterium sp.

Figure 5

In vivo infection rates of Hydra polyps recolonized by different bacterial isolates. (a) Experimental set-up for mono- and di-associated and conventionalized (conv) Hydra polyps used for fungal infection experiments. (b) Bacterial load of recolonized Hydra polyps. N/A indicates ‘not available' as Pseudomonas sp. shows swarming behavior and thereby overgrew Curvibacter sp., n≥4. (c) In vivo infection rates with Fusarium sp. after inoculation with spores. Statistical analyses were conducted by Fisher's exact test. Different lowercase letters indicate significant differences between treatments: ‘a' indicates significantly different from control (P<0.01), ‘b' indicates significantly different from GF (P<0.01), ‘c' indicates significantly different from control and GF (P<0.01). Fraction numbers indicate x infected cases per n replicates. Acid., Acidovorax sp.; Curv., Curvibacter sp.; Duga., Duganella sp.; Pelo., Pelomonas sp.; Pseu., Pseudomonas sp.; Undi., Undibacterium sp.