A mutualistic model bacterium is lethal to non-symbiotic hosts via the type VI secretion system

Abstract

What makes a bacterium pathogenic? Since the early days of germ theory, researchers have categorized bacteria as pathogens or non-pathogens, those that cause harm and those that do not, but this binary view is not always accurate. Vibrio fischeri is an exclusive mutualistic symbiont found within the light organs of Hawaiian bobtail squid. This symbiotic interaction requires V. fischeri to utilize a range of behaviors and produce molecules that are often associated with pathogenicity. This juxtaposition of employing "pathogenic" behaviors for a symbiotic relationship led the field to focus on how V. fischeri establishes a beneficial association with its host. In this study, we observe that V. fischeri induces mortality in zebrafish embryos and Artemia nauplii. Non-lethal doses of V. fischeri leads to zebrafish growth delays and phenotypes indicative of disease. Our data also provide evidence that the conserved type VI secretion system on chromosome I (T6SS1) plays a role in the V. fischeri-induced mortality of zebrafish embryos and Artemia nauplii. These results support the hypothesis that the V. fischeri T6SS1 is involved in eukaryotic cell interactions. Despite its traditional view as a beneficial symbiont, we provide evidence that V. fischeri is capable of harming aquatic organisms, indicating its potential to be pathogenic toward non-symbiotic hosts.

Keywords: Pathogenicity; Type IV Secretion System; V. fischeri; Zebrafish.

Figure 1:

V. fischeri is lethal to zebrafish embryos and Artemia nauplii. (A) Survival curve of zebrafish embryos immersed with V. fischeri ES114 over 48 h. Performed with 2 independent replicates of 24 zebrafish embryos per treatment. (B) Controls used to validate dependence on viable V. fischeri. Each point represents independent replicates of at least 16 embryos. (C) Enumeration of bacteria following homogenization of non-sterile embryos with and without streptomycin treatment. Each point represents independent replicates of 8 embryos. (D-I) Embryos at 72 hours post fertilization (hpf) imaged following 48 h of immersion in 3.0 × 10⁶ CFU/mL V. fischeri. (D) Untreated control. (E) Blood pooling in the tail (arrow) and no blood flow present throughout the heart. (F) Short length with delayed yolk sac absorption, small head, necrotic tissue, and blood pooling in the duct of Cuvier (arrow). (G) Short length with severe maldevelopment including bent tail (arrow) and necrotic tissue. (H) Short length with necrotic tissue and a small head. (I) Embryo with pericardial edema. (J) Survival curve of Artemia nauplii immersed with V. fischeri ES213 over 44 h. Performed with at least 2 independent replicates with 40–100 nauplii per treatment.

Figure 2:

Impact of the T6SS1 on immersed animal embryo mortality. (A) Survival percentage of starting population of Artemia nauplii immersed in 4.6 × 10⁶ CFU/mL V. fischeri. Performed with at least 3 independent replicates of 40–100 nauplii per treatment. (B) Survival of zebrafish embryos immersed in 3.0 x 10⁶ CFU/mL V. fischeri. Untreated control embryos all survived. Performed with 4 independent replicates of 24 zebrafish embryos per treatment. The statistical significance calculated between WT, ΔicmF1 mutants, and controls was calculated with a multiple comparisons two-way ANOVA. Asterisks show significant difference between WT and mutant pairs, daggers show significance with untreated controls, ns, no significant difference (p > 0.05), * p < 0.05, ** or †† p < 0.01, *** or ††† p < 0.001, **** or †††† p < 0.0001. (C) Model for V. fischeri effects on host health (pathogenic or beneficial) and the role of T6SS1, made with the help of Biorender.com.