A Metabolite Produced by Gut Microbes Represses Phage Infections in Vibrio cholerae

Abstract

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. Bacteriophages that prey on V. cholerae may be employed as phage therapy against cholera. However, the influence of the chemical environment on the infectivity of vibriophages has been unexplored. Here, we discovered that a common metabolite produced by gut microbes─linear enterobactin (LinEnt), represses vibriophage proliferation. We found that the antiphage effect by LinEnt is due to iron sequestration and that multiple forms of iron sequestration can protect V. cholerae from phage predation. This discovery emphasizes the significance that the chemical environment can have on natural phage infectivity and phage-based interventions.

Figure 1.

LinEnt represses ICP1 infection. (a) Growth curves of V. cholerae cultures infected with ICP1 phages (MOI = 10⁻⁶) supplemented with 20 μg of Streptomyces sp. metabolite extract or DMSO. (b) Growth curves of V. cholerae cultures infected with ICP1 phages (MOI = 10⁻⁶) supplemented with 200 μM LinEnt or DMSO. The growth curves represent the mean of three biological replicates, and the shaded area corresponds to the standard error of the mean. (c) The chemical structure of LinEnt. (d) The percentage of ICP1 phages adsorbed on V. cholerae cells treated with 200 μM LinEnt or DMSO. Bars represent the mean of three biological replicates, and the error bars correspond to the standard error of the mean. (e) Growth curves of V. cholerae PLE⁻ cultures infected with ICP1 CRISPR⁻ phages (MOI = 10⁻⁶) supplemented with 200 μM LinEnt or DMSO. The growth curves represent the mean of two biological replicates, and the shaded area corresponds to the standard error of the mean. (f) The change of ICP1 concentration within one round of infection under the treatment of 200 μM LinEnt or DMSO. Curves represent the mean of three biological replicates, and the error bars correspond to the standard error of the mean.

Figure 2.

Iron sequestration generally inhibits ICP1 infection. (a) Growth curves of V. cholerae cultures in the presence of Fe-LinEnt when infected by ICP1 phages (MOI = 10⁻⁶). The growth curves represent the mean of three biological replicates, and the shaded area corresponds to the standard error of the mean. (b) The chemical structures of Ent and EDDHA. (c) Growth curves of V. cholerae cultures infected with ICP1 phages (MOI = 10⁻⁶) supplemented with 200 μM LinEnt, 3.7 μM Ent, 22 μM EDDHA, or DMSO.

Figure 3.

The effective concentration ranges of different iron chelators. The growth curves of V. cholerae without adding phages under the treatment of (a) LinEnt, (c) Ent, or (e) EDDHA. The growth curves of V. cholerae infected by ICP1 phages (MOI = 10⁻⁶) in the presence of (b) LinEnt, (d) Ent, or (f) EDDHA. Curves represent the mean of three biological replicates, and the error bars correspond to the standard error of the mean.

Figure 4.

LinEnt also protects V. cholerae from the ICP3 phage. Growth curves of V. cholerae cultures infected with (a) ICP2 phages (MOI = 10⁻⁵) and (b) ICP3 phages (MOI = 10⁻⁵) after treatment with 200 μM of LinEnt or DMSO. The growth curves represent the mean of three biological replicates, and the shaded area corresponds to the standard error of the mean.