Gallocin A, an Atypical Two-Peptide Bacteriocin with Intramolecular Disulfide Bonds Required for Activity

Abstract

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip, is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Keywords: Streptococcus gallolyticus; antimicrobial peptides; bacteriocins; class IIb bacteriocin; disulfide bond; immunity peptide.

FIG 1

Gallocin A is a two-peptide bacteriocin. (A) The core operon encoding gallocin A peptides and the immunity protein in SGG strain UCN34. Gallocin genes are indicated in red and renamed gllA1 and gllA2 according to the nomenclature of Hill et al. (2). (B) Agar diffusion assay to test gallocin activity from supernatants of UCN34 wild-type (WT), ΔgllA1, ΔgllA2, and Δblp against gallocin-sensitive Streptococcus gallolyticus subsp. macedonicus (SGM) strain. One representative plate of three independent replicates is shown. (C and D) Growth curves of SGG Δblp, Streptococcus agalactiae A909 and Lactococcus lactis NZ9000 containing an empty plasmid (p) or a plasmid expressing gip (p-gip) in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) medium supplemented with supernatant of ΔblpS (a strain overproducing gallocin A, “+ gallocin”) or Δblp (gallocin A deletion mutant, “– gallocin”) and 0.01% of Tween 20. The mean of two independent replicates is shown.

FIG 2

Gallocin A is active against most streptococci, lactococci, and enterococci. Phylogenetic tree based on the 16S RNA sequence (from the Silva online database) of different bacterial species that are resistant (green) or susceptible (red) to gallocin A, as determined by agar diffusion assay (Fig. S2).

FIG 3

Gallocin A can permeabilize bacterial membranes and lipid vesicles. (A and B) Fluorescence of the voltage-sensitive DiBAC4(3) [bis-(1,3-dibutylbarbituric acid)trimethine oxonol] (A) or membrane-impermeant propidium iodide (PI) (B) after resuspension of Enterococcus faecalis OG1RF in supernatants of UCN34 WT, Δblp (– gallocin A), and ΔblpS (overexpressing gallocin A). One experiment representative of three independent replicates is shown. (C and D) Measure of the fluorescence corresponding to the release of ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid; excitation: 390 nm, emission: 515 nm) encapsulated in large unilamellar vesicles after addition of SGG supernatant or Triton X-100 (positive control). (C) At 60 s, Triton or the supernatant of SGG UCN34 WT, Δblp, WT 30× (concentrated 30 times), Δblp 30×, was added to the liposomes. (D) At 60 s (SN1), the supernatant of ΔgllA1 or ΔgllA2 was added to the lipid vesicle suspension. At 200 s (SN2), the supernatant of the other strain was added. AU, arbitrary unit.

FIG 4

Gallocin A peptides possess a disulfide bridge important for their structure and activity. (A) Agar diffusion assay to test bactericidal activity of purified nisin (25 μg/mL) and supernatants of SGG WT or ΔblpS supplemented or not with 50 mM dithiothreitol (DTT; left panel) or 100 mM β-mercaptoethanol (BME; right panel). One plate representative of three independent replicates is shown. (B) Schematic representation of the gallocin genomic locus and pBLAST domain identification in BlpT protein. (C) Recovered E. faecalis after coculture at a 1:1 ratio for 4 h with SGG WT, Δblp, ΔblpT, and WT revertant from blpT deletion (bWT). The mean and standard deviation of three independent replicates is shown. Asterisks represent statistical differences with ***, P < 0.001 as assessed using two-way analysis of variance in GraphPad Prism version 9.

FIG 5

Structural models of GllA1 and GllA2 predicted using ColabFold. (A and B) Pre-peptide and mature forms of GllA1 (A) and GllA2 (B) predicted using ColabFold, visualization was obtained with PyMOL (version 2.5.2 PyMOL Molecular Graphics System; Schrödinger, LLC). All representations are colored with a predicted local distance difference test (lDDT) score of 30% (red) to 100% (blue). For the pre-GllA1 and pre-GllA2, glycine doublet is colored in green. Disulfide bridges are represented as sticks.

FIG 6

Structural models of GIP and its interactions with GllA1 and GllA2. (A) ColabFold modeling of GIP and visualization with PyMOL. (B to D) ColabFold modeling of the interaction between GllA1/GllA2 (B), GIP/GllA1 (C), GIP/GllA2 (D), and GllA1/GIP/GllA2. (E) Interaction models aligned on the Cα of each GIP.

FIG 7

Gallocin A-resistant mutants (RSM) form aggregates and exhibit morphological defects compared to the parental gallocin A-sensitive strain SGM. Epifluorescence microscopy images of SGM WT and RSM-1 to -14 (excluding the two mutants that did not grow in THY supplemented with gallocin A) labeled with the wheat germ agglutinin-488, a fluorescent peptidoglycan dye. Scale bar (1 μm) is shown on the bottom right. Representative images from three independent experiments are shown.