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.

This paper is a very nice study of a novel type of class IIB bacteriocin possessing disulfide bonds, which the authors have named Gallocin A. Despite the lack of purified Gallocin A in their assays, they have provided appropriate controls for their experiments that I believe provide good evidence for their findings. I have a few comments for the author.
1) You mention that the strains of S. agalactiae you tested have differences in susceptibility to gallocin A. Based on the finding that the mechanism of action of gallocin is likely membrane permeabilization, do you think this is due to differences in membrane composition, or do you think that this is due to some other process? Is it possible that multiple mechanisms of gallocin killing could exist, similar to what has been found for some other bacterocins? 2) Do the authors know or have a hypothesis as to why Tween 20 was necessary for gallocin A activity in their vesicle membrane model? 3) Please show the data for the β-mercaptoethanol abolishing gallocin A activity, or take the data not shown out of the paper. 4) This is probably a bit outside the scope of the paper, as DTT/BME is shown to abrogate gallocin A activity in plates, but does mutation of the Cys in galA1/galA2 result in inability of the peptides to have antibacterial activity? In the same line, does deletion of blpT result in the lack of the oxidized disulfide bond observed by LC-MS/MS in galA1/galA2? 5) From the evidence shown, it appears that blpT is likely important in disulfide bond formation in galA1/galA2. Following this, if disulfide bond formation is important for galA1/galA2 activity, does deletion of blpT result in a phenotype similar to deletion of blp in the plate assays? 6) I know that purification of these types of peptides can be difficult, but can you comment on if you have examined isolation of these, and examined if they interact directly by ITC or the like? 7) Do you have an idea of how much gallocin A is produced by the WT strain? 8) You mention that the WalRK mutants exhibit abnormal morphology, this is clearly shown in Fig. 7 and is well documented in the literature for other Streptococci. Could you provide enumeration of the chain length in SGM WT vs. these resistant mutants, i.e. are they statistically different? One of two of your more severe mutants would be acceptable. 9) You mention in your methods that your control SGM WT strain had mutations compared to the reference sequence, and the RSM mutants did as well. Are these background mutations shared between WT and RSM mutants? Do you believe this is due to strain mutation/adaptation over time in the laboratory, as has been observed for other species?
Other minor comments: -Some of the pictures in Fig. S2 are quite pixelated. Could you provide higher resolution pictures for these? Also it would benefit to have multiple images, although I know this could make the figure quite busy and I don't think is absolutely necessary. -In Figure S5, control is spelled in French. Please change to English. -In Line 254, I believe the authors meant to refer to Table 2 instead of Table 1.
- Figure 4C, please outline what statistical test is used to compare the conditions. - Figure S5 is a little difficult to interpret, Could you present in a slightly different way to make the areas of mutation larger? -Please add scale bars to Figure 7 for the microscopy.
-Can you outline how many times each of the experiments were performed in the figure legends?
Reviewer #2 (Public repository details (Required)): Bacterial genome data Reviewer #2 (Comments for the Author): General comments: The authors studied the function of gallocin A, a bacteriocin of Streptococcus gallolyticus subsp. gallolyticus. The aim of the study is to characterize the mode of action and the mechanisms of resistance for gallocin A. The authors can demonstrate lipid bilayer disruption by gallocin A and they show by genetic approaches that gallocin A is a two peptide bacteriocin and that GIP functions as an immunity protein. The main novelty of the manuscript are the disulfide bonds proposed to be important for the bacteriocin activity. This hypothesis is confirmed by a loss of bacteriocin activity upon treatment with reducing agents and the detection of a thioredoxin domain in a gene of the bacteriocin cluster that upon knockout causes diminished bacteriocin activity. To substantiate this observation a mutation of cystein residues of gallocin A should be performed. Some information about the concentration at which gallocin A is killing bacterial target strains should also be included in the manuscript. Analysis of gallocin A resistant mutants suggest an involvement of the cell wall structure in the resistance mechanism but could not identify a specific protein receptor.
Specific comments: 1 Page 2 line 55 SGG is not only associated with asymptomatic colon tumors, please modify.
2 Page 12 line 297-304 Here the concentrations at which gallocin is active should be mentioned and that the spectrum of activity against closely related species is in line with previous investigations should be added. While the sensitivity of S. agalactiae has been tested. What about other pathogenic streptococci, like S. pyogenes or S. dysgalactiae subsp equisimilis?

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Reviewer #1 (Comments for the Author):
This paper is a very nice study of a novel type of class IIB bacteriocin possessing disulfide bonds, which the authors have named Gallocin A. Despite the lack of purified Gallocin A in their assays, they have provided appropriate controls for their experiments that I believe provide good evidence for their findings. I have a few comments for the author.
We thank you for your positive comment on our manuscript.
1) You mention that the strains of S. agalactiae you tested have differences in susceptibility to gallocin A. Based on the finding that the mechanism of action of gallocin is likely membrane permeabilization, do you think this is due to differences in membrane composition, or do you think that this is due to some other process?
This is a very good but very difficult question to answer. In the liposome assay, we do observe a direct membrane permeabilization by gallocin A. But access to the GBS membrane in living bacteria can be limited or promoted by strain-specific peptidoglycan associated factors and/or capsule composition. Besides that, we cannot discard the possibility suggested by the Reviewer about differences in membrane composition.
Is it possible that multiple mechanisms of gallocin killing could exist, similar to what has been found for some other bacterocins?
To our knowledge, all class IIb bacteriocins kill their target cells through membrane permeabilization. This is also what we have found for gallocin A. We cannot exclude the possibility of other mechanisms of action.
2) Do the authors know or have a hypothesis as to why Tween 20 was necessary for gallocin A activity in their vesicle membrane model?
Class IIb bacteriocin are known to adopt their tri-dimensional structure when exposed to hydrophobic environment (see Nissen-Meyer et al., 2010) and Tween 20 may provide this environment. We think that addition of small amount of Tween 20 in the supernatant helps to disperse GllA1/GllA2 complexes, thus allowing each peptide of Gallocin A to reach the target cell membrane and refold in their active state once in the membrane.
3) Please show the data for the β-mercaptoethanol abolishing gallocin A activity, or take the data not shown out of the paper.
We have added the data showing the effect of β-mercaptoethanol on gallocin A activity.
4) This is probably a bit outside the scope of the paper, as DTT/BME is shown to abrogate gallocin A activity in plates, but does mutation of the Cys in galA1/galA2 result in inability of the peptides to have antibacterial activity?
We agree with the reviewer that it would be a very good experiment to prove the importance of the cysteine residues in the gallocin A peptides. However, we are missing a tool to detect gallocin A peptides. All our attempts to tag the gallocin peptides abrogated the peptide activity. Thus, we have no simple way to check if the mutated GllA1/GllA2 peptide would be produced to the same quantity and not degraded in the presence of this mutation. Moreover, constructing mutants in Streptococcus gallolyticus is still quite challenging and time-consuming. We thought about introducing the mutations on the complementation plasmid, however since complementation is already only partial with the wild-type gene, it will be difficult to get unambiguous results.
In the same line, does deletion of blpT result in the lack of the oxidized disulfide bond observed by LC-MS/MS in galA1/galA2?
We have not sent ∆blpT supernatant for LC-MS/MS determination for the reasons stated in point 5 (see below).

5) From the evidence shown, it appears that blpT is likely important in disulfide bond
formation in galA1/galA2. Following this, if disulfide bond formation is important for galA1/galA2 activity, does deletion of blpT result in a phenotype similar to deletion of blp in the plate assays?
This is a very good question. Deletion of blpT did not produce any phenotype on plate assays while it had a striking effect in direct competition assay. We hypothesize that addition of Tween 20 and presence of oxygen could help spontaneous disulfide bond formation (Meehan et al., 2017, J Bacteriol.) during plate assays. We thus decided to perform the plate assay in anaerobic conditions, and we did observe reduction of gallocin activity in the blpT mutant as compared to the wild-type, but replicates were not always consistent. So, we decided not to show these results. Similarly, addition of a small amount of Tween 20 (0.1%) in the blpT mutant during direct competition assays in liquid medium increased killing activity of gallocin A but these results not being fully conclusive, we left them out.
LC/MS-MS on the ∆blpT supernatant is a great idea but should be performed in full anaerobic conditions, which remain a technical challenge and beyond the scope of the paper.
6) I know that purification of these types of peptides can be difficult, but can you comment on if you have examined isolation of these, and examined if they interact directly by ITC or the like?
Several experiments were carried out to purify these peptides but none of them was successful -Addition of a hemagglutinin or 6XHis tag to the C/N-terminal end of gllA1/gllA2 peptides resulted in loss of gallocin A activity. -Chemical synthesis of GllA1 and GllA2 peptides failed.
-Fusion of GllA1 and GllA2 to Glutathion S-transferase (GST) protein to lower their toxicity and hydrophobicity and for purification purposes was only partly successful and will need further improvement. -ITC is an excellent proposition, however, we would need pure samples equilibrated in the exact same buffer at high concentration of gllA1/gllA2 peptides.

7) Do you have an idea of how much gallocin A is produced by the WT strain?
Unfortunately, without purified GllA1 and GllA2, we cannot determine gallocin A concentration in the supernatant of the WT strain. However, what we do know is that production of gallocin A is a highly regulated process and sensitive to quorum-sensing as demonstrated in our previous study (Proutière et al., mBio, 2021).
8) You mention that the WalRK mutants exhibit abnormal morphology, this is clearly shown in Fig. 7 and is well documented in the literature for other Streptococci. Could you provide enumeration of the chain length in SGM WT vs. these resistant mutants, i.e. are they statistically different? One of two of your more severe mutants would be acceptable.
We would like to share with the reviewer the chaining phenotype observed below by flowcytometry on 10,000 bacteria for RSM1, RSM12 and RSM14. However, we feel that this quantification is a bit out of scope and that does not add much to the qualitative data presented in Fig. 7. 9) You mention in your methods that your control SGM WT strain had mutations compared to the reference sequence, and the RSM mutants did as well. Are these background mutations shared between WT and RSM mutants? Do you believe this is due to strain mutation/adaptation over time in the laboratory, as has been observed for other species?
Indeed, some of these mutations which were present in both WT and resistant mutants could indicate strain evolution over time in the laboratory. However, we do not think that these mutations are the results of strain adaptation but rather due to different sequencing methods: PacBio-long-reads for the reference SGM genome vs Illumina-short reads for SNP identification in the RSM mutants.
Other minor comments: -Some of the pictures in Fig. S2 are quite pixelated. Could you provide higher resolution pictures for these? Also it would benefit to have multiple images, although I know this could make the figure quite busy and I don't think is absolutely necessary.
A low-resolution merged PDF containing both manuscript and figures was provided at this first stage of submission. Naturally, we will provide higher resolution image in TIFF or PNG format for all the figures. This figure is already quite busy so we will not provide multiple images.
-In Figure S5, control is spelled in French. Please change to English.
-In Line 254, I believe the authors meant to refer to Table 2 instead of Table 1.
Thank you for noticing, we changed it.
- Figure 4C, please outline what statistical test is used to compare the conditions.
Asterisks represent statistical differences relative to WT strain UCN34 with ***p 0.001 as assessed by using two-way ANOVA in GraphPad Prism version 9.
- Figure S5 is a little difficult to interpret, Could you present in a slightly different way to make the areas of mutation larger? -Please add scale bars to Figure 7 for the microscopy. Ok -Line 423-OD is spelled incorrectly. Thank you for noticing, we changed it.
-Can you outline how many times each of the experiments were performed in the figure legends? Ok

Reviewer #2 (Comments for the Author):
General comments: The authors studied the function of gallocin A, a bacteriocin of Streptococcus gallolyticus subsp. gallolyticus. The aim of the study is to characterize the mode of action and the mechanisms of resistance for gallocin A. The authors can demonstrate lipid bilayer disruption by gallocin A and they show by genetic approaches that gallocin A is a two peptide bacteriocin and that GIP functions as an immunity protein. The main novelty of the manuscript are the disulfide bonds proposed to be important for the bacteriocin activity. This hypothesis is confirmed by a loss of bacteriocin activity upon treatment with reducing agents and the detection of a thioredoxin domain in a gene of the bacteriocin cluster that upon knockout causes diminished bacteriocin activity. To substantiate this observation a mutation of cystein residues of gallocin A should be performed. Some information about the concentration at which gallocin A is killing bacterial target strains should also be included in the manuscript. Analysis of gallocin A resistant mutants suggest an involvement of the cell wall structure in the resistance mechanism but could not identify a specific protein receptor.
We would like to thank Reviewer 2 for carefully reading our manuscript and for her/his comments.
As detailed in the answer #4 Reviewer 1 and pasted below "We agree with the reviewer that it would be a very good experiment to prove the importance of the cysteine residues in the gallocin A peptides. However, we are missing a tool to detect gallocin A peptides. All our attempts to tag the gallocin peptides abrogated the peptide activity. Thus, we have no simple way to check if the mutated GllA1/GllA2 peptide would be produced to the same quantity and not degraded in the presence of this mutation. Moreover, constructing mutants in Streptococcus gallolyticus is still quite challenging and time-consuming. We thought about introducing the mutations on the complementation plasmid, however since complementation is already only partial with the wild-type gene, it will be difficult to get unambiguous results".
Unfortunately, without purified GllA1 and GllA2, we cannot determine gallocin A concentration in the supernatant of the WT strain. Thus, it is difficult to estimate the concentration of gallocin A needed to kill target bacteria.
Specific comments: 1 Page 2 line 55 SGG is not only associated with asymptomatic colon tumors, please modify. We have removed the word "asymptomatic" from the sentence line 2. We hope that this answers your comment.
2 Page 12 line 297-304 Here the concentrations at which gallocin is active should be mentioned and that the spectrum of activity against closely related species is in line with previous investigations should be added.
As stated above, in the absence of purified peptides constituting gallocin A, we cannot determine the concentration of gallocin A. We have used in all our assays filtered bacterial supernatant of WT and mutant strains. While the sensitivity of S. agalactiae has been tested. What about other pathogenic streptococci, like S. pyogenes or S. dysgalactiae subsp equisimilis?
We have tested two S. pyogenes isolates and two S. dysgalactiae subsp equisimilis, and all of them were found to be susceptible to gallocin A. We have added one representative plate of each specie in Fig. S2.

End of Authors reply
Finally, we would like to thank the two Reviewers for their comments and suggestions that we believe helped us to improve the quality of the manuscript.
We hope that the revised manuscript will be found acceptable for publication.