Pathogen-targeting glycovesicles as a therapy for salmonellosis

Antibiotic therapy is usually not recommended for salmonellosis, as it is associated with prolonged fecal carriage without reducing symptom duration or severity. Here we show that antibiotics encapsulated in hydrogen sulfide (H2S)-responsive glycovesicles may be potentially useful for the treatment of salmonellosis. The antibiotics are released in the presence of Salmonella, which is known to produce H2S. This approach prevents the quick absorption of antibiotics into the bloodstream, allows localized targeting of the pathogen in the gut, and alleviates disease symptoms in a mouse infection model. In addition, it reduces antibiotic-induced changes in the gut microbiota, and increases the abundance of potentially beneficial lactobacilli due to the release of prebiotic xylooligosaccharide analogs.

In this study, They introduced a universal strategy for encapsulation of antibiotics by hydrogen sulfide (H2S) responsive glycovesicles to reduce salmonellosis. However, I do not see that the information provided is not significantly contributing to an advance in technology of cure the salmonellosis and new information of biology. In addition, there is not much of in vivo study to prove the direct evidence of Salmonella inhibition by this construct. It is very difficult to see the direct mechanism to inhibit the target organism specifically by this. It requres a set of precise annimal study to show the corelation between the organism and the molecule.
Reviewer #2 (Remarks to the Author): The manuscript by Mu et al. describes the use of a novel mode to deliver antibiotics (via glycovesicules) with the goal to treat Salmonellosis. The results presented by the authors are novel and promising, however the paper lacks proper descriptions of some aspects of the methology used. I have additional recommendations that I believe would strengthened the conclusions presented by the authors. My comments are below: 1) It is not clear to me how this mode of delivery would be specifically activated by Salmonella and not by other members of the microbiota that are also able to produce H2S. The authors show that Listeria doesn't induce the release of the vesicles contents as well as Salmonella, but they should also include another H2S producing bacteria to their assay. On a similar note, to show specificity to H2S inducing release, the authors should repeat the experiments with a Salmonella strain that cannot produce H2S and determine if that reduces the release of the vesicle contents.
2) The authors state that "Pathogen-inspired antimicrobial glycovesicles prevent antibiotic-induced damage to the gut microbiota". However their data shows that treatment with AM-CIP does significantly change the the composition of the gut microbiota (i.e.: Lactobacillus increase) and the authors don't have data to to determine if these changes are beneficial or not to the host. Therefore, the authors statement above is an over interpretation of the data presented and cannot be confirmed unless a functional analysis of the microbiota from all the treatments (i.e.: fecal transplant followed by challenged with Salmonella) is performed.
3) What is the intestinal concentration of the antibiotics (CIP and NEO) when delivered using the vesicles? This information would be very relevant to determine the mode of action of this treatment. 4) Overall the manuscript lacks a proper reference to the statistical methods used to assess differences in the experiments presented. The statistical tests used should be clearly stated on the figure legends and on the material and methods section. On a similar note the microbiota analysis methodology should be expanded to provide detailed information on the softwares and packages used to perform the analysis. This manuscript describes the glycovesicle system, which releases the antibiotics in response to hydrogen sulfide produced by salmonella. This vesicle system was developed to prevent quick absorption of the antibiotics into bloodstream and allow antibiotics to target H2S-producing colonies locally at the site of enteric infections. The authors demonstrated that this vesicle systems effectively reduced the Salmonella infections in gastrointestinal tract without significant altering gut microbiota. These results are impressive, but the reviewer feel that the authors should address the following points, which more strongly support the conclusions made by them and warrant publication of this manuscript.
1. The drug releasing experiment shown in Fig. 1d was conducted with 2 mM of hydrogen sulfide. Is this H2S level biologically relevant and not largely different from the concentration in the H2Sproducing colonies? The authors also should address H2S concentration-dependent drug releasing ability of AM-CIP. 2. The authors did not provide evidence that the drug concentration increase locally at the infection site and this localized concentration state is maintained for certain period in the gastrointestinal tract. This is the critical point of the study and thus should be experimentally proved. The fluorescence imaging might be useful to address this point. 3. The fluorophore release experiment should be done with non-infected mice and compare the data with that of the infected mice shown in Figure 2i. 4. The authors claimed that the cleaved xylooligosaccharide diffused in gastrointestinal tract and promoted the growth of the beneficial microbiota. On the other hand, the authors also claimed that released antibiotics locally remained at the pathogen colonies. The authors should give explanation about the difference of this molecular behavior. 5. In Fig 3d, AM administration cannot increase the relative abundance of Lactobacillaceae. This result seems to be strange since the vacant vesicle also can be cleaved by hydrogen sulfide and release xylooligosaccharide. The authors should explain this point. 6. In Fig 2b-2f, the drug dosages were not described.

Response to Reviewers' comments: Reviewer #1 (Remarks to the Author):
In this study, they introduced a universal strategy for encapsulation of antibiotics by hydrogen sulfide (H 2 S) responsive glycovesicles to reduce salmonellosis. However, I do not see that the information provided is not significantly contributing to an advance in technology of cure the salmonellosis and new information of biology. In addition, there is not much of in vivo study to prove the direct evidence of Salmonella inhibition by this construct. It is very difficult to see the direct mechanism to inhibit the target organism specifically by this. It requires a set of precise animal study to show the correlation between the organism and the molecule. Response Salmonellosis is one of the most frequently occurring intestinal diseases. Although antibiotics are a life-saving tool for the treatment of bacterial infections, antimicrobial therapy is not recommended for this disease. Antibiotic use does not shorten the length of diarrhea, reduces pathogen shedding only transiently, and involves the risk of adverse drug reactions. In this work, we introduce a versatile pathogen-inspired system for delivering absorbable or poorly absorbed antibiotics with a minimal risk of adverse outcomes to treat salmonellosis. This system includes the following advantages: 1) prevent quick absorption of oral antibiotics into the bloodstream; 2) allow antibiotics to target enteric pathogens locally and thereby alleviates disease symptoms; 3) greatly ameliorate antibiotic-induced damage to gut microbiota; and 4) increase beneficial mirobiota Lactobacillaceae by the releasable prebiotic xylooligosaccharide analogs. This study might open up a new avenue to develop pathogen-inspired antimicrobial nanovesicles to resolve enteric infections. In this new submission, we performed a set of experiment to address the reviewer's concerns. First of all, we compare the difference in the drug release behavior between Salmonella infected and uninfected mice by use of an AM vesicle loaded with Fluo (an H 2 S-responsive fluorescence probe). The ileum from infected mice, but not from normal ones elicits the strongest fluorescence intensity in a time-dependent manner (Fig. 2i), implying that 1) S. typhimurium triggers the release of the probe from AM vesicles and 2) there is higher relative abundance of S. typhimurium in ileum. Secondly, in vivo imaging experiments reveal AM vesicles are disassembled on-demand at the site of infection (Fig. 2j). Besides the direct killing of pathogenic bacteria, the increased abundance of Lactobacillaceae by the releasable prebiotic xylooligosaccharide analogs might also contribute to the resolution of Salmonellosis. We perform fecal transplant experiments and our data show that gut microbiota shaped by antibiotic-loaded glycovesicle is more beneficial to the host than that by antibiotic alone (Fig. S10).
We hope this reviewer would see our efforts in improving this manuscript and accept this version.

Reviewer #2 (Remarks to the Author):
The manuscript by Mu et al. describes the use of a novel mode to deliver antibiotics (via glycovesicles) with the goal to treat Salmonellosis. The results presented by the authors are novel and promising, however the paper lacks proper descriptions of some aspects of the methology used. I have additional recommendations that I believe would strengthened the conclusions presented by the authors. My comments are below: Response: we thank the reviewer for her/his insightful summary and constructive comments. Accordingly, we have made changes upon the comments and substantially improved the manuscript. All changes are tracked, and we hope the reviewer would accept this revised manuscript.

1) It is not clear to me how this mode of delivery would be specifically activated by Salmonella and not by other members of the microbiota that are also able to produce H 2 S. The authors show that Listeria doesn't induce the release of the vesicles contents as well as Salmonella, but they should also include another H 2 S producing bacteria to their assay. On a similar note, to show specificity to H 2 S inducing release, the authors should repeat the experiments with a Salmonella strain that cannot produce H 2 S and determine if that reduces the release of the vesicle contents.
Response: as suggested, we include two novel organisms, S. typhimurium (producing H 2 S) and C. freudii (another H 2 S-producing organism) in Fig.1f. As shown, There are much stronger fluorescence intensity in the probe-loaded AM vesicles when incubated with both S. typhimurium (producing H 2 S) than that of S. paratyphi A (Fig. 1f), an observation that AM vesicles can only be decomposed by H 2 S-producing Salmonella. S. typhimurium induces much stronger fluorescence than C. freudii does at the same concentration of bacterial cells. The fact that S. typhimurium is more abundant at the local sites of infected intestine than other H 2 S-producing bacteria will facilitate the disassembly of AM vesicles.
2) The authors state that "Pathogen-inspired antimicrobial glycovesicles prevent antibiotic-induced damage to the gut microbiota". However their data shows that treatment with AM-CIP does significantly change the composition of the gut microbiota (i.e.: Lactobacillus increase) and the authors don't have data to determine if these changes are beneficial or not to the host. Therefore, the authors statement above is an over interpretation of the data presented and cannot be confirmed unless a functional analysis of the microbiota from all the treatments (i.e.: fecal transplant followed by challenged with Salmonella) is performed.
Response: in order not to overinterprete the data, we change the sentence "Pathogen-inspired antimicrobial glycovesicles prevent antibiotic-induced damage to the gut microbiota" as "Pathogen-inspired antimicrobial glycovesicles ameliorate antibiotic-induced damage to the gut microbiota". We performed functional analysis of the microbiota through fecal transplant experiments, as following: To see whether AM-CIP shaped gut microbiota is beneficial to the host, we transfer the gut microbiota from AM-CIP or CIP treated Salmonella-infected donor mice to infected recipient mice. The recipient mice received with AM-CIP shaped gut microbiota have lower pathogen burden and tissue inflammation than that in mice received with CIP shaped one (Fig.S10).
3) What is the intestinal concentration of the antibiotics (CIP and NEO) when delivered using the vesicles? This information would be very relevant to determine the mode of action of this treatment. Response: the intestinal concentration of CIP is shown in Fig. 3d, and the intestinal concentration of NEO is shown in Fig. S11.

4)
Overall the manuscript lacks a proper reference to the statistical methods used to assess differences in the experiments presented. The statistical tests used should be clearly stated on the figure legends and on the material and methods section. On a similar note the microbiota analysis methodology should be expanded to provide detailed information on the softwares and packages used to perform the analysis. Response: we thank the reviewer's suggestion and we are very sorry for our carelessness. Now we include information about statistical tests in figure legends and method sections. As suggested, the microbiota analysis methodology is also expanded in the method section.

5) Authors should state in the Methodology which Salmonella Typhimurium strain was used in the experiments performed.
Response: in this work, three bacteria strains were used, including Salmonella enterica serovar typhimurium (SL1344), Salmonella paratyphi A (ATCC9150) and Citrobacter freundii (ATCC43864). We have added these information in the method section.

6) A quantification of the histopathology analysis of figure 2h should be included as it is hard to see differences in histopathological lesions/inflammation between the groups with the images presented.
Response: now we provide histopathology score of the H&E images in Fig. 2h. The criteria for histopathology scoring is listed in supplementary information.

7) Figure 1d-f: it is not clear how many times the experiments where performed as how many biological replicates (N)?
Response: we have added these information in the legend of Fig. 1.