Blocking Toxin Function and Modulating the Gut Microbiota: Caffeic Acid and its Derivatives as Potential Treatments for Clostridioides difficile Infection

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthened the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well-written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

This is really a manuscript about CAPE, not caffeic acid, and the title should reflect that. Also, a few details are missing from the description of the experiments. The authors should carefully revise the manuscript to ascertain that all details that could affect the interpretation of their results are presented clearly. Just as an example, the authors state in the results section that TcdB was incubated with compounds and then added to cells. Was there a wash step in between? Could compound carryover affect how the cells reacted independently from TcdB? This is just an example of how the authors should be careful with descriptions of their experimental procedures. Lastly, authors should be careful when drawing conclusions from the analysis of microbiota composition data. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Therefore, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Reviewer #2 (Public review):

Summary:

This work is towards the development of nonantibiotic treatment for C. difficile. The authors screened a chemical library for activity against the C. difficile toxin TcdB, and found a group of compounds with antitoxin activity. Caffeic acid derivatives were highly represented within this group of antitoxin compounds, and the remaining portion of this work involves defining the mechanism of action of caffeic acid phenethyl ester (CAPE) and testing CAPE in mouse C. difficile infection model. The authors conclude CAPE attenuates C. difficile disease by limiting toxin activity and increasing microbial diversity during C. difficile infection.

Strengths/ Weaknesses:

The strategy employed by the authors is sound although not necessarily novel. A compound that can target multiple steps in the pathogenies of C. difficile would be an exciting finding. However, the data presented does not convincingly demonstrate that CAPE attenuates C. difficile disease and the mechanism of action of CAPE is not convincingly defined. The following points highlight the rationale for my evaluation.

(1) The toxin exposure in tissue culture seems brief (Figure 1). Do longer incubation times between the toxin and cells still show CAPE prevents toxin activity?

(2) The conclusion that CAPE has antitoxin activity during infection would be strengthened if the mouse was pretreated with CAPE before toxin injections (Figure 1D).

(3) CAPE does not bind to TcdB with high affinity as shown by SPR (Figure 4). A higher affinity may be necessary to inhibit TcdB during infection. The GTD binds with millimolar affinity and does not show saturable binding. Is the GTD the binding site for CAPE? Autoprocessing is also affected by CAPE indicating CAPE is binding non-GTD sites on TcdB.

(4) In the infection model, CAPE does not statistically significantly attenuate weight loss during C. difficile infection (Figure 6). I recognize that weight loss is an indirect measure of C. difficile disease but histopathology also does not show substantial disease alleviation (see below).

(5) In the infection model (Figure 6), the histopathology analysis shows substantial improvement in edema but limited improvement in cellular infiltration and epithelial damage. Histopathology is probably the most critical parameter in this model and a compound with disease-modifying effects should provide substantial improvements.

(6) The reduction in C. difficile colonization is interesting. It is unclear if this is due to antitoxin activity and/or due to CAPE modifying the gut microbiota and metabolites (Figure 6). To interpret these data, a control is needed that has CAPE treatment without C. difficile infection or infection with an atoxicogenic strain.

(7) Similar to the CAPE data, the melatonin data does not display potent antitoxin activity and the mouse model experiment shows marginal improvement in the histopathological analysis (Figure 9). Using 100 µg/ml of melatonin (~ 400 micromolar) to inactivate TcdB in cell culture seems high. Can that level be achieved in the gut?

(8) The following parameters should be considered and would aid in the interpretation of this work. Does CAPE directly affect the growth of C. difficile? Does CAPE affect the secretion of TcdB from C. difficile? Does CAPE alter the sporulation and germination of C. diffcile?

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI

Strengths:

The results are really good, and the CAPE shows a good and promising alternative for treating CDI. The methodology and results are well presented, with tables and figures that corroborate them. It is solid work and very promising.

Weaknesses:

Some references are too old or missing.