Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant

ABSTRACT Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells. IMPORTANCE Clostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.


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Reviewer #1 (Comments for the Author): This manuscript by Wang et al describes the construction and characterisation of a 630erm slpA deletion strain.Over the years many groups have tried, without success, to construct such a strain, so this will be of great interest to the community.The data presented appears solid and interpretation is not over the top.However, I do have some concerns about how the authors have placed their work into the wider context of the field and, in particular, the downplaying of previous work on slpA in C. difficile.Although this is the first slpA deletion that I am aware of, it is not the first S-layer null mutant and many of the phenotypes described here have been described previously.Despite this, the similarities to previously published work are not highlighted by the authors.Kirk at al 2017 described colony morphology, growth, sporulation and toxin expression differences essentially identical to those described here but that is not acknowledged at all.Müh et al also observed the same colony morphology differences using CRISPRi knock down, confirmed the sporulation effects and also noted a propensity to cell lysis.Line 27: "was constructed" -as written this implies that you've built the CRISPR-Cas9 system rather than using a previously published vector.Please rephrase.
Line 28: "knocked out" -I would suggest changing this to deleted here and elsewhere.The novelty here is the true deletion of slpA.
Line 65: Reference 11 here is not the original source.The slpA frameshift mutant was first described in Kirk et al 2017.
Line 66: "while the frameshift was suppressed during infection" I think this is referring to the mutant being avirulent but it's really hard to make sense of this as written.Please rephrase.
Lines 68-69: Müh et al also described sporulation defects and propensity to lysis similar to what you describe in this manuscript.
Line 103: the indicated band is definitely not Cwp84, at least not solely.Cwp2, Cwp66 and Cwp84 co-migrate and are very difficult to separate on SDS PAGE.Of the three, Cwp84 is by far the least abundant.
Line 120: there is a difference in response to vancomycin here but the significance is hard to justify -a 0.2 ug/ml difference in MIC hardly seems worth reporting.
Line 122: I'm not sure why you are referring to this process as autolysis.Autolysis is an active process mediated by cell wall hydrolases and that does not appear to be what you are seeing here.
Lines 127-128: aggregation is not synonymous with biofilm formation as implied here.
Lines 136-138: I may be misunderstanding what's presented here but fluorescence intensity is not a good read out of biofilm thickness.As you have performed confocal microscopy you can presumably see the thickness in Z stacks.
Line 143: "direct proof for the key role of SlpA in host-cell adherence" -this is a significant over-interpretation.The slpA mutant is lacking the dominant surface protein, has a surface with completely different biochemical characteristics and has likely changed expression and localisation of multiple other cell surface proteins, several of which have been implicated in adherence.Ascribing a direct role for SlpA in adherence is not justified by these data.
Line 152: 11 is the wrong reference again.Line 153: "has not been charactered [sic] well" -this is simply not true.The previously described S-layer null mutant that you are referring to here has been extensively characterised in at least two papers (Kirk et al 2017 andOrmsby et al 2023), revealing many of the same phenotypes that you have described here, in addition to several others that you have not tested.
Lines 159-160: No, Kirk et al 2017 already demonstrated that slpA is not essential and this was subsequently supported by CRISPRi by Müh et al in 2019.
Line 161: "growth of which was not obviously affected" -not correct.Kirk et al noted that the slpA frameshift mutant reached a maximum stationary phase OD of 2.2 compared with 3.2 for the wild type strain.This is essentially identical to the 60% defect you describe here.
Line 178-180: If the 42 kDa band were indeed the C terminal fragment of CwpV then a similarly intense band for the larger N terminal fragment would also be apparent.
Line 197: the difference is length fairly small and I would not refer to these as filamentous.
Line 202-203: the reference to the D. radiodurans SlpA here is a little odd as the proteins have absolutely no similarity.
Line 213: again has not been considered essential for many years.
Line 238: References 27 and 28 are not the appropriate original sources for this method.Lines 257-258: "subcultured in... 10 continuously cycles" -I don't follow what you've done here.Can you please rephrase?Line 266 onwards: Amirkamali is not the original source for this method.
Line 290: 65C for how long?Line 294: How were these inoculated?Very important as that can have a dramatic effect on MIC."Growth... monitored" -only endpoints are shown so presumably all of these were in stationary phase?Line 337: again I would argue that this is not autolysis.The data presented in this paper is compelling, supporting and extending previous observations regarding the contribution of the SlpA on a wide variety of phenotypes including toxin production and the capacity of the strain to sporulate.However, to improve the paper, there is a need to contextualise the data presented -how can it be linked to the loss of the S layer and what additional data would need to be included to support these observations.Description of the results is currently very limited and needs to be extended to include a greater explanation of ALL the results presented and what they mean.This could be supported in the discussion with a further linkage to existing literature.If the aim of the paper is to highlight the potential and effective use of CrispR Cas as a mutational system, then generation of an additional mutant in a strain with a different SlpA type is warranted.
A review of the written English is required throughout as there are numerous spelling mistakes and a number of poorly written passages.
Review: Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene knockout mutant.This paper, by Shaohua Wang et.al., offers an insight into the role of SlpA in C. difficile pathogenesis through the deletion, via CrispR/Cas mutagenesis, of the entire slpA of 630 erm.This genetic modification resulted in the demonstrable loss of expression of both the post-translationary cleaved HMW and LMW proteins in low pH glycine extracts from this bacterium.The work then focuses on characterisation of differences between the parental and modified strain, in the context of several relevant features of pathogenesis including growth rates, toxin production, sporulation and biofilm formation.

Main Comments
The authors highlight that the generation of this mutant using CrispR/cas nullifies the argument that SlpA is essential for survival, a concept that was promoted due to the failure of other systems to generate a site-specific mutant.However, this should be qualified as while no site-specific mutant has been produced, a naturally occurring mutant has been isolated.Regarding its essentiality in vivo, this remains unclear from this manuscript as no testing was performed.Worryingly, data in Figure S2, suggests that there is a significant loss of mutant viability after 48h, which would indicate that the SlpA expression have relevance for even long-term survival in vitro.
While the authors report their direct observations regarding the behaviour of the mutants in vitro, the explanation and interpretation of the phenotypes observed is limited, especially in the context of the presence and absence of an S layer.It is reported that the mutant sporulates poorly and shows limited toxin production, however, there is no wider attempt to explain why this might be the case.Given that the mutant appears to show limited viability after 48h does this reflect the fact that a high proportion of the bacteria are no longer viable or they cannot sporulate effectively?Interestingly, several of the features described hereincluding reduction in colony size, toxin production, sporulation defects and increased sensitivity to lysis have been described elsewhere in the literature in the context of a naturally occurring SlpA mutant, R20291 FM2.5.In the reviewed manuscript, this mutant is dismissively described as poorly characterised, however, given the similarities in several features, acknowledging this work would strengthen the manuscript; given the additional data provided that may help to explain differences in toxin production and sporulation described here.
Other data is simply reported without contextualization.What is the relevance of loss of motility -Is it associated with loss of membrane stability -are the flagellar still made -can they be recovered from the supernatant of growth medium.There should be an expansion of either the description of these results, which are currently very limited, or additional speculation added to the discussion to provide greater overview of the observations.What is the relevance of enhanced susceptibility to killing by vancomycin and Triton X ?
In parallel, there is no real explanation as to why the OD values and viability of the mutant do not correlate.If the bacterial cells are smaller in size as indicated (and which data in Fig S1 supports), the higher density of bacteria as indicated by the viability counts would still be expected to increase light deflection by the culture.Statistically, is the difference in viability significant?The differences in Figure 2B are enhanced by the choice of a linear rather than logarithmic scale for this data.However, if true, the difference in OD/viability raises questions regarding the standardisation of inoculum for the assays described.For example, for the adhesion assay, the paper indicates mid exponential cells were added at a ratio of 20:1, however, it was not clear how this was standardised to provide the same starting inoculum.Similarly given the observed loss of viability of the mutant over time (48h) in vitro (Fig S2), could the reported differences in adhesion between the mutant and parental strain be linked to loss of viability and not differences in capacity to adhere?Was this considered?
For biofilm production, it would be useful to include electron micrographs of the biofilm structures generated by the mutant and parental strains.While data is very supportive of this phenotype, increased crystal violet staining could reflect enhanced active biofilm formation by the mutant, or modifications to the surface charge of the bacterial surface, resulting in aggregation rather than biofilm formation.While other methods are for biofilm measurement are included indicated either these are no standard for biofilm measurement (F p ) or often suffer from the capacity to penetrate the biofilm structure effectively (live/ dead stain).
Lastly, given that the highlight of this paper is the generation of a site directed SlpA mutant using Crisp/Cas, an attempt, or at least tried attempt to generate at least a second mutant from a strain expressing a different SlpA type would enhance the paper.The feasibility of this may be dependent on existing antibiotic resistances in the strains, but the generation and testing of an additional mutant would provide a broader evaluation of the technology and greater understanding of the essentiality of this gene.In particular, the ease by which similar mutations in other SlpA types could be generated could provide important information.
Minor points.The language used in the introduction, results and discussion is naïve and ambiguous in places.In contrast the methods are well written, although additional information on standardisation of the inoculum in various assays would be useful.There are multiple spelling and grammatical errors throughout the manuscript that require modification.
In the introduction, there is a strong statement indicating that adhesion is critical for C. difficile pathogenesis and yet this has not definitively been shown at least in vivo.
In a number of places, there are references to intercellular proteins.It is not clear what location this relates

Responses to revivers:
We appreciate the reviewers' time and dedication to giving feedback and are grateful for insightful suggestions, which have helped us improve our manuscript substantially.This revised version of the manuscript addresses all the comments provided by the reviewers.The changes made are highlighted in yellow within the manuscript.Please check below for the responses to each comment from the reviewers.
Reviewer #1 (Comments for the Author): This manuscript by Wang et al describes the construction and characterisation of a 630erm slpA deletion strain.Over the years many groups have tried, without success, to construct such a strain, so this will be of great interest to the community.The data presented appears solid and interpretation is not over the top.
Thanks for recognizing the novelty of our study, and your positive comments on our results.
However, I do have some concerns about how the authors have placed their work into the wider context of the field and, in particular, the downplaying of previous work on slpA in C. difficile.Although this is the first slpA deletion that I am aware of, it is not the first S-layer null mutant and many of the phenotypes described here have been described previously.Despite this, the similarities to previously published work are not highlighted by the authors.
We totally agree with the reviewer's concerns.We did include the previous studies in generating S-layer null mutant and slpA knockdown mutants.As suggested, in the revised version, we added more details highlighting and comparing phenotypes between those mutants.Kirk at al 2017 described colony morphology, growth, sporulation and toxin expression differences essentially identical to those described here but that is not acknowledged at all.Müh et al also observed the same colony morphology differences using CRISPRi knock down, confirmed the sporulation effects and also noted a propensity to cell lysis.
Thanks!We discussed similar findings in line 160-167.Line 27: "was constructed" -as written this implies that you've built the CRISPR-Cas9 system rather than using a previously published vector.Please rephrase.
Thanks for your careful check, we changed the description to "In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system." in line 27.
Line 28: "knocked out" -I would suggest changing this to deleted here and elsewhere.The novelty here is the true deletion of slpA.
Good suggestion, thanks!We have replaced "knocked out" with "deleted" in the manuscript.
Line 65: Reference 11 here is not the original source.The slpA frameshift mutant was first described in Kirk et al 2017.
Thanks, we have cited the direct reference from Kirk et al. 2017. in line 64.
Line 66: "while the frameshift was suppressed during infection" I think this is referring to the mutant being avirulent but it's really hard to make sense of this as written.Please rephrase.
Thanks for your good suggestions, we modified the sentence to "and the the framshift mutant is avirulent during infection".
Lines 68-69: Müh et al also described sporulation defects and propensity to lysis similar to what you describe in this manuscript.
We added these results in line 68-69 as "Based on this CRISPRi system, the expression of SlpA was knocked down, and the slpA-depleted cells demonstrated reduced sporulation and increased lysozyme sensitivity (12)."Line 103: the indicated band is definitely not Cwp84, at least not solely.Cwp2, Cwp66 and Cwp84 co-migrate and are very difficult to separate on SDS PAGE.Of the three, Cwp84 is by far the least abundant.
Appreciate your concerns.As we are not able to distinguish this band based on the size, we removed the indication for CWP84 decrease, and changed the description as "Along with disappearance of SlpA bands, other cell wall proteins including those around 50-75 kDa were lower expressed, and those around 15-50 kDa were higher expressed".Moreover, we modified the Fig. 1 and the legend accordingly.
Line 120: there is a difference in response to vancomycin here but the significance is hard to justifya 0.2 ug/ml difference in MIC hardly seems worth reporting.
We agree that the 0.2 ug/ml difference in MIC is slight.As the sensitivity to different vancomycin concentrations are still obvious, we modified the description to "C. difficile 630∆erm∆slpA exhibited a vancomycin MIC of 0.8 µg/ml, slightly (0.2 µg/ml) less than that for C. difficile 630∆erm, but C. difficile 630∆erm∆slpA was more susceptible to vancomycin at sub-MICs (Fig. 3B)." in line 119-121.
Line 122: I'm not sure why you are referring to this process as autolysis.Autolysis is an active process mediated by cell wall hydrolases and that does not appear to be what you are seeing here.
Thanks for your great question!Different from the spontaneous autolysis, we used Triton X-100 to stimulate autolysis.To be clear, we modified all the autolysis to "Triton X-100-induced autolysis".
Lines 127-128: aggregation is not synonymous with biofilm formation as implied here.
Thanks for your rigorous advice, we modified it as a potential indication, and added a citation to support this indication (line 129-130).
Lines 136-138: I may be misunderstanding what's presented here but fluorescence intensity is not a good read out of biofilm thickness.As you have performed confocal microscopy you can presumably see the thickness in Z stacks.
Thanks for your thoughts.CLSM images are used to support biofilm sessile cell information in both 3-D and 2-D modes.In 3-D mode, the biofilms appear to be uneven.Thus, z-axis height ratio is not a good quantitative measurement.Although fluorescence intensity is not very accurate, it is still widely used quantitatively by many researchers.To decrease the confusion, we removed "thickness" from the sentence, and focused on fluorescence intensity.Line 143: "direct proof for the key role of SlpA in host-cell adherence" -this is a significant overinterpretation.The slpA mutant is lacking the dominant surface protein, has a surface with completely different biochemical characteristics and has likely changed expression and localisation of multiple other cell surface proteins, several of which have been implicated in adherence.Ascribing a direct role for SlpA in adherence is not justified by these data.
Agree, the SlpA deletion induced changes on the cell surface should be the direct factors affecting the adherence.Then, we changed the description to "indicating the important role of SlpA in host-cell adherence of C. difficile" (line 143-144), and discussed more studies are needed in cell surface structures changes due to slpA deletion to reveal more targets involved in pathogen-host interaction (line 211-214).
Line 152: 11 is the wrong reference again.
We corrected it to the direct reference.Thanks!Line 153: "has not been charactered [sic] well" -this is simply not true.The previously described S-layer null mutant that you are referring to here has been extensively characterised in at least two papers (Kirk et al 2017 andOrmsby et al 2023), revealing many of the same phenotypes that you have described here, in addition to several others that you have not tested.
To avoid misunderstanding the truth, we modified the sentence to "However, the insertion mutant could not remove slpA completely, and more details about roles of SlpA in C. difficile pathogenesis need to be uncovered."in line 153-154.
Lines 159-160: No, Kirk et al 2017 already demonstrated that slpA is not essential and this was subsequently supported by CRISPRi by Müh et al in 2019.
We added more details about the insertion and knockdown mutants and modified the description as "The successful generation of slpA deletion mutant further negated the previous knowledge which considered slpA essential.Similar with the first S-layer-null insertion mutant of slpA (FM2.5)(11) and the CRISPRi knockdown of slpA in R20291 ( 12), deletion of slpA resulted in more transparent colony morphology, inhibited growth (Fig. 2A), attenuated toxin production (Fig. 2C and Supplementary Fig. S2B), and suppressed sporulation (Fig. 2D) of C. difficile 630∆erm∆slpA, verifying the important roles of SlpA in life cycle of C. difficile.Moreover, major pathogenic factors such as interaction with host cells and biofilm formation of the slpA deletion mutant were also illustrated in this study and discussed below."(line 160-167) Line 161: "growth of which was not obviously affected" -not correct.Kirk et al noted that the slpA frameshift mutant reached a maximum stationary phase OD of 2.2 compared with 3.2 for the wild type strain.This is essentially identical to the 60% defect you describe here.
Thanks for your careful check.As above, we discussed the similar results in line 161-167 as "Similar with the first S-layer-null insertion mutant of slpA (FM2.5)(11) and the CRISPRi knockdown of slpA in R20291 ( 12), deletion of slpA resulted in more transparent colony morphology, inhibited growth (Fig. 2A), attenuated toxin production (Fig. 2C and Supplementary Fig. S2B), and suppressed sporulation (Fig. 2D) of C. difficile 630∆erm∆slpA, verifying the important roles of SlpA in life cycle of C. difficile.Moreover, major pathogenic factors such as interaction with host cells and biofilm formation of the slpA deletion mutant were also illustrated in this study and discussed below.".Thank you!Line 178-180: If the 42 kDa band were indeed the C terminal fragment of CwpV then a similarly intense band for the larger N terminal fragment would also be apparent.
Thanks!It makes sense.Considering potential loss of some protein bands during cell wall proteins extraction, and the protein size of this band pointed us to CwpV N-terminal domain, we changed our description to "While further studies will be needed for identification of this band to verify the involvement of CwpV." (line 184-185) Line 197: the difference is length fairly small and I would not refer to these as filamentous.
Thanks for your good suggestions, we removed filamentous, and changed the sentence to "Disruption of the slpA gene resulted in shorter thin cells (Supplementary Fig. S1B) and increased biofilm production (Fig. 4) compared to the wild type…" (line 201) Line 202-203: the reference to the D. radiodurans SlpA here is a little odd as the proteins have absolutely no similarity.
Agree, and we replaced this reference with a recently published close one to emphasize the important roles of S-layer protein in biofilm formation.In the revised version, we changed the description to "and incubation of C. difficile with anti-S-layer monoclonal antibodies significantly reduced biofilm formation (25)" in line 206-207.Thanks!Line 213: again has not been considered essential for many years.
Thanks, and we modified this to "further providing solid proof against the essential role of slpA in C. difficile."(line 219-220) Line 238: References 27 and 28 are not the appropriate original sources for this method.
Thanks for your careful checking.We performed the conjugation based on our previous protocol (ref 27) and modified it with the optimized heat treatment to improve efficiency (we added the very original ref for this as 29).
Lines 257-258: "subcultured in... 10 continuously cycles" -I don't follow what you've done here.Can you please rephrase?Thanks for pointing out your confusion.To cure the plasmid (harboring antibiotic resistant markers) from the mutants, we cultivated mutants in BHIS medium without antibiotics, and transfer cultures into fresh BHIS medium after 8 h cultivation as a subculture cycle, after repeating 10 subculture cycles, the cultures were spread onto BHIS agar plates with and without antibiotics to check the curing of plasmid.
To reduce confusion, we rephrased the sentence as "Colonies demonstrating only the mutant band were picked and cultivated in BHIS medium without antibiotics.Subcultures were carried out every 8 h for 10 cycles, followed by being spread onto BHIS to obtain single colonies, which were then replica plated onto BHIS agar and BHIS agar with Tm plates."(263-265) Line 266 onwards: Amirkamali is not the original source for this method.
We followed the modified protocol from Amirkamali et al.To acknowledge the original reference, we also cited the original reference from Calabi et al. (line 273) Thanks!Line 290: 65C for how long?
We added 30 min which we used for heat treatment (line 297).Thanks!Line 294: How were these inoculated?Very important as that can have a dramatic effect on MIC."Growth... monitored" -only endpoints are shown so presumably all of these were in stationary phase?
For inoculation: fresh log phase cultures were used for inoculation, and each 96-well contains an initial culture of ~10 5 CFU.
We used OD absorbance at 10 h cultivation (early stationary phase) to compare their sensitivity to vancomycin.We added this detail as "and growth after 10 h cultivation was used to compare their sensitivity to vancomycin".(line 305) Line 337: again I would argue that this is not autolysis.
We changed it to "Triton X-100-induced autolysis".Thanks!Thanks.More details were added, and OD reading instead of survival was provided for Fig. 3B.Thanks for the good questions.Fig. 4B provides qualitative support to biofilm health using a biofilm test kit.C. difficile is a corrosive biofilm.Its biofilm harvests electrons from the stainlesssteel coupon underneath it in our test kit.When Fe 0 losses electrons to become Fe 2+ , corrosion occurs.More sessile cells harvest more electrons, causing more severe corrosion.Using two separate biofilm test kit units, we compared R p (corrosion resistance) responses of two different biofilms.The R p responses support other data indicating better biofilm growth of C. difficile 630∆erm∆slpA strain (showing lower R p , i.e., higher corrosivity).The R p responses are reliable qualitative indicator of corrosivity and thus biofilm strength.Regarding error bars for the R p response, we did not provide them because the R p sequence in Fig. 4B is consistent with other biofilm data (Fig. 4 A and C).It is common to skip replicates in using qualitative corrosion data used to support core data, after all, the Rp are meant to be qualitative, rather than the quantitative data which require error bars to provide statistical significance.
Like we explained above, CLSM images are used to support biofilm sessile cell information in both 3-D and 2-D modes.In 3-D mode, the biofilms appear to be uneven.Thus, we did not apply z-axis height ratio which is not a good quantitative measurement.Although fluorescence intensity is not very accurate, it is still widely used quantitatively.
Therefore, we modified the figure legend to explain the single measurements and CLSM results.Thank you!We changed cell size to cell length as suggested.The images were taken under brightfield-transmitted light under same conditions but with auto exposure time.The same microscope also allowed us to try phase contrast, but we were not able to get clear images.To improve the images, we slightly decreased the contrasts of both images with the same adjustment degree.The data presented in this paper is compelling, supporting and extending previous observations regarding the contribution of the SlpA on a wide variety of phenotypes including toxin production and the capacity of the strain to sporulate.
We gratefully appreciate your positive comments!However, to improve the paper, there is a need to contextualise the data presentedhow can it be linked to the loss of the S layer and what additional data would need to be included to support these observations.Description of the results is currently very limited and needs to be extended to include a greater explanation of ALL the results presented and what they mean.This could be supported in the discussion with a further linkage to existing literature.If the aim of the paper is to highlight the potential and effective use of CrispR Cas as a mutational system, then generation of an additional mutant in a strain with a different SlpA type is warranted.

Thanks for your constructive suggestions!
To explain and extend our results, we modified the discussion section with: 1) We added the comparison with the insertion and CRISPRi knockdown slpA mutants about roles of SlpA on phenotypes of C. difficile.2) We discussed the changes of other CWPs due to slpA deletion and their potential involvement in CDI pathogenesis.And this studying is ongoing in our lab.3) Based on current reports on roles of Cwp84, we discussed slpA deletion induced cwp84 expression change may be related to biofilm formation of slpA deleted C. difficile mutant.4) We discussed roles of SlpA in pathogen-host interaction based on the adhesion results and indicated that detection of cell wall structures due to slpA deletion will help uncovering more targets in CDI pathogenesis.
Thanks for your valuable suggestions!Although we deleted the slpA gene for the first time with the CRISPR-Cas9 system, we are not focused on this system in this manuscript.
A review of the written English is required throughout as there are numerous spelling mistakes and a number of poorly written passages.
Thanks for your careful check!The manuscript has been double-checked, and the typos and grammar errors we found have been corrected.
Re: Spectrum04005-23R1 (Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant) Dear Dr. Shaohua Wang: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Sincerely, Meera Unnikrishnan Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): This manuscript is a resubmission of a paper that I reviewed earlier this year.When I first read this manuscript, I had concerns about the connection to previous work in the field.The authors have done an admirable job of addressing these concerns and I believe the current version is significantly improved.Overall, I can identify no significant issues in this version and I think this paper will be of great interest in the field.
Figures and legendsNumber and type of replicates should be added to all legends.

Figure 1 :
Figure 1: As noted above this is likely not Cwp84.

Figure 2 :
Figure 2: Very little detail in the legend.More here would save the reader referring back and forth to the methods and would make the figure much more understandable.

Figure 3 :
Figure3: very little detail here.What does the dotted line in B represent?Also the measurement here is presumably max growth expressed as a percentage of the control.Survival is not correct when talking about MIC.

Figure 4 :
Figure 4: B has no error bars -single measurements?On rereading C I can't figure out if this is actually representing intensity across a Z stack or not.The legend needs a lot more information to aid interpretation.

Figure
Figure S1: B is cell length not size presumably.Are these images phase contrast?If so they are extremely over-saturated.

Figure
Figure S2: panel A would be much better merged with Fig. 2B.What is shown in S2B?The legend only says toxins [sic] production at 24 h and 48 h cultivation -not nearly enough detail incidentally.Fig 2C apparently also shows "less toxins production" at 24 and 48 h.So it seems these are showing the same thing, yet the values are completely different -there's > an order of magnitude difference in the wt at 48 h.Merging Figure 2B and S2A would help reader understanding.

Figure 1 :
Figures and legends Number and type of replicates should be added to all legends.Number and replicates were added when applicable.Thanks! Figure 1: As noted above this is likely not Cwp84.Cwp84 related information was removed.Thanks! Figure 2: Very little detail in the legend.More here would save the reader referring back and forth to the methods and would make the figure much more understandable.Thanks for your good suggestions.More details were added into the figure legend.

Figure 3 :
Figure3: very little detail here.What does the dotted line in B represent?Also the measurement here is presumably max growth expressed as a percentage of the control.Survival is not correct when talking about MIC.

Figure 4 :
Figure 4: B has no error barssingle measurements?On rereading C I can't figure out if this is actually representing intensity across a Z stack or not.The legend needs a lot more information to aid interpretation.

Figure
Figure S1: B is cell length not size presumably.Are these images phase contrast?If so they are extremely over-saturated.

Figure
Figure S2: panel A would be much better merged with Fig. 2B.What is shown in S2B?The legend only says toxins [sic] production at 24 h and 48 h cultivationnot nearly enough detail incidentally.Fig 2C apparently also shows "less toxins production" at 24 and 48 h.So it seems these are showing the same thing, yet the values are completely differentthere's > an order of magnitude difference in the wt at 48 h.Merging Figure 2B and S2A would help reader understanding.Thanks for your suggestions!Because Fig. SA and B referred to cell concentration and toxins production, respectively, we think it is helpful to keep them separate for the description.Appreciate your careful thoughts.Fig. S2B is the intercellular toxins result, and Fig. 2C in the main text is for the extracellular ones.We added more details in the figure legend to avoid the confusion.Minor typographical issues Line 35: hospital acquired infection Line 45: spore forming Line 54: SLPs are derived Line 101: loss of Line 110: slpA not SlpA Line 114: toxin production Line 175, 183 and 185: CWPs Line 189: cwp84 italics Line 221: coli