Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597
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Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline–proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile . Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. The work presented is clear and the arguments well formed. The manuscript is well written and contributes to the literature. Thank you for addressing all reviewers comments satisfactorily and in a timely manner.
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Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.
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Comments to Author
The authors have described the characterisation of CD1597, a PPEP-1 like protein. However, they did not identify any proteolysis activity, growth or sporulation defects associated with this protein. Overall, most experiments have been done well with appropriate controls, however some of the interpretations need to be clarified. It is not clear from the legends how many independent experiments or how many biological replicates or independent experiments the data represents- this need to be included. Where applicable statistical analysis (even if not significant can be included). Main concerns are listed below. * Need to use the new 630 gene annotations for CD1597, CD630_15970 * It looks like most of the 150 proteins were from E. coli, which is not surprising- please clarify. I think though that the …
Comments to Author
The authors have described the characterisation of CD1597, a PPEP-1 like protein. However, they did not identify any proteolysis activity, growth or sporulation defects associated with this protein. Overall, most experiments have been done well with appropriate controls, however some of the interpretations need to be clarified. It is not clear from the legends how many independent experiments or how many biological replicates or independent experiments the data represents- this need to be included. Where applicable statistical analysis (even if not significant can be included). Main concerns are listed below. * Need to use the new 630 gene annotations for CD1597, CD630_15970 * It looks like most of the 150 proteins were from E. coli, which is not surprising- please clarify. I think though that the purification section and Fig 2 can be moved to supplementary data. * Fig 8- Its good to have a control for ClosTron mutants, but would be important to show/ discuss what changes in the tcdC ::Ct mutant compared to the WT 630 strain. Furthermore, one cannot conclude that it's a ClosTron effect based on comparisons between the tcdC and cd1597 mutants? The authors claim that they have looked at other datasets and see similar overlaps, and yes, this would be more convincing if they see similar profiles across different ClosTron mutants which they do not show. However, based on what they have shown its hard to make any strong conclusions about ClosTron effects. * For the proteomics analysis shown here and later- how many replicates were run? This is important to know as there is much experiment to experiment variation. * Fig 9- did you look at growth in presence/ absence of sodium taurocholate- * Spore proteome: given that they authors think there is vegetative cells contamination not sure they can be so sure that CD1597 is in the spore (as mentioned in the discussion as well)? Again they mention 'as shown by other proteomic analyses' (line 603) without showing data. * I think PPEP-1 also has been shown to cleave host extracellular matrix proteins and that this could be a substrate for this enzyme. Worth including in the discussion that this could be a possibility. Although this protein does not have the classical secretion signal it may be shed or secreted through non classical mechanisms. Minor points Line 66: Gut wall- replace with gut mucosa Line 348 Although the purified protein samples predominantly contained the CD1597 constructs, reword Line 412- not deletion- disruption Line 443- gene names should be italics-
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The study by Claushuis et al. is a careful, rigorous analysis of the potential function of a putative Pro-Pro endopeptidase in Clostridioides difficile, an important pathogen. The authors have previously shown that a related protease, PPEP-1, that is involved in cleaving adhesion proteins from C. difficile's cell surface. Interestingly, CD1597 does not include a signal sequence and carries an N-terminal extension relative to PPEP-1, which is predicted to fold into an independent domain. The authors first attempt to biochemically characterize the substrate specificity of CD1597 using a library of FRET-based substrates designed for analyzing Pro-Pro endopeptidase activity. Unfortunately, they did not observe any activity by CD1597. They then make a ClosTron disruption of CD1597 and analyze the mutant's …
Comments to Author
The study by Claushuis et al. is a careful, rigorous analysis of the potential function of a putative Pro-Pro endopeptidase in Clostridioides difficile, an important pathogen. The authors have previously shown that a related protease, PPEP-1, that is involved in cleaving adhesion proteins from C. difficile's cell surface. Interestingly, CD1597 does not include a signal sequence and carries an N-terminal extension relative to PPEP-1, which is predicted to fold into an independent domain. The authors first attempt to biochemically characterize the substrate specificity of CD1597 using a library of FRET-based substrates designed for analyzing Pro-Pro endopeptidase activity. Unfortunately, they did not observe any activity by CD1597. They then make a ClosTron disruption of CD1597 and analyze the mutant's phenotype (growth rate, cell length, sporulation frequency), including its resulting proteome during vegetative growth and in spores. Unfortunately, again, they do not observe compelling phenotypes for any of these analyses. Their transcriptional analyses suggest that cd1597 is expressed at low levels, and indeed, they did not detect CD1597 in vegetative cells in their proteomic analyses, although they were able to detect it in spores. They also analyze the localization patterns of CD1597, which appears to be cytosolically localized as might be expected. Related to this last point, in my opinion, the images that they show at 48 hrs are not particularly useful because there is so much cell death. I wonder if CD1597 might be auto-inhibited by the N-terminal domain. Have the authors tested the catalytic activity of just the protease domain? If the N-terminal domain could be involved in substrate recognition or autoinhibition, just using the catalytic domain could allow for detection of activity using the FRET-based substrates. Similarly, does over-expression of just the catalytic domain impact C. difficile's physiology? The manuscript does not need to include these experiments per se given the amount of effort that went into this manuscript. However, it could yield some interesting results if they work. Related to these points, have the authors tried to align that N-terminal domain using FoldSeek? What is the conservation of CD1597 in other organisms - is the N-terminal extension unique? Is the gene cd1597 induced during sporulation? (keeping in mind that it could be post-translationally regulated) Have they looked at the transcriptional induction of the gene under stressful conditions e.g. in a sigB mutant? The protease may be important in stressful conditions.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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