Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

Round 1

Reviewer 1 Report

This is a well-written manuscript and will be interesting to those who are working in this field. A minor revision for language is required (please see the enclosed manuscript).

Comments for author File: Comments.pdf

Author Response

Reviewer 1 comment:
This is a well-written manuscript and will be interesting to those who are working in this field. A minor revision for language is required (please see the enclosed manuscript).

Reply to the comment.
Thank you so much for your critical reading. All suggestions were corrected in the revised manuscript accordingly. Please see below in detail.

Line 49, "mammal" was changed to "mammals".
Line 50, "infection in mammal is" was changed to "mammal infection are".
Line 59, "difficult" was changed to "challenging".
Line 63, "here is still room to further simplify the assay" was changed to "there are still rooms to simplify the assay further".
Line 104, "temperature sensitive" was changed to "temperature-sensitive".
Line 154, "Number" was changed to "The number".
Line 157, "significant" was changed to "a significant".
Line 172, "than that" was changed to "than".
Line 174, "were" was changed to "was".
Line 174, ", although" was changed to ". However,".
Line 177, ", and three" was changed to ". Three".
Line 178, "group " was changed to "group ,".
Line 183, "liquid assay" was changed to "the liquid assay".
Line 184, "The value for MG1655 as a model strain for fecal isolate was 1.17, and that for UTI89 as a model strain for UTI associated isolate was 1.75." was inserted.
Line 219, "Furthermore, although" was changed to "Although".
Line 222, "when compared " was changed to "compared".
Line 222, "the finding" was changed to "and the finding".
Line 246, "toxin" was changed to "the toxin".
Line 251, "be not" was changed to "not be".
Line 255, "number " was changed to "the number".
Line 258, "mammal" was changed to "a mammal".
Line 267, "mammal " was changed to "mammals".
Line 278, "involvement " was changed to "the involvement".
Line 282, "mammal" was changed to "a mammal".
Line 301, "iron" was changed to "the iron".
Line 303, "resulted in decreasing" was changed to "decreased".
Line 312, "pathogenic solid" was changed to "the solid pathogenic".
Line 315, "and" was changed to ", and".
Line 328, "wonderful " was changed to "his wonderful ".

Reviewer 2 Report

In this manuscript, Hashimoto et al. analyzed pathogenic potential of E. coli strains that were isolated from urinary tract infections and fecal samples. The authors show that E. coli that there are clear phylogenetic differences and that strains isolated from UTI generally have more virulence factors than those from fecal isolates. Moreover, the authors show that iron acquisition genes in these ExPEC strains are key virulence factors in a C. elegans infection model. The development of the liquid killing assay was a very innovative aspect of the manuscript. The results shown by the author mostly support their conclusions. Overall, some comments are listed below, which should be considered:

 

Comments

1. K-12 MG1655 should have been included as a control in the elegans liquid and solid killing assays. Including this control is key to comparing the pathogenic potentials of the E. coli isolates. This is particularly necessary to understand the contribution of iron acquisition systems between different E. coli strains because K12 does not have a heme uptake system.

 

2. The observation that iron acquisition is required for virulence is not unexpected. However, it seems important to answer one key question. Do the iron acquisition genes contribute to virulence of UTI isolates and fecal isolates to similar amount? Fig S1 shows that relative pathogenicity is lower in fecal isolates, however many of these fecal isolates still have the iron acquisition genes (Table 1). It would be very interesting to see if deletion of the iron acquisition genes in one of these fecal isolates reduces virulence in elegans? The requirement of iron acquisition is likely conserved across most strains since iron is an essential nutrient.

 

3. The urosepsis isolates exhibit lower pathogenicity than the other UTI isolates. Were any obvious differences in virulence factors or growth characteristics between these strains?

Author Response

Reviewer 2 comments:

In this manuscript, Hashimoto et al. analyzed pathogenic potential of E. coli strains that were isolated from urinary tract infections and fecal samples. The authors show that E. coli that there are clear phylogenetic differences and that strains isolated from UTI generally have more virulence factors than those from fecal isolates. Moreover, the authors show that iron acquisition genes in these ExPEC strains are key virulence factors in a C. elegans infection model. The development of the liquid killing assay was a very innovative aspect of the manuscript. The results shown by the author mostly support their conclusions. Overall, some comments are listed below, which should be considered:

Comments
1: K-12 MG1655 should have been included as a control in the elegans liquid and solid killing assays. Including this control is key to comparing the pathogenic potentials of the E. coli isolates. This is particularly necessary to understand the contribution of iron acquisition systems between different E. coli strains because K12 does not have a heme uptake system.
2: The observation that iron acquisition is required for virulence is not unexpected. However, it seems important to answer one key question. Do the iron acquisition genes contribute to virulence of UTI isolates and fecal isolates to similar amount? Fig S1 shows that relative pathogenicity is lower in fecal isolates, however many of these fecal isolates still have the iron acquisition genes (Table 1). It would be very interesting to see if deletion of the iron acquisition genes in one of these fecal isolates reduces virulence in elegans? The requirement of iron acquisition is likely conserved across most strains since iron is an essential nutrient.
3: The urosepsis isolates exhibit lower pathogenicity than the other UTI isolates. Were any obvious differences in virulence factors or growth characteristics between these strains?

Reply to the comments.
Thank you so much for your critical reading. The comments are very helpful to improve the manuscript.

Answer to comment 1.
As described in line 83, MG1655 is included in the studied strains. Relative pathogenicity of MG1655 in the liquid assay is 1.21, which is very close to the mean value (1.17). In addition, that for UIT89 was 1.75. It has been described in the figure legend (line188). The MG1655 for solid killing assay is described below.

Answer to comment 2.
Many genes for iron acquisition are identified in E. coli, and E. coli strains even commensal have multiple genes. UTI89 (the strain used as UPEC in solid killing assay) has more than the three genes, such as efe, sit, fec, feo, fet systems, and so on. To elucidate involvement of iron acquisition in pathogenicity in C. elegans, we are constructing mutants for the iron acquisition in UIT89 and MG1655, and going to challenge the animal experiment. We would like to show the data in our future work.

Answer to comment 3.
As shown in Fig S2, strains associated with urosepsis have larger number of virulence factors than fecal isolates. However, we haven't found any significance for virulence factors between urosepsis and the other pathogenic strains. Alternatively, the number of strains for urosepsis, which is 13 (Table S3), is probably not good enough to see the significance.