The impact of global warming on the signature virulence gene, thermolabile hemolysin, of Vibrio parahaemolyticus

ABSTRACT Global warming is increasing human exposure to pathogens and has already had an impact on human health. Vibrio parahaemolyticus, a major pathogen causing foodborne illness, accumulates in numerous aquatic organisms and can be affected by environmental stressors such as increased replication errors or DNA damage resulting in point mutations. This could lead to an elevation in the mutation rate, which influences the expansion of Vibrio spp. habitats and the spread of associated diseases. In this study, a total of 241 strains were isolated from aquatic products imported or exported through China Customs between 2005 and 2010. The whole genomes of those strains were sequenced, revealing a highly significant level of genetic diversity. Our analysis identified 27 new sequence types (STs) ranging from ST2950 to ST2976. The global temperature trend since 1950 affects the thermolabile hemolysin gene (tlh) found in all Vibrio parahaemolyticus leading to mutant sites exhibiting similar trends as temperatures rise; seven high-frequency mutation hotspots (A180G, T552G, G657T, T858C, C1062T, A1137G, and T1179C) were identified along with two clinically specific sites (T259C and A951T) that may indicate adaptation over time due to climate change, leading to increased virulence potential for this bacterial species. These results provide insight into the genetics of Vibrio parahaemolyticus and provide a reference for subsequent research, identification, and monitoring efforts related to its spread. IMPORTANCE In this study, Vibrio parahaemolyticus strains were collected from a large number of aquatic products globally and found that temperature has an impact on the virulence of these bacteria. As global temperatures rise, mutations in a gene marker called thermolabile hemolysin (tlh) also increase. This suggests that environmental isolates adapt to the warming environment and become more pathogenic. The findings can help in developing tools to analyze and monitor these bacteria as well as assess any link between climate change and vibrio-associated diseases, which could be used for forecasting outbreaks associated with them.


Results
-Figure 3 should probably be in supplemental material -Figure 4 is difficult to read as the panels are dense with small font text.Consider placing panel A above panel B so that both panels can be larger in the published document.
-Figures 5 and 6 need a title for the y-axis.Also, consider merging these two figures into one with an A and B panel.
-For Figure 7, consider placing panel A above panel B so that both panels can be larger in the published document.
-Consider merging Figures 8 and 9  -Tables 1and 2 are far too large to be included in the manuscript body.They need to go into supplemental material.-Change the orientation of the current Table 3 to vertical so that it can all fit on a single page in the body of the manuscript.Also, some of the white space can be removed.
-line 136: change "were analyzed" to "was used to analyze" -line 149: This information is not presented in Figure 3. How was it determined that isolates were dispersed and non-regionally, and no host was related?-line 200: Reference Figure 6 here?-lines 202-204: Include references for these statements -line 681: COGs were defined in the first sentence of this legend for Fig 2

Discussion
-line 282: It is unclear what the end of this sentence means -line 295: "mutated" rather than "mutant"?

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Review comments on "The impact of global warming on the signature virulence gene thermolabile hemolysin gene of Vibrio parahaemolyticus" The methods of the paper are robust, and the tie-in to global warming is prescient.Minor misspelling or grammar corrections are required, as well as clarifications on methods or results.
Line 68: Change "aggregated" to "aggravated".We appreciate the reviewers for their positive response and interest in our work.We are sincerely grateful to the reviewers for their invaluable feedback, which helps us improve our manuscript entitled "The impact of global warming on the signature virulence gene thermolabile hemolysin gene of Vibrio parahaemolyticus".In response to the reviewers' comments, the manuscript, in its current form, has been edited and modified extensively and proof-read.
Our point-by-point response to the comments is given below.

Response to reviewer #1:
The methods of the paper are robust, and the tie-in to global warming is prescient.
Minor misspelling or grammar corrections are required, as well as clarifications on methods or results.
Thank you for the correction.The word is changed as suggested (Page 4, line 68 of the maked up manuscript (pdf version)).

Line 89: Clarify why risk of infection is indirect?
Thank you for pointing out the misunderstanding.We would like to clarify that our intention was to highlight the fact that while Vibrio parahaemolyticus thrives in warmer waters, there have been reports of outbreaks occurring in colder regions, such as Alaska.
It appears that rising temperatures have been implicated in one of the largest known V. parahaemolyticus outbreaks.However, it is important to note that temperature alone does not directly cause infection or result in clinical symptoms.We have revised the manuscript to clarify our point as follows: "Despite V. parahaemolyticus thriving in warmer waters, outbreaks of V. parahaemolyticus have been reported in colder regions, including Alaska, where rising temperature plays an important role in contributing to (Reference 12 in the revised manuscript).

Line 197: Remove italics from "strains"
Thank you for pointing out the error.The word is modified as suggested (Page 10, line 199 of the maked up manuscript (pdf version)).

Line 226-228: Clarify if other unlabeled high-frequency mutation hotspots in
Figure 10 not chosen for further analysis were initially considered, but had a protein-protein binding probability of less than 50%.
Thank you for pointing out the misunderstanding.Initially, we identified the mutation hotspots with high frequency and subsequently conducted additional analyses.To provide further clarity, we made the following modifications: "We performed a comprehensive analysis of all tlh full-length sequences (1,257 bp), including 778 clinical strains, 739 environmental strains, and 241 strains collected in our study.These sequences were compared with the tlh gene of the reference strain (RIMD2210633).
Through this analysis, we identify a total of 48 SNPs.We calculated the frequency of mutations at each site and determined sites with a mutation frequency higher than 50% as high-frequency mutation points.We found 7 high-frequency mutation hotspots: A180G, T552G, G657T, T858C, C1062T, A1137G, and T1179C (Fig. 7).Additionally, we identified 2 specific sites, T259C and A951T, which were exclusively present in the clinical isolate (highlighted in red in Fig. 7).These mutations exhibited a high frequency of occurrence exceeding 15% among clinical strains.To further analyze the potential significance of these mutations, we calculated the protein-protein binding probability for the identified mutation sites (Table S5).Our analysis revealed substantial variation in the ability of different sites to engage in protein-protein interactions, with probabilities ranging from 0.8% to 59.3% (Table S5).Notably, 2 mutation sites were predicted to be associated with a high probability of protein-protein binding sites (PPBS) (Table S5).These findings suggest a potential link between these mutations and pathogenicity through protein-protein interactions.However, experimental validation 9. S1: Does this analysis take an increase in surveillance or strain collection into account?
Yes, our analysis takes into account an increase in surveillance and strain collection over time.We collected data from the NCBI database, which includes records from 1950 to 2020.We then examined the relationship between the number of SNPs in the tlh and temperature and visualized this relationship using a generalized linear model.

Response to reviewer #2:
The manuscript aims to provide further genomic understanding of Vibrio parahaemolyticus and to specifically develop understanding of diversity in its virulence factors and how the mutation potential of some of the key factors correlates with ambient temperatures.
The general approach of the study was to sequence the genomes of 241 strains that were isolated from aquatic products imported or exported from China Customs between 2005-2010.Specific emphasis was placed on variation in the thermolabile hemolysin gene (tlh) gene in the context of temperature.
The conclusion of the study was that the tlh gene is likely to be an important marker of the pathogenic potential of environmental isolates of V. parahaemolyticus as the environment continues to warm.

General Comments:
The manuscript was well-written.The arguments were easy to follow, well-reasoned, and supported by the data.I have relatively minor comments, mostly focusing on improving the quality of the figures and tables.
Minor comments: -Some further grammatical editing is needed -Write out all abbreviations at first appearance in the manuscript We sincerely appreciate your support of our paper.We have addressed the grammar-related issue and expanded the abbreviations by providing their full forms upon their first mention in the modified manuscript.

line 325: "HiSeq"
Thank you for the correction.We have made the correction, changing it to "Hiseq", as suggested (Pages 16-17, lines 340-341 of the maked up manuscript (pdf version))

line 345: Reword this sentence fragment to improve clarity
Thank you for your suggestion.We change the sentence into "To gain insight into the clonal relationships among the analyzed V. parahaemolyticus isolates, we conducted an analysis of their STs using goeBURST (v1.2.1) available at http://goeburst.phyloviz.net.
The analysis focused on identifying isolates belonging to the same clonal complex, which was determined by the presence of a minimum of five out of seven identical STs." (Pages 17-18, lines 359-363 of the maked up manuscript (pdf version))

line 349: Reword this sentence to improve clarity
Thank you for your suggestion.We modify the sentence into "Whole-genome sequences (WGS) were obtained from 241 V. parahaemolyticus strains (Table S2), along with 3 additional strains (VppAsia RIMD221063, VppUS2 FDA-R31, and VppX 10329 representing the Asian region, American region, and Pacific coast, respectively).
WGS-SNP analysis was performed using KSNP (v3.1) with a kmer length of 20 to analyze these sequences.The resulting data were utilized to construct maximum likelihood phylogenetic trees using iqtree (v2.0.3).Model Finder was employed with 1000 iterations to perform optimal model selection for this analysis."(Page 18, lines 365-372 of the maked up manuscript (pdf version))

line 377: "was completed using SWISS-MODEL"
Thank you for pointing out the error.We change the sentence into "The prediction of tlh protein structure was completed using SWISS-MODEL (https://swissmodel.expasy.org/)."(Page 19, lines 399-400 of the maked up manuscript (pdf version))

Figure 3 should probably be in supplemental material
Thank you for your advice.We have included it in the supplemental material, named Thank you for your advice.We have added titles to the y-axis in both Figures 5 and 6 to enhance clarity and comprehension.Furthermore, we have merged these two figures into a single figure, labeled as Figure 4 in the revised manuscript.
12. For Figure 7, consider placing panel A above panel B so that both panels can be larger in the published document.
Thank you for your suggestion.We have made modifications to Figure 7  14.Tables 1 and 2 are far too large to be included in the manuscript body.They need to go into supplemental material.
Thank you for your advice.In consideration of the size of Tables 1 and 2, we have relocated them to the supplemental material.They can now be found in Table S2 and Table S4 of the the revised manuscript, respectively.
15. Change the orientation of the current Table 3 to vertical so that it can all fit on a single page in the body of the manuscript.Also, some of the white space can be removed.
Thank you for your suggestion.We made an effort to change the table orientation to a vertical layout.However, despite our attempts, we encountered difficulties in achieving the aim to fit the entire table on a single page in the body of the manuscript.
Nevertheless, we have implemented alternative modifications to address this concern effectively: 1.In order to enhance clarity and efficiency, we have implemented abbreviations in the table header.Specifically, we explored abbreviations for transition (TS), transversion (TV), non-synonymous mutations (NSM), and synonymous mutations (SM).
2. To optimize space utilization, we have minimized unnecessary white space within the table, ensuring a more compact presentation.
We believe with these adjustments, we have effectively resolved the issue of accommodating the table within a single page in the revised manuscript.It can now be found in Table 1.17. line 149: This information is not presented in Figure 3. How was it determined that isolates were dispersed and non-regionally, and no host was related?
We agree with your point regarding the absence of information on isolates dispersed and host-relatedness.We have revised the sentence as follows: "The analysis of the strains collected in this study revealed a dispersed distribution without regional Thank you for your advice.We modify the sentence into "And as predicted by ScanNet (http://bioinfo3d.cs.tau.ac.il/ScanNet/),T259C and A951T may be particularly important for bacterial binding to the receptor since their protein-protein binding can be up to about 50%." (Pages 14-15, lines 299-301 of the maked up manuscript (pdf into one Figure with an A and B panel. these outbreaks."(Page 5, lines 87-90 of the maked up manuscript (pdf version)).To enhance accuracy, we have also updated the reference in question.Reference 12 now provides more precise information: "McLaughlin JB, DePaola A, Bopp CA, Martinek KA, Napolilli NP, Allison CG, Murray SL, Thompson EC, Bird MM, Middaugh JP. 2005.Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters.N Engl J Med. 353(14):1463-70.https://doi.org/10.1056/NEJMoa051594." is necessary to confirm these predictions."(Pages 11-12, lines 225-242 of the maked up manuscript (pdf version)).5. Line 324: We used, or, Trimmomatic was used Thank you for your suggestion.We change the sentence into "Trimmomatic was used to intercept adapters from sequencing reads generated by Illumina Hiseq" as suggested (Pages 16-17, lines 340-341 of the maked up manuscript (pdf version)).6. Line 327: We used Prokka, or, Prokka was used.Thank you for your suggestion.We change the sentence into "We used Prokka (v1.13.3) to annotate the genome and Prodigal (v2.6.3) to identify open-reading frames" (Page 17, lines 343-344 of the maked up manuscript (pdf version)).7. Line 295: Remove space between Fig, to stay consistent in the paper Thank you for your suggestion.We remove the space between "Fig" as suggested.(Page 15, line 314 of the maked up manuscript (pdf version)).8. Line 673: Typo, change to Figure Legend.Thank you for the correction.The word is changed as suggested (Page 33, line 696 of the maked up manuscript (pdf version)).

1. line 32 :
Write out ST here Thanks for your suggestion.We change the sentence into "In this study, a total of 241 strains were isolated from aquatic products imported or exported through China Customs between 2005-2010.The whole genomes of those strains were sequenced, revealing a highly significant level of genetic diversity.Our analysis identified 27 new sequence types (STs) ranging from ST2950 to ST2976." as suggested (Page 2, lines 30-33 of the maked up manuscript (pdf version)) 2. line 73: Resolve the referencing issue Thanks for the correction, we have resolved it (Page 4, line 73 of the maked up manuscript (pdf version)) 3. line 324: "Trimmomatic was used ..." Thank you for your suggestion.We change the sentence into "Trimmomatic was used to intercept adapters from sequencing reads generated by Illumina Hiseq" as suggested (Pages 16-17, lines 340-341 of the maked up manuscript (pdf version)).
figure now includes an A panel and a B panel, providing improved organization and presentation of the data.
16. line 136: change "were analyzed" to "was used to analyze"Thank you for pointing out the error.We change the sentence into "Gonzalez-Escalona et al. described a multilocus sequence typing (MLST) scheme for V. parahaemolyticus, which relies on 7 housekeeping loci(recA, dnaE, gyrB, dtdS, pntA, pyrC, and tnaA)    spanning both chromosomes of V. parahaemolyticus.This protocol was used to analyze the 241 isolates screened in this study."(Page 7, lines 133-136 of the maked up manuscript (pdf version)).
clustering." (Page 7, lines 148-150 of the maked up manuscript (pdf version)).18. line 200: Reference Figure 6 here?Thank you for your comment.We have included the reference to the figure in question and made adjustments accordingly.As a result, the designated reference figure has been updated from Figure 6 to Fig.4B of the maked up manuscript (pdf version).(Page 10, line 202 of the maked up manuscript (pdf version)).19.lines 202-204: Include references for these statements Thank you for your comment.To address the identified issue, we have made the necessary correction and additionally included reference 29 of the maked up manuscript (pdf version) "Mora C, McKenzie T, Gaw IM, Dean JM, von Hammerstein H, Knudson TA, Setter RO, Smith CZ, Webster KM, Patz JA, Franklin EC. 2022.Over half of known human pathogenic diseases can be aggravated by climate change.Nat Clim Chang.12(9):869-875.https://doi.org/10.1038/s41558-022-01426-1." as requested (Page 10, line 206 of the maked up manuscript (pdf version)).20.line 681: COGs were defined in the first sentence of this legend for Fig 2 Thank you for your advice.In response to your advice, we have removed the sentence "COGs indicate clusters of orthologous groups."from the legend for Figure 2 of the maked up manuscript (pdf version).(Page 33, lines 700-703 of the maked up manuscript (pdf version)).21. line 282: It is unclear what the end of this sentence means.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred