Antigenic variation is caused by long plasmid segment conversion in a hard tick-borne relapsing fever Borrelia miyamotoi

PLOS Pathogens

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David Skurnik

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PLOS Pathogens

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript by Waugh et al. addresses the effects of Treponema pallidum exposure on the gene transcript levels and other cellular properties in immortalized human cortical endothelial cell cultures. While the manuscript contains a lot of data, the presentation of the data and its interpretation is overly complex, making it challenging for the reader to understand the results and verify the conclusions by examining the data themselves. Suggestions are made to try to improve the presentation and other aspects.

Reviewer #2: (No Response)

Reviewer #3: The authors provide a comprehensive analysis detailing the comparative repertoires and arrangements of vmp archival gene cassettes from the genome sequences of Japanese B. miyamotoi strains. The authors use this information to demonstrate antigenic switching from reisolates obtained from needle infected mice. Significantly, the authors provide new information showing that gene cassette switching involves replacing the expression locus and downstream cassettes with a long segment from another archival plasmid that is a different mechanism from soft tick relapsing fever borrelia variation. This report is an important advancement for analyzing the mechanisms of antigenic variation in this hard tick relapsing fever spirochete that was initiated in the seminal studies by the Hovius and Gilmore laboratories.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The Summary does not address the protein expression or cellular morphology results in Figs. 3 and 7.

2. The final paragraph of the Introduction contains information that should instead be described in the Results and Discussion.

3, The manuscript would benefit from a diagram figure showing the experimental procedure and time course. Important elements of the study (T. pallidum source used, number of T. pallidum, IEC control) are not described adequately. It is unclear why a medium control was not also included in the transcriptome analysis.

4. The Results contains an excessive amount of verbiage describing the transcript abundance trends, and refers to massive supplemental tables where it is nearly impossible for the reader to find the related data. It would be better to include additional figures in which data for some of the most important regulatory networks are extracted and clearly shown; the detailed text descriptions should be removed or reduced considerably. As an example, much emphasis is placed on increased Snai1 transcript levels; however, that information is buried in Table S1 and not easily accessible. A figure showing its expression and that of genes in its regulon would be helpful.

5. There seems to be a disconnect between the transcript results, in which significant changes are generally seen at 12 and 24 hours, and the cellular changes shown in Fig. 7, which are maximal at 1 hour and dissipate by 2 hours. This result seems to indicate that signal transduction and cellular changes precede the transcript level changes. This point needs to be better described in the Results and incorporated into the Discussion.

6. The term ‘expression’ is frequently used to describe transcript levels, which of course is not equivalent to either transcription (or degradation) rates or protein expression levels. This aspect is further amplified by a tendency to overinterpret the results. The manuscript should be revised fairly extensively to correct these issues.

7. l. 614. It is stated here that either T. pallidum preparations from infected rabbits or in vitro cultured T. pallidum were utilized to inoculate the hCMEC/d3 cell cultures. This is an important difference, in that the rabbit tissue extract would be expected to contain inflammatory products produced in response to the T. pallidum infection. Also, the RNA-seq results presented appear to be from a single experiment with 5 replicates per time point and VTP or IEC condition. Therefore, it is important to clearly indicate which preparation was used in each experiment.

8. l. 632. hCMEC/d3 is an immortalized cell line, not primary or secondary cultures of human cerebral endothelial cells. This aspect should be stated clearly here, in the abstract, and in the results section. Also, the possibility that the observed transcriptional responses of these immortalized cells may differ from those of cerebral endothelial cells that have not been immortalized should be addressed in the Discussion.

9. L. 640-644. It is first stated that the hCMEC/d3 cells were lysed by the RNAprotect treatment, and later indicated that the suspensions were centrifuged to pellet the cells. Please clarify. Also, perhaps it should be said that motility was used as a measure of viability of T. pallidum (rather than “observed for motility and viability”); it is unclear how viability would be examined by means other than motility.

10. The T. pallidum viability assessment results should be reported in the Results section.

11. The titles and descriptions for the supplementary tables should be included within each file, along with a key of abbreviations.

Reviewer #2: (No Response)

Reviewer #3: I have no concerns or issues regarding the data or the conclusions or interpretations thereof. The amount and presentation of the data is impressive, although some areas could be slightly modified to guide the readership better (see suggestions below). Overall, an important body of work that should be of interest to the research community.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: See above

Reviewer #2: (No Response)

Reviewer #3: Minor comments for consideration:

1. Paragraph beginning with line 127; would be helpful to include the GenBank accession number here for the genome sequence of the strains. The accession numbers given in the supplementary tables were difficult to find on the NCBI search (at least for me).

2. Line 247; this was an area of confusion regarding which supplementary Tables to check. Suggest referencing Table S3 on line 248 instead of having both S2 and S3 on line 252. Also suggest a separate column on Table S3 stating the original expressed vmp gene.

3. Figure 5; suggest arrows to indicate the bands with the expression plasmid.

4. Lines 400-401; add the reference Hoornstra et al., iScience 2024. Genome sequences of 21 isolates were documented.

5. Line 464; Is there a reference that defines the ingredients of BSK-M? I’m not familiar with this media or how if differs from BSK-II, BSK-H, or BSK-R. If not, consider adding the specifics here.

6. Line 522; “Two microliters of extracted DNA…”, replace microliters with micrograms to show quantity used.

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Reproducibility:

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Dear Prof Takano,

We are pleased to inform you that your manuscript 'Antigenic variation is caused by long plasmid segment conversion in a hard tick-borne relapsing fever Borrelia miyamotoi' has been provisionally accepted for publication in PLOS Pathogens.

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Michael Malim

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Reviewer Comments (if any, and for reference):