Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission

Reviewer #1 (Public Review):

In this study, Sapiro et al sought to develop technology for a transcriptomic analysis of B. burgdorferi directly from infected ticks. The methodology has exciting implications to better understand pathogen RNA profiles during specific infection timepoints, even beyond the Lyme spirochete. The authors demonstrate successful sequencing of the B. burgdorferi transcriptome from ticks and perform mass spectrometry to identify possible tick proteins that interact with B. burgdorferi. This technology and first dataset will be useful for the field. The study is limited in that no transcripts/proteins are followed-up by additional experiments and no biological interactions/infectious-processes are investigated.

Critiques and Questions:

This study largely develops a method and is a resource article. This should be more directly stated in the abstract/introduction.

Details of the infection experiment are currently unclear and more information in the results section is warranted. State the species of tick and life-stage (larval vs nymphal ticks) used for experiments. For RNA-seq, are mice are infected and ticks are naïve or are ticks infected and transmitting Borrelia to uninfected mice?

What is the limit of detection for this protocol? Experimental data should be provided about the number of B. burgdorferi required to perform this approach.

More information regarding RNA-seq coverage is required. Line 147-148 "read coverage was sufficient"; what defines sufficient? Browser images of RNA-seq data across different genes would be useful to visualize the read coverage per gene. What is the distribution of reads among tRNAs, mRNAs, UTRs, and sRNAs?

My lab group was excited about the data generated from this paper. Therefore, we downloaded the raw RNA-seq data from GEO and ran it through our RNA-seq computational pipeline. Our QC analysis revealed that day 4 samples have a different GC% pattern and that a high percentage of E. coli sequences were detected. This should be further investigated and addressed in the paper: Are other bacteria being enriched by this method? Why would this be unique to day 4 samples? Does this affect data interpretation?

Comprehensive data comparisons of this study and others are warranted. While the authors note examples of known differentially expressed genes (like lines 235-241), how does this global study compare to other global approaches? Are new expression patterns emerging with this RNA-seq approach compared to other methods? What differences emerged from day 1 to day 4 ticks compared to differences observed in unfed to fed ticks or fed ticks to DMC experiments? Directly compare to the following studies (PMID: 11830671; PMID: 25425211; PMID: 36649080).

Details about the categorization of gene functions should be further described. The authors use functional analysis from Drechtrah et al., 2015, but that study also lacks details of how that annotation file was generated. Here, the authors have seemed to supplement the Drechtrah et al., 2015 list with bacteriophage and lipoprotein predictions - which are the same categories they focus their findings. Have they introduced a bias to these functional groups? While it can be noted that many lipoproteins are upregulated (or comment on specific genes classes), there are even more "unknown" proteins upregulated. I argue that not much can be inferred from functional analysis given the current annotation of the B. burgdorferi genome.

Reviewer #2 (Public Review):

This manuscript documents the study of the transcriptome of Borrelia burgdorferi at 1, 2, 3 and 4 days post-feeding in nymphs of Ixodes scapularis. The authors use antibody-based pull-downs to separate bacteria from tick and mouse cells to perform an enrichment. The data presented support that the transcriptome of B. burgdorferi changes over time in the tick. This work is important as until now, only limited information on specific genes had been collected. This is the first study of its kind and is valuable for the field.

The manuscript is overall well written and easy to follow. The data are compelling and support the conclusions.