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- W4384339675 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Methods Data availability References Peer review Author response Article and author information Metrics Abstract Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle. eLife assessment In this Tools and Resources article, the authors overcome the challenge of low Borrelia burgdorferi numbers during infection for analyses such as RNA-sequencing or mass spectrometry. They do so by physically enriching for spirochetes, which is important, as it provides technical advances for the study of global transcriptomic changes of B. burgdorferi during tick feeding, helping to build on the knowledge already collected by the field. The evidence presented is compelling, and the strategy described here could benefit researchers in the field and possibly also support broader applications. https://doi.org/10.7554/eLife.86636.3.sa0 About eLife assessments Introduction Vector-borne microbial pathogens are transmitted by the bite of arthropods and have evolved sophisticated ways to adapt to vastly different environments as they move between vector and host. Uncovering their adaptive mechanisms can not only open avenues for disrupting pathogen transmission but also provide fundamental insights into vector physiology and microbial symbioses (Shaw and Catteruccia, 2019). Lyme disease, the most reported vector-borne disease in North America, is caused by the bacterial pathogen Borrelia burgdorferi (Bb) (Rosenberg et al., 2018; Steere et al., 2016). Its primary vector, the blacklegged tick Ixodes scapularis, acquires and transmits Bb through two separate multi-day bloodmeals, one in which Bb is acquired from an infected vertebrate host and a second, during the subsequent life stage, in which Bb is transmitted to a new host (Tilly et al., 2008). During the transmission bloodmeal, Bb proliferates in the tick midgut before a subset of these cells disseminate to the salivary glands (Dunham-Ems et al., 2009). Bb is deposited into the new host via the tick saliva extruded into the bite site (Ribeiro et al., 1987). Given the prolonged nature of I. scapularis feeding, the high specificity of vector-pathogen relationships, and the complicated array of events needed for successful Bb transmission, the tick bloodmeal provides an opportune intervention point for preventing pathogen spread. However, we do not currently have a clear understanding of the molecular mechanisms involved in this process. Bb must adapt to dramatically different environments as it cycles from tick to vertebrate host, and understanding the genes involved in this process will enable the identification of key interactions to target to prevent transmission. When an infected tick feeds, Bb responds to bloodmeal-induced environmental changes and undergoes cellular modifications driven by key transcriptional circuits, including the RpoN/RpoS sigma factor cascade and the Hk1/Rrp1 two-component system (Radolf et al., 2012). In vitro analyses of Bb cells cultured in tick- or mammal-like growth conditions have pointed to additional genetic determinants of tick-borne transmission by revealing widespread transcriptome remodeling during host switching (reviewed in Samuels et al., 2021). However, it is not fully clear how these in vitro expression changes correspond to complex in vivo changes over the course of a transmission bloodmeal. Capturing comprehensive, longitudinal data on Bb gene expression from inside its vector has been hampered by technical challenges due to the dynamic nature of the bloodmeal and the general low abundance of bacterial cells relative to the tick (Samuels et al., 2021). Some progress has been made in making transcriptome-wide measurements from the tick. Bb sequence enrichment coupled with microarrays identified large scale changes in Bb gene expression between the first and second tick bloodmeals (Iyer et al., 2015). More recently, enriching Bb sequences from infected tick RNA-seq libraries through TBDCapSeq has provided clearer resolution into differences in Bb gene expression as it cycles between the fed tick and mammalian host (Grassmann et al., 2023). Still, we have limited temporal resolution into the molecular transitions that happen across key steps of tick feeding. This problem necessitates novel approaches to capture the transcriptomic changes of Bb within the natural tick environment. While the full landscape of Bb transmission determinants is not yet known, we do have a growing knowledge of functional processes that are critical during the tick stage, such as motility, metabolism, and immune evasion (Kurokawa et al., 2020; Phelan et al., 2019). These functions often rely on the unique protein-rich Bb outer surface. Notably, several specific tick–Bb protein–protein interactions are important for Bb survival, migration, or transmission to the next host. Bb encodes an extensive outer surface protein (Osp) family with members that are differentially expressed during host switching. One of these proteins, OspA, binds a tick cell surface protein, tick receptor for OspA (TROSPA), which is required for successful Bb colonization of the tick midgut during the first acquisition bloodmeal (Pal et al., 2004). Several other proteins have also been linked to Bb migration within the tick (Pal et al., 2021). For example, BBE31 binds a tick protein TRE31, and disruption of this interaction decreases the number of Bb cells that successfully migrate from the tick gut to salivary glands (Zhang et al., 2011). However, these interactions alone are not sufficient to block Bb growth or migration in ticks, suggesting there are likely additional molecular factors from Bb and ticks at play during tick-borne transmission. To provide a more comprehensive set of Bb determinants driving tick-borne transmission of this important human pathogen, we developed a novel sequencing-based strategy for ex vivo transcriptomic profiling of Bb populations within infected nymphal I. scapularis ticks as they transmit Bb to a mouse host. We used this method to longitudinally map genome-wide Bb expression changes for bacterial cells isolated from ticks during the transmission bloodmeal from 1 to 4 days after attachment. We identified 192 highly differentially expressed genes, including genes previously implicated in Bb transmission as well as many others. Genes upregulated during tick transmission included many outer surface lipoproteins, suggesting Bb dramatically remodels its cell envelope as it migrates through the tick. Mass spectrometry analyses revealed dramatic changes in the tick environment over feeding, identifying new potential determinants of a more extensive and diverse set of tick–microbe molecular interactions than previously appreciated. The Bb enrichment method and resulting datasets serve as a community resource to facilitate further investigations into the key determinants of Bb transmission. Results A two-step enrichment process facilitates robust transcriptional profiling of Bb during the tick bloodmeal To gain a more comprehensive understanding of Bb gene expression throughout the tick phase of the transmission cycle, we developed an experimental approach to characterize the Bb transcriptome of spirochetes isolated from nymphal I. scapularis ticks during a days-long bloodmeal in which Bb is transmitted to a vertebrate host. We aimed to establish a longitudinal transcriptional profile encompassing key pathogen transmission events each day of feeding after ticks attached to their mouse bloodmeal hosts (Figure 1A). We fed Bb-infected nymphal ticks on naive mice and collected the feeding ticks at daily intervals after the start of feeding until 4 days after attachment, at which time the ticks had fully engorged and detached from the mice. The major bottleneck for such an RNA sequencing (RNA-seq) approach is capturing sufficient quantities of Bb transcripts from complex multi-organism samples in which pathogen transcripts represent a very small minority. Our initial attempts to uncover Bb mRNA by simply removing tick mRNA with polyA-depletion and removing tick rRNA sequences using Depletion of Abundant Sequences by Hybridization (DASH; Dynerman et al., 2020; Gu et al., 2016) were unsuccessful. This approach resulted in an average of only 0.09% of RNA-seq reads mapping to Bb mRNA – approximately 10-fold less than we estimated would be needed to feasibly obtain robust transcriptome-wide differential gene expression analysis (Haas et al., 2012). Figure 1 with 2 supplements see all Download asset Open asset A two-step enrichment process facilitates robust transcriptional profiling of Bb during the tick bloodmeal. (A) Schematic of Bb during nymphal I. scapularis feeding. Bb in the nymphal tick midgut respond to the nutrient-rich bloodmeal by multiplying and changing their transcriptional state (Ouyang et al., 2012; de Silva and Fikrig, 1995). At the same time, the tick gut undergoes numerous changes to digest the bloodmeal (Caimano et al., 2015; Sonenshine and Anderson, 2014). After two to three days of feeding, a small number of Bb leave the midgut and enter the salivary glands (blue), while the majority are left behind in the gut after engorgement (Dunham-Ems et al., 2009). (B) Schematic of Bb enrichment process from feeding ticks. Whole ticks are dissociated, αBb antibodies are added to lysates, and antibodies and Bb are captured magnetically. RNA is extracted and RNA-seq libraries are prepared. DASH is then used to remove rRNA before sequencing. This process increases Bb reads in the resulting sequencing data. (C) RT-qPCR results showing the percentage of Bb flaB and I. scapularis gapdh RNA in the enriched versus depleted fractions after the enrichment process. Data come from 4 replicates each from day 2, day 3, and day 4, mean +/-SE. ****p-value <0.0001, paired t test. Nearly all Bb flaB RNA was found in the enriched fraction. (D) The percentage of reads mapping to rRNA before and after DASH. n=4. Data are shown as mean +/-SD. ****p-value <0.0001, paired t test. rRNA reads are drastically reduced after DASH. (E) The percentage of reads in RNA-seq libraries mapping to Bb. Bb mRNA reads make up a larger proportion of libraries than without enrichment. n=4. Data are shown as mean +/-SD, see Figure 1—source data 1. (F) The number of reads in millions (M) mapped to Bb for each day. n=4. Data are shown as mean +/-SD. An average of 4.3 million reads per sample mapped to Bb genes, covering 92% of annotated genes with at least 10 reads. Figure 1—source data 1 Overview of mapping statistics from 16 Bb sequencing samples. https://cdn.elifesciences.org/articles/86636/elife-86636-fig1-data1-v1.xlsx Download elife-86636-fig1-data1-v1.xlsx To dramatically increase Bb transcript representation in our libraries, we physically enriched Bb cells from tick lysates prior to library preparation by adding an initial step of immunomagnetic separation (Figure 1B). We took advantage of a commercial antibody previously generated against whole Bb cells (αBb, RRID: AB_1016668). By western blot analysis, we confirmed that αBb specifically recognized several Bb proteins, including surface protein OspA (Figure 1—figure supplement 1A), which is highly prevalent on the Bb surface in the tick (Ohnishi et al., 2001). In addition, immunofluorescence microscopy with αBb showed clear recognition of Bb cells from within the tick at each day of feeding (Figure 1—figure supplement 1B). After collecting infected nymphal ticks from mice one, two, three, and four days post-attachment, we used αBb and magnetic beads to enrich Bb cells from the tick material in the lysates. We tracked relative Bb enrichment through RT-qPCR of Bb flaB RNA and tick gapdh RNA in the separated samples. Measuring Bb flaB RNA from both Bb-enriched samples and their matched Bb-depleted fractions, we found over 95% of total Bb flaB RNA was present in enriched fractions (Figure 1C), suggesting our approach captured the vast majority of Bb transcripts from the tick. Total RNA recovered from the enrichment process was used to create RNA-seq libraries that subsequently underwent depletion of highly expressed rRNA sequences from tick, mouse, and Bb using DASH, which targets unwanted sequences for degradation by Cas9 (Gu et al., 2016; Ring et al., 2022). DASH reduced unwanted sequences from 94% to 9% of our total libraries, greatly increasing the relative abundance of Bb transcripts (Figure 1D). For resulting libraries generated across feeding, between 0.6% and 3.4% of reads mapped to annotated Bb genic regions (Figure 1E), which translated to an average of 4.3 million Bb genic reads per sample (Figure 1F and Figure 1—source data 1). As expected, for samples pulled from the mice one, two, and three days after attachment, the majority of the remaining sequencing reads mapped to the I. scapularis genome (72–83%; Figure 1—source data 1). On day 4, when ticks were fully engorged and were recovered from mouse cages rather than pulled from the mice, we found that fewer reads mapped to the I. scapularis genome (24–44%). Only a small percentage of reads from all samples mapped to the host Mus musculus genome (1–3%). To identify the source of the remaining reads in the day 4 samples, we ran the data through a publicly available computational pipeline that identifies microbes in sequencing datasets, CZ ID (Kalantar et al., 2020). This analysis led us to discover that a large percentage of day 4 reads mapped to bacterial species Pseudomonas fulva (41–64%) (Figure 1—source data 1), which may have been present in our mouse cages. While these samples had a lower percentage of reads that mapped to Bb, broad transcriptome coverage was still obtained by increasing total sequencing depth. Across all samples from the 4 days, at least 10 reads mapped to 92% of annotated Bb protein coding genes and pseudogenes. The median number of reads per gene in each sample varied from 338 to 1167 reads (Figure 1—source data 1). This coverage was sufficient for statistically significant downstream differential expression analyses for the vast majority of Bb genes. To evaluate whether our approach introduced any major artifacts in Bb expression, we sequenced and compared RNA-seq libraries from in vitro cultured Bb cells before and after immunomagnetic enrichment. We found minimal expression differences (29 genes with p<0.05, fold changes between 0.83 and 1.12; Figure 1—figure supplement 2 and Figure 1—figure supplement 2—source data 1), suggesting experimental enrichment did not significantly alter global transcriptome profiles for Bb. Thus, our enrichment approach enabled genome-wide analysis of Bb population-level expression changes that occur within the feeding nymph as Bb is transmitted to the host. Global ex vivo profiling of Bb reveals extent and kinetics of transcriptional changes To provide a broad overview of Bb expression changes in the tick during the nymphal I. scapularis transmission bloodmeal, we performed principal component analysis (PCA) on the Bb transcriptome data from one, two, three, and four days after attachment (n=4). We reasoned that if many longitudinal expression changes were occurring across Bb populations, we would observe greater data variability between time points than between biological replicates. Indeed, we found replicates from each day grouped together, whereas distinct time points were largely non-overlapping. The first principal component, which explained 64% of the variance in our data, correlated well with day of feeding (Figure 2A). The global pattern suggested that Bb gene expression changes generally trended in the same direction over the course of feeding with the most dramatic differences between flanking timepoints on day 1 and day 4. Figure 2 with 2 supplements see all Download asset Open asset Global ex vivo profiling of Bb reveals extent and kinetics of transcriptional changes. (A) Principal component analysis of normalized read counts from samples from across feeding, see Figure 2—source data 1. PC1 correlates strongly with day of feeding. (B) Schematic depicting how data was analyzed, as pairwise comparisons between the first day after attachment and all other days. (C–E) Volcano plots of differentially expressed genes comparing day 2 versus day 1 (C), day 3 versus day 1 (D), and day 4 versus day 1 (E). The total number of upregulated genes is shown in the top right and the number of downregulated genes is shown in the top left. Yellow dots are genes that first change expression between day 1 and day 2, red dots are genes that first change expression between day 1 and day 3, and purple dots are genes that first change expression between day 1 and day 4. Two genes with log2 fold changes >4 are shown at x=4, and five genes with -log10(padj)>60 are shown at y=60. Only genes with p-value <0.05 from Wald tests and at least a twofold change are highlighted, see Figure 2—source data 2. n=4. By day 4 of feeding, 153 genes are upregulated and 33 genes are downregulated from day 1 baseline levels. Figure 2—source data 1 DESeq2 normalized counts for all genes across all samples. https://cdn.elifesciences.org/articles/86636/elife-86636-fig2-data1-v1.xlsx Download elife-86636-fig2-data1-v1.xlsx Figure 2—source data 2 Transcriptome-wide differential expression analysis results from Bb across tick feeding timepoints. https://cdn.elifesciences.org/articles/86636/elife-86636-fig2-data2-v1.xlsx Download elife-86636-fig2-data2-v1.xlsx Using day 1 (early attachment) as a baseline, we performed differential expression analysis for all Bb genes at subsequent time points (day 2, day 3, day 4; Figure 2B and Figure 2—source data 2). We examined changes with p-values <0.05 when adjusted for multiple hypothesis testing and fold changes above a twofold threshold (listed in Figure 3—source data 1). These analyses mirrored the global longitudinal expression pattern predicted by the PCA. The total number of differentially expressed (DE) genes when compared to day 1 increased with each subsequent timepoint to day 4. By day 4, there were 186 DE genes, including 153 upregulated and 33 downregulated (Figure 2C–E). Across all later time point comparisons to day 1, DE genes were highly overlapping and largely changed in the same directions. For example, of the DE genes that increased on day 2, 29 of 30 were still increased on day 3, and 29 of 30 were still increased on day 4. In the day 2, day 3, and day 4 comparisons to the day 1 baseline, we found 192 DE genes in total (Figure 3—source data 1). We observed some differences between gene expression patterns, such as the overall timing and kinetics of expression changes. Transcript levels for some DE genes changed suddenly over the course of feeding, while others were more gradual. To our knowledge, this is the first comprehensive report of global Bb expression changes over multiple stages of a tick feeding. To assess the integrity of our dataset, we first examined expression profiles of previously characterized targets of major transcriptional programs activated at the onset of the bloodmeal: the RpoN/RpoS sigma factor cascade and the Hk1/Rrp1 two-component system (Caimano et al., 2015; Grassmann et al., 2023). Between day 1 and day 4, the expression of rpoS increased (Figure 2—figure supplement 1A), as expected (Hübner et al., 2001), and the majority of genes activated by RpoS in the feeding tick (Grassmann et al., 2023), including canonical targets ospC and dbpA, were also significantly upregulated (79/89, p<0.05, Wald tests; Figure 2—figure supplement 1B). The majority of genes activated or repressed by Rrp1 in vitro (Caimano et al., 2015) also trended in the expected direction ex vivo between day 1 and day 4 (111/148 upregulated, 37/57 downregulated, p<0.05, Wald tests, Figure 2—figure supplement 1C). We also examined the expression trends of genes regulated by RelBbu as part of the stringent response, another major transcriptional program active in Bb in the tick during nutrient starvation (Drecktrah et al., 2015). About half of RelBbu-regulated genes changed in the direction expected if the stringent response was active during this time (129/251 upregulated, 111/226 downregulated, p<0.05, Wald tests) (Figure 2—figure supplement 1D); however, more of the genes that were twofold downregulated over feeding are regulated by RelBbu than either RpoS or Rrp1, suggesting it may play a role during this time frame. We then compared the 192 twofold DE genes in our longitudinal time course to those identified in two previous studies that measured Bb gene expression changes from culture conditions approximating the unfed tick and fed tick through modulation of temperature and/or pH (Ojaimi et al., 2003; Revel et al., 2002). 31% of the DE genes upregulated from day 1 (49/158) were more highly expressed in ‘fed tick’ conditions compared to ‘unfed tick’ conditions in one or both studies, while 24% of the DE genes downregulated from day 1 (8/24) were more highly expressed in ‘unfed tick’ conditions in one or both studies (Figure 2—figure supplement 2A and Figure 3—source data 1). The studies become more concordant when focusing on the DE genes that were upregulated on day 2, which were generally the genes that changed the most dramatically in the time course. 70% of DE genes upregulated on day 2 (21/30) were more highly expressed in ‘fed tick’ conditions in these previous studies, suggesting that the majority of the most dramatic gene expression changes we saw across feeding agree with what was observed in these previous studies. We also compared the DE genes to two studies that assessed Bb gene expression differences in fed nymphs versus dialysis membrane chambers (DMCs), which mimic Bb conditions in the mammal (Grassmann et al., 2023; Iyer et al., 2015). 63% of all upregulated DE genes (100/158) were differentially expressed between fed nymphs and DMCs in one or both studies (Figure 2—figure supplement 2B and Figure 3—source data 1). The genes that were more highly expressed in nymphs were most concentrated amongst the day 2 DE genes (17/30, 57%), while the genes more highly expressed in DMCs were concentrated amongst the day 3 and day 4 DE genes (55/128, 43%). These comparisons suggested that the timing and magnitude of gene expression changes during feeding may indicate whether gene expression will peak in the tick or continue rising once Bb is transmitted to the host. Through comparisons to these previous studies, we were able to verify that our data captured many expected transcriptional trends occurring during tick feeding. Nevertheless, 14% of the twofold DE genes were not previously found to change expression in these different tick-feeding contexts (Grassmann et al., 2023; Iyer et al., 2015; Ojaimi et al., 2003; Revel et al., 2002) or identified in these RNA-seq studies as dependent upon RpoS, Rrp1, or RelBbu (Caimano et al., 2015; Drecktrah et al., 2015; Grassmann et al., 2023), which are three known Bb regulatory programs active in the tick (Samuels et al., 2021). These additional genes highlight the necessity of measuring transcription in the tick environment and suggest we uncovered gene expression changes specific to the tick stage of the Bb enzootic cycle. The nature and dynamics of these changes provide insights into potential genetic determinants of Bb survival, proliferation, and dissemination in the tick during transmission. Bb genes upregulated during feeding are found predominantly on plasmids Bb has a complex, highly fragmented genome (Barbour, 1988; Figure 3A), including numerous plasmids that are necessary during specific stages of the enzootic cycle (Schwartz et al., 2021) suggesting they contain genes that are crucial for pathogen transmission and survival. In fact, many genes found on the plasmids have been previously shown to alter expression upon environmental changes or in different host environments (Iyer et al., 2015; Ojaimi et al., 2005; Revel et al., 2002; Tokarz et al., 2004). Thus, we reasoned that many of the 192 DE Bb genes that change expression from day 1 to any later feeding time point (Figure 3—source data 1) would reside on the plasmids, and we examined their distribution throughout the genome. Consistent with these previous reports, we found that most of the upregulated genes were located on the plasmids (143/158; 90%), while fewer were found on the chromosome (15/158; 10%; Figure 3B), which is home to the majority of metabolic and other housekeeping genes. In contrast, the majority of the downregulated genes were found on the chromosome (27/34, 79%; Figure 3C). Figure 3 Download asset Open asset Bb genes upregulated during feeding are found predominantly on plasmids. (A) Schematic of the chromosome and plasmids in the Bb B31-S9 genome. Plasmid names denote whether the plasmid is linear (lp) or circular (cp) and the length of plasmids in kilobases (kb). For example, lp17 is a 17 kb linear plasmid. Genome is shown approximately to scale. (B–C) The number of genes from each chromosome or plasmid that increased (B) or decreased (C) expression twofold during feeding, see Figure 3—source data 1 for gene information. Upregulated genes are distributed across plasmids, while most downregulated genes are found on the chromosome and lp54. Figure 3—source data 1 Twofold differentially expressed Bb genes from across tick feeding timepoints. https://cdn.elifesciences.org/articles/86636/elife-86636-fig3-data1-v1.xlsx Download elife-86636-fig3-data1-v1.xlsx Several plasmid-encoded genes that were longitudinally upregulated in our dataset have known roles during the tick bloodmeal or in mammalian infection. Linear plasmid 54 (lp54), which is an essential plasmid present in all Bb isolates (Casjens et al., 2012), contained the largest number of upregulated genes. Many of the genes on lp54 are regulated by RpoS during feeding, including those encoding adhesins DbpA and DbpB, which are important for infectivity in the host (Blevins et al., 2008). This set also included five members of a paralogous family of outer surface lipoproteins BBA64, BBA65, BBA66, BBA71, and BBA73. BBA64 and BBA66 are necessary for optimal transmission via the tick bite (Gilmore et al., 2010; Patton et al., 2013). These findings indicate our dataset captures key Bb transcriptional responses known to be important for survival inside the tick during a bloodmeal. Many upregulated genes were also encoded by cp32 plasmid prophages. Bb strain B31-S9 harbors seven cp32 isoforms that are highly similar to each other (Casjens et al., 2012). When cp32 prophages are induced, phage virions called ϕBB1 are produced (Eggers and Samuels, 1999). In addition to phage structural genes, cp32 contain loci that encode various families of paralogous outer surface proteins (Stevenson et al., 2000). Amongst the cp32 genes that increased over feeding were members of the RevA, Erp, and Mlp families, which are known to increase expression during the bloodmeal (Gilmore et al., 2001). We also found several phage genes that were upregulated, including those encoding proteins annotated as phage terminases on cp32-3, cp32-4, and cp32-7 (BBS45, BBR45, and BBO44). Some cp32 genes have been shown to change expression in response to the presence of blood (Tokarz et al., 2004) and as a part of the stringent response regulated by RelBbu (Drecktrah et al., 2015), while BBD18 and RpoS regulate prophage production in the tick midgut after feeding (Wachter et al., 2023). Our data suggest that some prophage genes are upregulated over the course of tick feeding, raising the possibility that cp32 prophage are induced towards the end of feeding. Overall, our data support the long-held idea that the Bb plasmids, which house many genes encoding cell envelope proteins, proteins of unknown function, and prophage genes, play a critical role in the enzootic cycle during the key transition period of tick feeding. Bb genes encoding outer surface proteins are highly prevalent among upregulated genes To gain a better overall sense of the types of genes that changed over feeding and the timing of those changes, we grouped DE genes into functional categories. Since a high proportion of plasmid genes encode lipoproteins within the unique protein-rich outer surface of Bb, genes of unknown function, and predicted prophage genes (Casjens et al., 2000; Fraser et al., 1997), we expected that many of the DE genes would fall int" @default.
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- W4384339675 title "Reviewer #1 (Public Review):: Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission" @default.
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