Ethics statement

Mice were bred and maintained in the Tufts University Animal Facility. All experiments were performed following the guidelines of the American Veterinary Medical Association (AVMA) as well as the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were performed with approval of the Tufts University Institutional Animal Care and Use Committee (IACUC, Protocol# B2015-159). Euthanasia was performed in accordance with guidelines provided by the AVMA and was approved by the Tufts University IACUC.

Bacterial strains and growth conditions

B. burgdorferi strains were grown in Barbour-Stoenner-Kelley II (BSK-II) medium in sealed tubes at 32°C with 1% CO2. Escherichia coli strains (Top10) for plasmid preparation were grown on Lysogeny broth (LB) agar plates or in LB broth at 37oC. E. coli cultures contained either 50 μg/ml spectinomycin or 10 μg/ml gentamicin. The parental strain of the Tn library, the infectious B. burgdorferi strain 5A18NP1, was used as the wild-type strain in all studies and lacks two plasmids (lp56 and lp28-4) [45]. The following antibiotics were used for selection in cultures of B. burgdorferi when appropriate: kanamycin at 200 μg/ml, gentamicin at 40 μg/ml, and streptomycin at 50 μg/ml. Tn mutants were obtained from the arrayed B. burgdorferi library [45]. All individual Tn mutants used in this study were screened by polymerase chain reaction (PCR) at the locus of interest to confirm pure populations as previously described [42, 43]. In cases where mixed populations were identified (i.e. two PCR products indicating the presence of both a wild-type and Tn disrupted locus), the strain was plated for single colonies in semi-solid agarose overlays. Individual colonies were then selected and re-screened to confirm pure populations. All Tn mutants were subsequently plasmid- typed to identify the loss of any plasmids required for murine or tick infection [46, 47]. A description of all individual Tn mutants used in this study is available in Table 1. Other B. burgdorferi strains as well as plasmids used in this study are also described in Table 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Strains and plasmids used in this study.

https://doi.org/10.1371/journal.ppat.1007644.t001

Feeding of ticks with B. burgdorferi by immersion

Infection of ticks with B. burgdorferi from the mutant library was performed using a method previously described [48]. Briefly, before immersion, spirochete cell density was determined by dark field microscopy. Cell suspensions were centrifuged for 10 min at 8,000 x g and were resuspended at the desired cell density and in the desired medium. I. scapularis larvae were obtained from the National Tick Research and Education Resource at Oklahoma State University and were maintained in a humid tick incubating chamber at room temperature. Larvae were used within 4 months of emergence.

Before immersion, I. scapularis larvae were removed from the chamber and allowed to sit at ambient humidity in an air-conditioned room for 2h. The larvae were then transferred with a small brush into 1.5 ml microcentrifuge tubes. B. burgdorferi culture suspension of 108 bacteria in 1 ml was then added to the ticks. For the Tn-seq studies, the suspension consisted of the B. burgdorferi Tn mutant library [42, 43]. For confirmatory experiments, individual mutants, mixtures of mutants and complemented mutants, or mixtures of mutants and controls were used in the suspension. The tubes were gently vortexed to suspend larvae in the culture and incubated at 32°C for 1 h. Tubes were gently vortexed every 15 min to redistribute ticks in the culture. After incubation at 32°C, tubes were centrifuged at 200 x g for 1 min. The supernatant was removed, and ticks were washed once with phosphate-buffered saline (PBS).

Larvae were then transferred from the microcentrifuge tube to a sealed mouse restraining device. The mice were of C57BL/6 background and were all females aged 4-6 weeks old. A mouse was placed into the restrainer containing the larvae, and the larvae were allowed to attach. Mice were removed from the restrainer after 30 min and transferred to cages suspended over water moats. Engorged larvae were collected from the moats 3 to 5 days after placement and transferred into a tick incubation chamber.

Tick processing for Tn-seq analysis

Ticks were collected as they completed feeding from the animals. Cages were checked daily to collect ticks. Ticks were batched and processed for Tn-seq up to 48 hrs after collection [48]. Briefly, ticks were washed in 3% hydrogen peroxide, 70% ethanol, and finally in PBS. The ticks were allowed to dry before placement in 500 μl of BSK-II medium with kanamycin and gentamicin. To isolate spirochetes from the ticks, the ticks were crushed with a plastic pestle (Fisherbrand RNase- Free Disposable Pellet Pestles). These tick homogenates were inoculated into 5 ml of BSK-II containing kanamycin and gentamicin. The cultures were incubated for two days to allow outgrowth. Following this, the spirochetes were pelleted by centrifugation for 10 min at 8,000 x g. Bacterial pellets were washed once in PBS, and then the dry pellet was stored at -80°C until further processing for Tn-seq.

Preparing libraries for Illumina sequencing

Genomic libraries for sequencing were constructed as described previously [42,43]. Chromosomal DNA was sheared using the M220 Focused-ultrasonicator (Covaris) in microTUBEs with a target peak at 350 bp. The first round of PCR amplification was performed using a modified primer with optimized annealing to the Tn (pMargent1A, 5'-ggtaccttaggagaccgggg-3')[43]. Libraries were multiplexed and pooled for analysis. Sequencing was performed on an Illumina HiSeq 2500 at the Tufts University Core Facility as 50-bp single-end reads, as described previously [42,43].

Tn-seq data analysis

Sequenced reads were clustered by barcode sequence. Data analysis were performed using the Galaxy platform and followed a previously published protocol [43]. We obtained an average of 1.2 x 10⁷ reads per barcode, with 1.7 x 10⁶ reads per condition for analysis after removal of low quality sequences [43]. Reads were mapped to the B. burgdorferi B31 genome using Bowtie, and a custom script was used to count the number of sequence reads corresponding to each insertion site in the genome. Sequence reads were analyzed “by-site” and “by-gene”. Only Tn mutants that were represented by at least ten sequence reads in both input samples were included in the “by site” analysis. In contrast, the “by-gene” analysis included all sequence reads mapping within a particular gene. Genes represented by less than ten sequence reads in both untreated samples were excluded from the “by-gene” analysis. Tn mutants with zero reads in the output samples were assigned a value of one for the purpose of calculation. The frequency of each Tn mutant in a particular condition was determined by dividing the number of sequence reads corresponding to each Tn mutant by the total number of sequences in the barcode. A frequency ratio was then determined by dividing the frequency of a Tn mutant in the output (bacteria recovered from the ticks) sample by its frequency in the input population. For the purposes of prioritizing mutants for follow-up, frequency ratios between 0.5 and 2 were considered neutral.

Generation of B. burgdorferi mutant and complemented strains

A plasmid for complementation of the Tn::bb0164 mutant was generated previously by overlap PCR [43]. A plasmid for directing cis complementation of Tn::bb0412 via allelic exchange was generated from three PCR fragments: intact bb0412 with 995 bp upstream sequence (F1), PflgB-aadA for antibiotic selection (F2), and 1022 bp downstream of bb0412 (F3). Primers were designed with approximately 30 bp on the 5' end for overlap, with the numerically assigned PCR products assembled in order into the final construct. See S1 Table for a list of all primer sequences used in this study. Individual PCR fragments were amplified with AccuPrime Pfx (ThermoFisher Scientific, MA) per the manufacturer's instructions. An overlap PCR was performed with equal volumes of the appropriate number of PCR fragments with AccuPrime Pfx reagents per the manufacturer's recommendations. Following PCR, the products were resolved by agarose gel electrophoresis and gel-purified (Zymoclean Gel DNA Recovery Kit, Zymo Research, Irvine, CA) for cloning into pCR-Blunt (ThermoFisher Scientific, Grand Island, NY) following the manufacturer's protocol. The bb0412 complementation vector was designated pAK412 (Table 1).

Plasmid pMR05 was constructed for trans complementation of the Tn::bb0017 mutant was generated by amplifying a DNA sequence containing the bb0017 open reading frame and the 298-bp upstream region from 5A18NP1 genomic DNA via PCR using the primers bb0017FLAG-F-SacI and bb0017FLAG-R-XbaI (S1 Table). The resulting PCR products as well as pKFSS1 were digested with SacI and XbaI, and the resulting PCR product and pKFSS1 fragments were ligated together using T4 DNA Ligase (New England Biolabs), generating pMR05 (Table 1).

Plasmid pBS01 was constructed to direct allelic exchange at the bb0017 locus, resulting in deletion of the bb0017 open reading frame (Table 1). Plasmid pBS01 contains 1835 bp of sequence upstream of bb0017 (amplified from genomic DNA using primers bb0017del1 and bb0017del2), followed by a sequence containing the constitutive PflgB promoter and a streptomycin resistance gene (aadA, amplified from pKFSS1 using primers bb0017del3 and bb0017del4), followed by 1999 bp of sequence downstream of bb0017 (amplified from genomic DNA using primers bb0017del5 and bb0017del6). PCR products were purified using the Qiagen PCR purification kit and were subsequently assembled into the pBlueScript cloning vector using the NEBuilder HiFi DNA Assembly Cloning Kit.

Plasmid pBS02 was constructed to direct expression of bb0017 in the Δbb0017 mutant. Plasmid pBS02 is identical to pMR05, except for replacement of the streptomycin resistance cassette by a gentamicin cassette (Table 1). The streptomycin cassette was excised from pMR05 by restriction digest with AatII and NdeI. The gentamicin cassette had been previously subcloned into the pCR2.1cloning vector. The gentamicin resistance gene was excised from this vector using the same restriction enzymes as above, and the resulting fragment was ligated with the digested pMR05 backbone (Table 1).

Plasmid pMH88R was used to constitutively express the glp operon in the Δbb0017 mutant. Plasmid pMH88R was a kind gift of Dr. Frank Yang [33]. All completed plasmids were verified by restriction digest and dideoxy sequencing.

Plasmids were introduced into B. burgdorferi by transformation as previously described [50,51]. Cis complementation vector pAK412 was transformed into B. burgdorferi Tn::bb0412, and transformants were designated AK102 (Table 1). The complemented strain was screened by PCR for allelic exchange using the forward primer of fragment 1 and the reverse primer for the PflgB- aadA cassette for each construct. The bb0017 deletion construct pBS01 was transformed into B. burgdorferi strain 5A18NP1, generating strain Δbb0017 (Table 1). In order to complement the bb0017 mutation, plasmid pBS02 was introduced into the Δbb0017 background, generating strain Δbb0017 + bb0017 (Table 1). The overexpression of the glp operon construct pMH88R was transformed into the Δbb0017 mutant and was used to generate the Δbb0017 + glpFKD strain (Table 1). Potential transformants were confirmed by PCR with primers designed to detect either a replicating plasmid or a double crossover event, as appropriate, followed by dideoxy sequencing of the PCR product to confirm the expected nucleotide sequence.

Tick competition assays

To evaluate survival in the tick host, individual Tn mutants of interest were combined with their respective complemented strain or wild type bacteria in a 1:1 mixture. Each strain was grown independently. Cell density was determined by microscopy, and 5 x 10⁷ B. burgdorferi were harvested by centrifugation. The pellets from both cultures were then resuspended in the same 1 ml of BSK-II medium to a final overall density of 1 x 10⁸ cells/ml. Ticks were submerged in the cultures as described, placed on mice for feeding, and collected over three days as described above. Each tick was washed successively with 3% hydrogen peroxide, 70% ethanol and PBS, then crushed into 250 μl of BSK-II containing kanamycin and 5 μg/ml amphotericin B. The cultures were allowed to acclimate in liquid medium for a period of 2 h before plating, to allow the bacteria to escape from the crushed tick into the medium. Plating in semi-solid agarose overlay was performed as previously described [50]. These plates were then sealed in plastic bags and placed at 32°C for 10 days. The plates were removed from the incubator and colonies were enumerated. The ratio of the wild-type or complemented strains to the Tn mutant was determined by counting the colonies on the appropriate antibiotic selective plates. A competitive index was calculated for these experiments by dividing the amount of mutant recovered by the amount of complement or WT that was recovered. In the case where no mutant was recovered, its value was set to one for the purposes of calculation of the competitive index.

RNA-sequencing (RNA-seq) library preparation and data analysis

Two independent cultures of the 5A18NP1 and Tn::bb0017 strains were grown to mid-logarithmic phase, washed once in PBS, and resuspended in BSK II medium at a concentration of 6 × 107 bacteria/ml. Cultures were incubated at 32°C with 1% CO2 for 1 h. The bacterial pellet was harvested by centrifugation at 9500 × g for 5 min at 4°C, washed once in ice-cold PBS, and frozen on dry ice. RNA was isolated using the miRNeasy kit (Qiagen). The TURBO DNA-free kit (Invitrogen) was used to remove contaminating genomic DNA. Library preparation for RNA-seq analysis was conducted at the Tufts University Genomics Core Facility. Samples were depleted of rRNA using the Gram-Negative Ribo-Zero rRNA Removal Kit (Illumina). Strand-specific libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina). Sequencing was performed on an Illumina HiSeq 2500 at the Tufts University Core Facility as 50-bp single-end reads.

Data analysis were performed using the Galaxy platform [43]. Sequence reads were aligned to the B. burgdorferi B31 genome using TopHat for Illumina v1.4.1 with the default settings. Differences in transcript expression were determined using CuffDiff, again using the default settings.

Reverse transcriptase quantitative PCR (qRT-PCR)

Total RNA was extracted from bacterial cells grown to mid-logarithmic growth phase at 32°C using TRIzol (Invitrogen) following the manufacturer’s instructions. RNA samples were treated with the TURBO DNA-free kit (Invitrogen) to remove contaminating DNA. cDNA was prepared using random hexamers (Promega) and the ImProm-II Reverse Transcription System (Promega). Control reactions were performed in the absence of reverse transcriptase to control for the presence of genomic DNA. Sequences of the primers used to determine the differential expression of target genes are listed in S1 Table, and expression levels were normalized against those of the B. burgdorferi housekeeping gene flaB.

Quantification of target genes from cDNA was performed using the iTaq Universal SYBR Green Supermix (BioRad)[43]. Samples were run in duplicate or triplicate. Analysis of the RT-qPCR data was conducted using the ΔCT method. Data collection was performed using the CFX Connect Real- Time PCR Detection System (BioRad).

Western blots

Strains were grown in BSK-II at 32°C/1% CO2 until cultures reached early stationary phase. A volume corresponding to 1 × 10⁸ bacteria was harvested by centrifugation at 9500 × g for 5 min at 4°C. The bacterial pellet was frozen at -80°C until processing. To lyse the cells, the bacterial pellet was resuspended in approximately 100 μl of 1X NuPAGE buffer (ThermoFisher) and boiled for 5 min. A 20 μl volume of each lysate was electrophoresed in 4-15% gradient SDS-PAGE gels (BioRad). Proteins were transferred to a polyvinyldifluoride (PVDF) membrane (Trans-Blot Turbo BioRad). Membranes were blocked in 5% milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS). Primary antibodies were diluted as follows in TTBS: anti-RpoS (1:50, courtesy of Dr. Frank Yang), anti-BosR (1:500, courtesy of Dr. Frank Yang), anti-FlaB (1:1000, courtesy of Dr. Xin Li), anti-OspC (1:10,000, courtesy of Dr. Xin Li), anti-OspA (1:1000, Rockland Immunochemicals) and anti- DbpA (1:1000, Rockland Immunochemicals). Appropriate horseradish peroxidase (HRP)- conjugated secondary antibodies were used at a 1:10,000 dilution in TTBS. Detection was performed using the Luminata Forte substrate (Millipore), followed by exposure to film (Denville Scientific) or imaging using a ChemiDoc XRS+ Imager.

In vivo Tn-seq screen in larval ticks

In order to use Tn-seq to determine genes involved in B. burgdorferi fitness for survival in ticks, we first needed to decide the number of ticks to include in the experiments. Using immersion feeding, it has previously been shown that approximately 103 B. burgdorferi could be recovered per tick [48,52]. However, these experiments examined a single strain of bacteria, and since bacteria were allowed to replicate within the tick before recovery, the actual number of bacteria entering to establish colonization was likely less than 10³. We were also concerned that the bottleneck may be further exacerbated by plasmid loss in the parental strain of the transposon library. It has previously been reported that the mean number of spirochetes per tick with 5A18NP1 lacking both lp28-4 and lp56 plasmids was lower than that in ticks infected with B. burgdorferi harboring all plasmids [53]. The ordered transposon library contains insertions into 45.5% of the predicted protein encoding genes [45]. To ensure sufficient coverage of the pooled library of around 4,000 mutants we chose to target approximately 150 ticks per experiment. If only half the predicted number of bacteria established infection (500 bacteria per tick), this approach should still provide approximately 20- fold coverage of the mutants within our library.

In each of two independent experiments, approximately 300 larval ticks were immersed in a culture solution containing the entire Tn library and then fed on mice. This resulted in the collection of approximately 160 fed ticks per experiment. Fed ticks were processed and cultured in BSK medium for 2 days. The input culture was also cultured for an additional 2 days to match the tick cultures. Bacteria from both cultures were harvested and sequencing libraries prepared.

Reproducibility was high between the two input libraries (Fig 1A, Pearson coefficient R2=0.98). The correlation between the Tn frequencies of the populations recovered from the two groups of tick larvae was also high with a Pearson coefficient R2= 0.85 (Fig 1B). A frequency ratio was calculated for each Tn mutant in the library by comparing its frequency in the output library to its frequency in the input library. For analysis, we included only Tn mutants represented by at least 10 sequence reads (out of a total of approximately 1.7 x 10⁶ sequence reads per experiment) in both replicates of the input libraries. This number was chosen to reduce the risk of stochastic loss after selection in the ticks. Tn mutants that were represented in the input library but had zero reads in the output library were assigned a value of one read in order to be able to calculate a frequency ratio. A frequency ratio less than one indicates that the Tn mutant decreased in frequency after recovery from fed ticks suggesting that the disrupted gene is involved in survival in the larval host. A frequency ratio greater than one indicates that the Tn mutant increased in frequency after larval colonization suggesting that the disruption of the corresponding gene provides a fitness advantage. An overall frequency ratio was also calculated for each gene by aggregating all of the sequence reads mapping to Tn insertions within the same gene (S1 File). A complete list of mutant fitness for larval colonization by gene and by site is provided in S1 File.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Correlation between replicates of the larval tick survival Tn-seq experiment.

(A) Correlation between the mutant compositions of the two replicate cultures of the input Tn library. (B) Correlation between the mutant compositions of the two replicate cultures of the Tn mutants recovered from the fed larvae (output library). Each data point represents the frequency of an individual Tn mutant in relation to the total input or output respectively. Each axis represents a different biological replicate.

https://doi.org/10.1371/journal.ppat.1007644.g001

In order to validate our screen, we began by analyzing genes that have been previously shown to be essential for tick survival, to ensure that these genes had been identified in the screen. Borrelial genes that have been described in the literature as critical to tick survival for which mutants are present in the library include: bb0419 and bb0420, respectively encoding a response regulator designated Rrp1 and a histidine kinase designated Hk1 [30,35]; guaA and guaB, two genes involved in the purine salvage pathway [37]; glpD, encoding a glycerol 3-phosphate dehydrogenase [33,38]; and bptA, a surface-expressed lipoprotein [8]. In the Tn-seq experiment, consistent with previously published results, insertional mutants in each of these genes except glpD showed attenuated ability to survive in the tick, with median frequency ratios of <0.1 (Fig 2A). GlpD has been shown to be important following the molt from larvae to nymph when carbon sources are less abundant and the organism begins to utilize glycerol, which may explain why Tn::glpD mutants did not exhibit phenotypes in our screen [35].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Fitness of selected genes required for Borrelia burgdorferi survival in larval ticks.

(A) Frequency ratios of individual Tn mutants with insertions into the indicated genes known to be important in B. burgdorferi survival in the tick. The frequency ratios of individual Tn mutants with insertions in these genes are shown for both replicates. The median is indicated with a bar. An open shape indicates that no reads were recovered for this mutant and for the purposes of calculation the read number was set to one. (B) Individual Tn mutants were mixed in a one to one ratio with the parental strain 5A18NP1 or the respective complemented strain, and the mixtures were used to infect larvae. After a blood meal these larvae were crushed and plated with appropriate antibiotics to distinguish between the Tn mutant and the complemented strain (or parental strain). Competitive Indices (CI) were determined. Closed shapes indicate that the Tn mutant was recovered following the competition experiment. Open shapes indicate no Tn mutants were recovered following competition experiment. In these cases, the number of Tn mutants recovered was set to one for the purposes of calculation. Statistical significance was determined by one sample t test (***, P<0.0001). (Statistics performed on GraphPad Prism version 7, GraphPad Software, LaJolla California).

https://doi.org/10.1371/journal.ppat.1007644.g002

Further analysis was performed to identify new genes involved in tick survival. A large number of mutants (N=309) had a frequency ratio of less than 0.5 compared with the input library. In order to prioritize mutants with the strongest phenotypes for follow-up analysis, we focused on mutants with a fitness ratio of less than 0.1. Mutants with insertions in 102 genes had an average overall frequency ratio below 0.1 in both experiments (S1 File). However, this group of 102 mutants included some with insertions into genes that have been shown not to be required for tick colonization (e.g. ospC). While many of this group of 102 genes may be involved in tick survival as it includes many of the genes previously identified as involved in tick colonization, to further reduce the chance of false discovery by the screen, we increased the stringency of our criteria and focused on the subset of 46 genes that had >100 reads in the input library but were completely absent in the processed ticks from both experiments (Table 2). These genes would be predicted to have the greatest impact on fitness for survival in larval ticks. Of these 46 genes, many have no predicted function and have not been previously characterized (Fig 3). Approximately 22% of the genes are predicted lipoproteins, while 7% are involved in carbohydrate transport (Fig 3).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Functional classification of genes required for tick colonization.

A cluster of orthologous gene analysis was performed on 46 genes that were found at a frequency of greater than 100 reads in the input libraries and were unrecovered in all output populations. Classifications were based on predicted functions.

https://doi.org/10.1371/journal.ppat.1007644.g003

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. List of Borrelia burgdorferi transposon mutants not recovered from larval ticks.

https://doi.org/10.1371/journal.ppat.1007644.t002

Confirmation of gene involvement in tick survival

To confirm the results of the Tn-seq screen, we chose mutants with insertions in five genes (bb0017, bb0164, bb0412, bb0050 and bb0051) that showed the strongest fitness defects, and that have not previously been reported to be involved in tick survival. Each of these genes was well represented by insertion mutants in the input library and had an overall frequency ratio of less than 0.1 following the ingestion of a blood meal by larval ticks. Competition assays were conducted to assess the capability of each individual mutant to survive the larval blood meal (Fig 2B).

Three of the transposon mutants were competed against a complemented strain, while the remaining two transposon mutants were competed against the parental strain (Fig 2B). The Tn::bb0017, Tn::bb0164, Tn::bb0412, Tn::bb0050 and Tn::bb0051 mutants were all outcompeted by the parental or respective complemented strains, confirming a role for all five genes in blood meal survival (Fig 2B). We were also able to further confirm this phenotype when competing a bb0017 clean deletion strain against its complemented strain. (Fig 2B). The complemented strain greatly outcompeted the deletion strain confirming the role of bb0017 in surviving the blood meal (Fig 2B).

Identifying the role of identified genes in tick survival

The Tn-seq and competition experiments do not distinguish between the possibilities that 1) the identified genes are required for initial entry into the tick during immersion; or 2) they are required for surviving the blood meal taken by the tick. The competition experiment that was described previously was modified so that the ticks were not allowed to take a blood meal after immersion in the culture containing the two competing strains, allowing us to separate fitness defects due to uptake from fitness defects due to blood meal survival. The ticks were crushed after two hours of immersion feeding or kept overnight and crushed 24 hours post-immersion. The relative frequencies of the mutant and complemented or parental strains were then determined as before. However, in contrast to the competitive defect exhibited by all five Tn mutants after the blood meal, we were able to recover all Tn mutants after immersion feeding in equal or greater numbers compared to the WT or complemented mutants. However, in the absence of a blood meal, as expected, the numbers of bacteria were greatly reduced and B. burgdorferi was not recovered from all individual ticks. The results of these studies are shown in Table 3. These data support a role for these five borrelial genes in surviving changes associated with the blood meal. Also, importantly, because several of these genes have identified roles in ROS resistance and hydrogen peroxide was used in washing the ticks, these experiments confirm that the hydrogen peroxide wash did not affect selection of these mutants.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Competition in the absence of a blood meal.

https://doi.org/10.1371/journal.ppat.1007644.t003

In silico analysis predicts BB0017 is a membrane-associated signal transduction protein

To begin to better understand the mechanisms by which the genes identified in the Tn-seq screen contribute to tick-phase survival, we performed further investigation into one of the genes identified as critical for survival of the blood meal: bb0017. The gene bb0017 was previously identified in a screen for genes that confer resistance to ROS [43]. BB0017 is highly conserved among both B. burgdorferi sensu stricto and other sensu lato strains (>99% and >94% identity at the amino acid level, respectively). BB0017 homologues are also conserved in the relapsing fever strains (>80% identity) [54]. In the B. burgdorferi strain B31, bb0017 is annotated an integral membrane protein of the YitT family. BB0017 contains four predicted transmembrane domains as well as a C-terminal soluble domain and contains the conserved domain of unknown function DUF2179 (S1A & S1B Fig) [55]. A structure-based similarity search using Phyre2 suggested that the C-terminal domain of BB0017 is structurally similar to PII and PII-like proteins, despite low overall sequence identity (<27% identity) [56]. No high confidence predictions were made for the N-terminal domain of BB0017.

PII proteins are a broadly conserved class of signal transduction proteins found in bacteria, archaea, and plants and are generally small cytoplasmic proteins involved in nitrogen metabolism [57–59]. PII proteins generally function as trimers and control the activity of their regulatory targets through direct protein-protein interactions in response to both post-translational modifications (such as uridylylation) and ligand binding (including ADP, ATP, and 2-oxoglutarate). The long, flexible T- loop mediates interactions with regulatory targets, while a conserved motif in the shorter B-loop is involved in ligand binding (S1C Fig, GlnKEc). More recently, several PII-like families of proteins have been identified in bacteria, including a family of proteins in Gram-positive bacteria that bind cyclic diadenylate monophosphate (c-di-AMP) as well as a broadly conserved family of proteins (CutA) that confer copper tolerance in Escherichia coli and bind acetylcholinesterase in mammals [60–66].

While the PII and PII-like proteins share a common ferredoxin-like fold, the lengths of the T and B loops differ significantly between the different protein families. In the case of the PII-like c-di-AMP binding proteins, the lengths of the B and T loops are reversed relative to the PII proteins and are referred to as the B´ and T´ loops (PstASa, S1C Fig). Structural data suggests that the functions of the B´ and T´ loops are also reversed relative to the PII proteins, with the short T´ loop being involved in ligand binding and the long flexible B´ loop possibly involved in effector binding [61,62,64]. In the case of the copper tolerance protein CutA1, both the B and T loops are truncated, and the same is true for BB0017. Interestingly, BB0017 appears to lack conserved residues involved in ligand binding by both the PII and PII-like protein families. The presence of the N-terminal transmembrane domain also distinguishes BB0017 from the PII and PII-like protein families and suggests that membrane localization may be important for BB0017 function.

BB0017 downregulates expression of mammalian-phase lipoproteins

Because BB0017 contains a putative signal transduction domain, we hypothesized that the Tn::bb0017 mutant would exhibit global differences in gene expression compared to the parental strain. Total RNA was isolated from the parental and Tn::bb0017 strains, and RNA sequencing (RNA-seq) was used to compare the transcriptomes of both strains. We identified 16 genes that were significantly downregulated more than twofold in the Tn::bb0017 mutant compared to the parental strain and 25 genes that were significantly upregulated more than twofold (S2 Table and Fig 4). It is important to note that bb0017 does not appear in S2 Table. While expression of bb0017 was significantly different between the Tn::bb0017 and parental strains, the difference was less than twofold, and bb0017 expression was actually higher in the Tn::bb0017 mutant compared to the parental strain. Sequence coverage maps confirm that transcription in the Tn::bb0017 mutant is abrogated downstream of the Tn insertion as expected (S2 Fig). However, increased numbers of sequence reads mapped in the 5’ portion of bb0017, likely due to transcription from the strong PflaB promoter contained within the Tn (S2 Fig). It is unclear whether there is translation of the 5’ portion of bb0017 in the Tn::bb0017 mutant, resulting in a truncated protein, but if a truncated protein is produced in the Tn::bb0017 mutant, these results could suggest that the C-terminal portion of BB0017 is the critical portion for survival in the tick.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Genes differentially expressed in the Tn::bb0017 mutant compared to parental strain in RNA-seq experiment.

Volcano plot showing differential gene expression between the Tn::bb0017 mutant and the parental strain. Genes that were significantly different are labeled by name in the graph. Purple circles indicates that the gene is repressed by rpoS while orange circles indicates that a gene is activated by rpoS. The gene abbreviation system assigns a letter based on the plasmid location.

https://doi.org/10.1371/journal.ppat.1007644.g004

Strikingly, the putative bb0017 regulon overlaps significantly with that of RpoS, a key regulator of virulence gene expression in B. burgdorferi [15,16,19,20]. RpoS is directly responsible for upregulating a number of genes required for survival in the mammalian host, including dbpA, dbpB, ospC, and bbk32 and repressing expression of genes important for tick survival such as glpD [15,19,20,38,67]. The dbpA, dbpB, ospC, and bbk32 genes are all upregulated in the Tn::bb0017 mutant (S2 Table, Fig 4). Several genes known to be subject to RpoS-mediated repression, including genes located within a glycerol utilization operon important for tick infectivity, are downregulated in the Tn::bb0017 mutant (bb0240-bb0243, S2 Table) [38]; however, other regulators such as c-di-GMP may also affect expression of these genes.

RNA-seq validation

To confirm the results of the RNA-seq screen, we generated a mutant lacking the entire bb0017 open reading frame. A survey of available B. burgdorferi genome sequences revealed two different annotated start sites (S1 Fig). We chose to delete the region encompassing the first start site, which includes a putative 71 bp small RNA (SR0011) in the bb0016-bb0017 intergenic region [68] (S1 Fig). We restored bb0017 expression including the upstream SR11 intergenic region under the control of the native promoter from a replicating plasmid in the Δbb0017 mutant, and confirmed expression by qRT-PCR (S3 Fig).

We performed qRT-PCR on the Δbb0017 mutant as well as the complemented strain to validate the results of the RNA-seq using the transposon insertion strain. We selected a subset of differentially regulated genes from the RNA-seq, as well as some representative regulatory proteins of B. burgdorferi that showed no change in expression. bosR and rpoS levels were not significantly different in the RNA-seq and this phenotype was reproduced in the deletion strain as well as the complement by qRT-PCR (Fig 5). We then confirmed six genes, ospC, dbpA, glpD, bba37, bba25, and bbk32 that were differentially expressed by RNA-seq in the transposon mutant strain. Each showed a similar pattern of expression in the clean deletion strain with recovery in the complemented mutant strain, with the exception of glpD (Fig 5). Transcription of glpD was decreased in the transposon mutant and its complement as well as the deletion strain and its complement in comparison to the wild type making it unlikely that this difference was due to secondary site mutations or polar effects as each of the mutants and complements were created from separate isolations from the parental strain. Of note is that all the changes are small (fourfold) compared to the other genes tested by qRT-PCR. Given the lack of involvement of glpD involvement in tick survival as assayed by the Tn-seq and its established role at a different stage in tick survival, it is likely that the change is not physiologically relevant.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Validation of RNA-seq by RT-qPCR.

RT-qPCR for expression of a panel of genes that were of significance from the RNA-seq in the parental strain 5A18NP1, the Δbb0017 mutant, and the respective complemented strain. A) dbpA, B) ospC, C) bosR, D) rpoS, E) bba25, F) bbk32, G) glpD, H) bba37. Expression of the target genes was normalized to expression of the B. burgdorferi housekeeping gene flaB using the ΔCT method. Statistics were performed using one way ANOVA with Tukey’s test for multiple comparisons.(*= P<0.05, **= P<0.005)(Black Star= P<0.05 from parental, Black circle= P<0.05 from glpFKD over expressing strain) (Statistics performed on GraphPad Prism version 7, GraphPad Software, LaJolla California).

https://doi.org/10.1371/journal.ppat.1007644.g005

Overexpression of the glycerol utilization operon in the Δbb0017 mutant does not restore the ability to survive the blood meal

To ensure that we were not missing a role for GlpD in mediating effects of the Δbb0017 mutant, we created a strain that overexpresses the entire glp operon, including glpFKD, in the Δbb017 deletion strain. This construct has previously been used to successfully overexpress GlpD [33]. We confirmed that glpD was successfully transcriptionally over-expressed by qRT-PCR (Fig 5). Using this strain, we then performed a competition experiment between the glp operon overexpressing strain and the Δbb0017 strain. We were not able to recover either strain from this experiment following selective plating from six collected fed larvae. This indicates that increasing the ability for glycerol utilization is not sufficient to rescue the Δbb0017 mutant and the defect in tick colonization is unlikely to result strictly from decreased expression of glpD.

BB0017 represses lipoprotein expression in a BosR-and RpoS-dependent manner

We performed immunoblots for OspC and DbpA in the Tn::bb0017 and Δbb0017 mutants. Levels of both DbpA and OspC were elevated in the Tn::bb0017 and Δbb0017 mutants, confirming the results of the RNA-seq screen (Fig 6A). Restoration of bb0017 expression from a replicating plasmid (which also contains SR0011) in both mutants decreased DbpA and OspC levels to those of the parental strain (Fig 6A). The fact that both the Tn::bb0017 mutant (in which SR0011 remains intact) and the Δbb0017 mutant (in which SR0011 is disrupted) exhibit increased lipoprotein expression, suggests that bb0017 is required for the phenotype, although these results to do not exclude the possibility that SR0011 may also be involved. Expression of OspA, a surface lipoprotein required for infectivity in the tick, was not affected by the absence of bb0017 under the conditions tested.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6.

Immunoblot analysis of (A) lipoprotein expression and (B) regulatory protein expression across different B. burgdorferi bb0017 mutant and complemented strains. A Tn::oppA1 mutant was also included in these experiments as a further control strain. Bacterial cultures were grown at 32°C with 1% CO2 until early stationary phase. A volume corresponding to 1×10⁸ bacteria was harvested by centrifugation, cells were washed and lysed, and lysate corresponding to approximately 2×10⁵ cells was subjected to immunoblot analysis. FlaB levels were analyzed as a loading control.

https://doi.org/10.1371/journal.ppat.1007644.g006

The expression of dbpA and ospC requires the alternative sigma factor RpoS [15,20]. The regulation of rpoS in turn involves a second alternative sigma factor RpoN (σ54), the enhancer binding protein Rrp2, the Borrelia oxidative stress response regulator BosR, and the small regulatory RNA DsrA [15,16,24,25,69,70]. We hypothesized that BB0017 mediates the repression of ospC and dbpA indirectly by affecting the expression of an upstream regulator. We therefore investigated RpoS levels in the Tn::bb0017 and Δbb0017 mutants. RpoS levels were increased in both mutants, and restoration of bb0017 expression resulted in decreased RpoS levels (Fig 6B). To understand the mechanism by which BB0017 affects RpoS expression, we next investigated production of BosR, a positive regulator of RpoS [24,70]. As was the case for RpoS, BosR levels were increased in the Tn::bb0017 and Δbb0017 mutants, and complementation of bb0017 restored levels to those similar to the parental strain (Fig 6B).