A two-step enrichment process facilitates robust transcriptional profiling of Bb during the tick bloodmeal.

(A) Schematic of Bb during tick feeding. Bb in the nymphal tick midgut respond to the bloodmeal by multiplying and changing their transcriptional state. At the same time, the tick gut undergoes numerous changes to digest the bloodmeal. 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. (B) Schematic of Bb enrichment process from feeding ticks. Whole ticks are dissociated, anti-Bb 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. (C) RT-qPCR results showing the percentage of Bb flaB and tick 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. Nearly all Bb flaB RNA was found in the enriched fraction, while the majority of tick gapdh was found in the depleted fraction. (D) The percentage of reads mapping to rRNA before and after DASH. Data are shown as mean +/− SD. 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 small but sufficient proportion of libraries for transcriptomics. Data are shown as mean +/− SD. (F) The number of reads in millions (M) mapped to Bb for each day. Data are shown as mean +/− SD. An average of 4.3 million reads per sample mapped to Bb genes, covering 92% of genes with at least 10 reads.

Global ex vivo profiling of Bb reveals extent and kinetics of transcriptional changes.

(A) Principal component analysis of samples from across feeding. 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 of day 2 versus day 1 after attachment (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 downregulated genes is shown in the top left. Yellow dots are genes that change expression between day 1 and day 2, red dots are genes that change expression between day 1 and day 3, and purple dots are genes that change expression between day 1 and day 4. Two genes with log2 fold changes >4 are shown at 4, and five genes with −log10(padj) >60 are shown at 60. Only genes with at least a 2-fold change are highlighted. p-value < 0.05, Wald tests. By day 4 of feeding, 153 genes are upregulated and 33 genes are downregulated from day 1 baseline levels.

Bb genes upregulated during feeding are found predominantly on plasmids.

(A) Schematic of the chromosome and plasmids in the Bb B31-S9 genome. (B-C) The number of genes from each chromosome or plasmid that increased (B) or decreased expression (C) twofold during feeding. Upregulated genes are distributed across plasmids, while downregulated genes are found on the chromosome and lp54.

Bb genes encoding outer surface proteins are enriched among upregulated genes.

(A) The number of Bb genes that change over the course of tick feeding sorted into functional categories. Genes that first change 2 days after attachment are shown in yellow, 3 days after attachment in red, and 4 days after attachment in purple. A majority of upregulated genes fall into cell envelope and unknown categories. (B) Schematic of the outer membrane of Bb showing the location of outer surface lipoproteins. (C) Heat map of the average Transcripts Per Million (TPM) of all genes encoding outer surface lipoproteins across the 4 days of tick feeding. Genes in blue were twofold upregulated and genes in pink twofold downregulated over feeding. A majority of genes encoding outer surface proteins increased in expression throughout feeding, while having different magnitudes of expression.

Identification of candidate tick interaction partners of Bb cells ex vivo.

(A) Schematic of experiment to determine candidate tick proteins interacting with Bb over the course of feeding. Ticks were collected 1 day after attachment and 4 days after attachment. Uninfected ticks at the same time points were mixed with cultured Bb as controls. Bb was enriched with anti-Bb antibody as in RNA-seq experiments and then subjected to mass spectrometry to identify tick proteins present in the samples. Venn diagram depicts the proteins enriched in day 1 and day 4 samples over controls. Tick proteins that are enriched with Bb vary greatly over the course of feeding. (B) Tick proteins uniquely identified one day after attachment that are annotated as extracellular matrix (ECM) proteins. (C) Tick proteins uniquely identified four days after attachment that are annotated as low-density lipoprotein receptors. ECM and membrane proteins may be good candidates for Bb-interacting proteins.

Anti-Bb antibody recognizes ospA and binds Bb in the tick throughout the bloodmeal.

(A) Western blot with anti-Bb on lysate from cultured Bb: wildtype (A3, left), a mutant lacking ospA (ospA1), and the mutant with ospA restored (ospA+B1). Anti-Bb recognizes ospA among other proteins. (B) Immunofluorescence microscopy with anti-Bb (green) and propidium iodide (PI) (DNA, red) on each day of feeding (yellow is merge). Anti-Bb antibody recognizes Bb in the tick across the bloodmeal.

Enrichment process does not induce large scale gene expression changes in in vitro cultured Bb.

Log2 fold changes vs. mean normalized number of counts comparing cultured Bb input and samples after enrichment with anti-Bb. n=3. Red dots, p-value < 0.05, Wald tests. The gene expression changes induced during processing are much smaller than those observed between days of feeding.

Ex vivo RNA-seq corroborates key transcriptional programs in the tick.

(A) Volcano plot of DE genes comparing day 4 to day 1, with Rrp1-upregulated (blue) and downregulated (pink) genes. Rrp1-regulated genes correlate well with up and downregulated genes during feeding. (B) Volcano plot of DE genes comparing day 4 to day 1, with RpoS-upregulated (blue) and downregulated (pink) genes. Genes upregulated by RpoS in mammals increase during feeding, but genes repressed by RpoS in mammals are not repressed in the tick. (C) Tukey style boxplot of Transcripts Per Million (TPM) on each day for rpoS. Black dots represent replicates. ****p-value < 0.00001, Wald test. rpoS expression increases over the course of feeding. (D) Volcano plot of DE genes comparing day 4 to day 1, with RelBbu-upregulated (blue) and downregulated (pink) genes. About half of RelBbu genes change in the expected direction over feeding.