The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine

Introduction Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. Method Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. Results We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. Conclusion Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine

Structure of the <i>I</i>. <i>scapularis</i> Tudor-SN.

<p>Tudor-SN amino acid sequence alignment between <i>I</i>. <i>scapularis</i> (B7QIP4_IXOSC), <i>I</i>. <i>ricinus</i> (V5GZ29_IXORI) and <i>Danio rerio</i> (Q5RGK8_DANRE). Conserved domains in <i>I</i>. <i>scapularis</i> Tudor-SN contain conserved staphylococcal nuclease homologues (SN, cd00175; red) and Tudor domain (cd04508; blue).</p

Overrepresented k-mers in <i>I</i>. <i>scapularis</i> tick transcriptome.

<p>Profile of k-mers in transcriptomics data generated from uninfected and <i>A</i>. <i>phagocytophilum</i>-infected <i>I</i>. <i>scapularis</i> nymphs and adult female guts and salivary glands. Overrepresented T-rich (red arrows) and/or G-rich (black arrows) sequences were identified in the central part (nucleotides 10–70 of 100) of all samples. The overall %GC content for the transcriptome in all samples was 48±6. Data shown represents results of two replicates. Produced by <u>FastQC</u> (version 0.9.1; <a href="http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/" target="_blank">http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/</a>).</p

Phylogenetic position of the <i>I</i>. <i>scapularis</i> Tudor-SN.

<p>Amino acid sequences of Tudor-SN orthologs from several taxa (coloured branches) were aligned with <i>I</i>. <i>scapularis</i> Tudor-SN (red asterisk) using MAFFT. Phylogenetic analyses using ML (shown) and NJ were conducted with similar results. Numbers on internal branches are bootstrapping values. Only bootstrap values higher than 50 are shown.</p

Effect of <i>Tudor-SN</i> knockdown on LGTV infection.

<p>(A) <i>I</i>. <i>scapularis</i> IDE8 cells were treated with <i>Tudor-SN</i>, <i>Rs86</i> or <i>GFP</i> control dsRNAs and infected with LGTV at MOI 0.1. LGTV RNA levels were determined after 24h and 48h by <i>NS5</i> real-time PCR normalizing against tick <i>β-actin</i> mRNA levels and are presented relative to the <i>GFP</i> dsRNA control. Relative LGTV RNA levels were compared between groups by two-way ANOVA and were not statistically different between <i>Tudor-SN</i> and <i>GFP</i> dsRNA-treated cells (P>0.05; N = 8). (B) <i>I</i>. <i>scapularis</i> IDE8 cells were treated with <i>Tudor-SN</i>, <i>Rs86</i> or <i>GFP</i> control dsRNAs and infected with LGTV at MOI 0.1. Virus titers in the cell supernatants were determined by plaque assay after 24h and 48h and are presented relative to the <i>GFP</i> dsRNA control. Relative LGTV titers were compared between groups by two-way ANOVA and were not statistically different between <i>Tudor-SN</i> and <i>GFP</i> dsRNA-treated cells (P>0.05; N = 8). (C) <i>I</i>. <i>ricinus</i> IRE/CTVM20 cells were treated with <i>Tudor-SN</i>, <i>Rs86</i> or <i>GFP</i> control dsRNAs and infected with LGTV at MOI 0.1. LGTV RNA levels were determined after 24h and 48h by <i>NS5</i> real-time PCR normalizing against tick <i>β-actin</i> mRNA levels and are presented relative to the <i>GFP</i> dsRNA control. Relative LGTV RNA levels were compared between groups by two-way ANOVA (P≤0.05; N = 8). (D) <i>I</i>. <i>ricinus</i> IRE/CTVM20 cells were treated with <i>Tudor-SN</i>, <i>Rs86</i> or <i>GFP</i> control dsRNAs and infected with LGTV at MOI 0.1. Virus titers in the cell supernatants were determined by plaque assay after 24h and 48h and are presented relative to the <i>GFP</i> dsRNA control. Relative LGTV titers were compared between groups by two-way ANOVA and were not statistically different between <i>Tudor-SN</i> and <i>GFP</i> dsRNA-treated cells (P>0.05; N = 8). For each tick cell line, two independent experiments with 4 replicates each were conducted and combined after normalization.</p

Structure of the <i>I</i>. <i>scapularis</i> Tudor-SN.

<p>Tudor-SN amino acid sequence alignment between <i>I</i>. <i>scapularis</i> (B7QIP4_IXOSC), <i>I</i>. <i>ricinus</i> (V5GZ29_IXORI) and <i>Danio rerio</i> (Q5RGK8_DANRE). Conserved domains in <i>I</i>. <i>scapularis</i> Tudor-SN contain conserved staphylococcal nuclease homologues (SN, cd00175; red) and Tudor domain (cd04508; blue).</p

<i>Tudor-SN</i> expression in response to pathogen infection.

<p>(A) Differential expression/representation of Tudor-SN and Ago genes/proteins in <i>I</i>. <i>scapularis</i> nymphs, adult female guts and salivary glands in response to infection with <i>A</i>. <i>phagocytophilum</i>. Data was obtained from transcriptomics and proteomics analyses and values are shown as infected/uninfected Log2-fold ratio (P<0.05). Abbreviations:-, down-regulated/under-represented in infected ticks; +, up-regulated/over-represented in infected ticks; NS, no significant difference between infected and uninfected ticks; NF, not found in trascriptomics or proteomics data. (B) <i>Tudor-SN</i> mRNA levels in <i>I</i>. <i>scapularis</i> ISE6 cells in response to early (day 6) and late (day 13) infections with <i>A</i>. <i>phagocytophilum</i>. Five independent experiments were conducted for each early and late infection. <i>Tudor-SN</i> mRNA levels were determined by real-time RT-PCR in uninfected and infected cells and the infected-to-uninfected ratio of Ct values normalized against tick <i>16S rRNA</i> and <i>cyclophilin</i> are shown in arbitrary units (Ave+SD). Normalized <i>Tudor-SN</i> mRNA levels were compared by Student’s t-test with unequal variance and were not statistically different between infected and uninfected cells (P = 0.05; N = 5). (C) <i>Tudor-SN</i> mRNA levels in <i>I</i>. <i>ricinus</i> ticks during TBEV infection. <i>Tudor-SN</i> mRNA levels were determined by real-time RT-PCR in the guts and salivary glands of female ticks (N = 10 ticks for each time point) uninfected and artificially infected with TBEV and fed on mice for 0, 1, 3 and 5 days. <i>Tudor-SN</i> Ct values normalized against tick <i>16S rRNA</i> are shown in arbitrary units (Ave±SD) and were used to calculate infected/uninfected ratios and compared between Days 1–5 and Day 0 by Student’s t-test with unequal variance (*P<0.05; N = 10).</p

Effect of <i>Tudor-SN</i> knockdown on tick feeding.

<p>(A) <i>Tudor-SN</i> mRNA levels were analyzed using RNA extracted from <i>I</i>. <i>scapularis</i> eggs (three batches of approximately 500 eggs each), fed and unfed larvae (three pools of 50 larvae each), fed and unfed nymphs (three pools of 15 nymphs each), and fed and unfed male and female adult tick tissues (4 ticks each) by real-time RT-PCR and normalizing against tick <i>cyclophilin</i> and <i>ribosomal protein S4</i>. Normalized Ct values were compared between unfed and fed ticks by Student's t-test with unequal variance (*P<0.05). (B) <i>Tudor-SN</i> mRNA levels in <i>I</i>. <i>ricinus</i> ticks during tick feeding. <i>Tudor-SN</i> mRNA levels were determined by real-time RT-PCR in the guts and salivary glands of uninfected female ticks (N = 10 ticks for each time point) fed on mice for 0 (unfed ticks), 1, 3 and 5 days. <i>Tudor-SN</i> Ct values normalized against tick <i>16S rRNA</i> are shown in arbitrary units (Ave±SD) and were compared between Days 0–3 and Day 5 by Student's t-test with unequal variance (*P<0.05). (C) <i>I</i>. <i>scapularis</i> female ticks were injected with dsRNA and fed on sheep. Tick weights (Ave+SD) were compared between groups by Student's t-test with unequal variance (*P<0.05; N = 20 ticks per group). Panels show representative images at day 5 of tick feeding.</p