<i>I</i>. <i>ricinus</i> infestations on rIr86-1, rIr86-2 or rIr86-combination vaccinated rabbits.

<p>Rabbits were infested with 50 <i>I</i>. <i>ricinus</i> adult females and 50 <i>I</i>. <i>ricinus</i> adult males two weeks after the second boost (t = 56) in a pilot experiment (experiment A) and a confirmatory experiment (experiment B). Each column on the x-axis represents one animal in experiment A (A and B) or experiment B (C and D). <b>A.</b> Post feeding female tick weight in experiment A, each dot represents one tick. <b>B.</b> Egg mass from experiment A. Fed adult female ticks were stored individually. After 6 weeks, egg mass was weighted. Each dot represent egg mass from one female tick. <b>C.</b> Post feeding female tick weight in experiment B. <b>D.</b> Egg mass from experiment B. Error bars represent mean ± SEM. Mean values significantly different in a one-way analysis of variance (ANOVA) with the Tukey-Kramer multiple-comparison test for multi-group comparisons are indicated by two asterisk (p < 0.01) or three asterisks (p < 0.001).</p

Specificity and IgG response against rIr86-1 and rIr86-2 in experiment A (pilot experiment) and B (confirmatory experiment).

<p>IgG in serum (1:10,000) was assessed with ELISA using 1 μg/mL of rIr86-1 and rIr86-2 on anti-V5 coated plates. <b>A and B</b>. IgG response in serum from animals in experiment A vaccinated with rIr86-1 (circle), rIr86-2 (triangle), rIr86-combination (diamond) or ovalbumin (square) (n = 1 for each vaccine) reacting to rIr86-1 (A) or rIr86-2 (B). <b>C.</b> Mean IgG response in serum (1:10,000) from three animals vaccinated against rIr86-combination in experiment B measuring IgG response to rIr86-1 (circle) or rIr86-2 (triangle). <b>D.</b> Mean IgG titer in serum from three animals vaccinated against rIr86-combination in experiment B, diluted 1:10² to 1:10⁷ on ELISA coated with rIr86-1 (circle) or rIr86-2 (triangle). Error bars represent mean ± SEM.</p

Expression of recombinant Ir86-1 and Ir86-2 and detection of Ir86-1 and Ir86-2 in <i>I</i>. <i>ricinus</i> gut lysate.

<p><b>A.</b> Purified <i>Drosophila</i>-expressed recombinant Ir86-1 (Lane 1) and Ir86-2 (Lane 2) electrophoresed on SDS 7.5% polyacrylamide gel and stained with Coomassie blue. <b>B.</b> Gut lysate from 20 adult female <i>I</i>. <i>ricinus</i> tick guts electrophoresed on SDS 10% polyacrylamide gel stained with Coomassie blue (Lane 1) or transferred to a PVDF membrane which was probed with antiserum (1:200) from rabbits vaccinated against rIr86-1 (Lane 2), rIr86-2 (Lane 3) or with antiserum from the control rabbit vaccinated against ovalbumin (Lane 4).</p

A Tick Gut Protein with Fibronectin III Domains Aids <i>Borrelia burgdorferi</i> Congregation to the Gut during Transmission

<div><p><i>Borrelia burgdorferi</i> transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with <i>Borrelia</i> membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between <i>Borrelia</i> and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut.</p></div

<i>I. scapularis</i> gut yeast surface display library screening identifies four potential <i>Borrelia</i>-interacting tick gut proteins.

I<p>database of <a href="http://www.vectorbase.org" target="_blank">www.vectorbase.org</a>,</p>II<p>as stated on Genbank annotations,</p>III<p>SMART domain prediction (<a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>),</p>IV<p>PSORT (<a href="http://psort.hgc.jp/" target="_blank">http://psort.hgc.jp/</a>),</p>V<p>SignalP analysis (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>),</p>VI<p>Theoretical molecular weight (MW) in kDa using ExPASy proteomics server (<a href="http://web.expasy.org/compute_pi/" target="_blank">http://web.expasy.org/compute_pi/</a>).</p><p>*Based on Ixofin3D sequence, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004278#ppat-1004278-g002" target="_blank">Figure 2</a>. ORF: Open Reading Frame.</p

Yeast Surface Display (YSD) approach to identify tick gut proteins that interact with <i>B. burgdorferi</i> membrane proteins.

<p><b>A.</b> EBY-100 yeast cells transformed with an <i>Ixodes scapularis</i> salivary gland cDNA library were induced overnight before magnetic sorting. The following day, surface tick protein expression as fusion proteins with the yeast agglutinin protein Aga2p was confirmed with antibodies against the Xpress epitope using flow cytometry. <b>B.</b> After each magnetic sort, binding of Alexa488-labeled <i>B. burgdorferi</i> membrane protein extract to EBY-100 yeast cells was analysed using flow cytometry. <b>C.</b> Flow cytometric analysis of individual yeast displayed clones from Sort 4. <i>Borrelia</i>-binding clones 1, 2 and 3 and one representative non-binding clones are shown. In panels <b>B</b> and <b>C</b> cell populations within the gate represent yeast-displayed clones bound to Alexa488-labeled <i>B. burgdorferi</i> membrane protein extract. <b>D.</b> Quantitative RT-PCR assessment of the mRNA expression profiles of the genes encoding clones 1, 2 and 3 in salivary glands (SG) and guts (MG) of <i>B. burgdorferi</i>-infected and uninfected nymphs at 24 and 72 hours post tick attachment. Error bars represent mean ± SEM and mean values significantly different in a two-tailed non-parametric Mann-Whitney test (<i>p</i><0.05) indicated by one asterisk (<i>p<0.05</i>) or two asterisks (<i>p</i><0.01).</p

Ixofin3D localization in the tick gut.

<p><b>A.</b> Purified <i>Drosophila</i>-expressed recombinant Ixofin3D-PF electrophoresed on SDS 12% polyacrylamide gel and <b>Lane 1</b>, Coomassie blue stained; <b>Lane 2</b>, Periodic Acid-Schiff stained; and <b>Lane 3</b>, rIxofin3D-PF immunoblotted and probed with polyclonal rabbit anti-Ixofin3D-PF serum. <b>B</b>. Confocal microscopy of PFA-fixed guts of 24 and 72h fed uninfected and <i>B. burgdorferi</i>-infected nymphs. Gut nuclei, <i>B. burgdorferi</i> and Ixofin3D stained with TO-PRO-3 (blue), anti <i>B. burgdorferi</i> (FITC-green) and anti-rIxofin3D-PF serum (TRITC-red) respectively. Magnification ×20. Guts stained with anti-Ovalbumin IgG (TRITC-red) served as antibody control. <b>C</b>. Mean pixel intensities of regions of interest in the TRITC channel (representing anti-rIxofin3D-PF serum binding to Ixofin3D) of the confocal images obtained in <b>B</b>. Each data point represents one region of interest. Error bars represent mean ± SEM and mean values significantly different in a one-way ANOVA with Tukey's multiple comparison test indicated by two asterisks (<i>p</i><0.01) or indicated by three asterisks (<i>p</i><0.0001).</p

<i>In vitro</i> analysis of Ixofin3D-<i>Borrelia burgdorferi</i> interaction.

<p><b>A.</b> Imunofluorescence microscopy of PFA-fixed <i>in vitro</i> grown <i>B. burgdorferi</i> to assess binding to rIxofin3D-PF. <i>B. burgdorferi</i> was detected with FITC-conjugated <i>B. burgdorferi</i> antisera (FITC-green), rIxofin3D-PF (Panel 1) was detected using rabbit anti-Ixofin3D-PF IgG (TRITC-red), and rIxophilin (Panel 2) was detected using mouse anti-rIxophilin IgG (TRITC-red). Magnification ×20. <b>B.</b> ELISA assessment of dose-dependent binding of rIxofin3D-PF to <i>B. burgdorferi</i> membrane protein extract-coated plates compared to rIxophilin, a tick protein that does not bind to <i>Borrelia</i>.</p

RNA-mediated knockdown of <i>ixofin3D</i> expression results in decreased aggregation of <i>Borrelia burgdorferi</i> on the gut.

<p><b>A.</b> Confocal microscopy of PFA-fixed guts from 72 h fed <i>B. burgdorferi</i>-infected nymphs injected with ds <i>ixofin3D</i> or ds <i>gfp</i> RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-<i>B. burgdorferi</i> (N40) IgG (FITC-green) respectively. <b>B</b>. Mean pixel intensities of regions of interest in the FITC channel (representing anti-<i>B. burgdorferi</i> serum binding to spirochetes) of the confocal images obtained in <b>A</b>. Each data point represents one region of interest. <b>C</b>. Confocal microscopy of PFA-fixed salivary glands from 72 h fed <i>B. burgdorferi</i>-infected nymphs injected with ds <i>ixofin3D</i> or ds <i>gfp</i> RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-<i>B. burgdorferi</i> (N40) IgG (FITC-green) respectively. In <b>A</b> and <b>C</b> magnification ×40. <b>D</b>. Spirochetes in each salivary gland pair counted manually. Each data point represents one salivary gland pair. <b>E</b>. Confocal microscopy of guts from 72 h fed <i>B. burgdorferi</i>-infected nymphs injected with ds <i>ixofin3D</i> or ds <i>gfp</i> RNA washed to remove unbound <i>Borrelia</i>, and PFA fixed prior to staining. Nuclei were stained with propidium iodide (red), and spirochetes with anti-<i>B. burgdorferi</i> (N40) IgG (FITC-green). Magnification ×40. <b>F</b>. qRT-PCR assessment of <i>Borrelia</i> burden in washed tick guts. Each data point in B represents a pool of 3 guts. <b>G</b>. Mean pixel intensities of regions of interest in the FITC channel (representing anti-<i>B. burgdorferi</i> serum binding) of the confocal images obtained in <b>E</b>. Each data point represents one region of interest. Error bars in <b>B</b>, <b>D</b>, <b>F</b> and <b>G</b> represent mean ± SEM. Mean values significantly different in a two-tailed non-parametric Mann-Whitney test indicated by one asterisk (<i>p</i><0.05) or indicated by two asterisks (<i>p</i><0.01).</p

Impact of passive and active immunization against Ixofin3D-PF on <i>B. burgdorferi</i> burden in ticks and in murine skin.

<p><b>A–C:</b> Rabbit anti-rIxofin3D-PF serum or rabbit anti-Ovalbumin serum was passively transferred into each mouse 24 h prior to <i>B. burgdorferi</i>-infected tick challenge (4–5 ticks/mouse). <b>A.</b> Engorgement weights of repleted ticks; <b>B.</b> qRT-PCR assessment of <i>Borrelia</i> burden in tick guts and salivary glands; <b>C.</b> qPCR assessment of <i>Borrelia</i> burden in murine skin at 7 and 14 days post-tick feeding. <b>D–F:</b> Mice actively immunized with rIxofin3D-PF or Ovalbumin and challenged with <i>B. burgdorferi</i>-infected ticks (4–5 ticks/mouse). <b>D.</b> Engorgement weights of repleted ticks; <b>E.</b> qRT-PCR assessment of <i>Borrelia</i> burden in tick guts and salivary glands; <b>F.</b> qPCR assessment of <i>Borrelia</i> burden in mice skin at 7 and 14 days post-tick feeding. Each data point in A and D represents one tick. Each data point in B and E represents a pool of 2–3 guts or salivary glands. Each data point in C and F represents one mouse. Error bars represent mean ± SEM and mean values significantly different in a two-tailed non-parametric Mann-Whitney test (<i>p</i><0.05) indicated by an asterisk. A representative of three experiments is shown.</p