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A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission.

Narasimhan S, Coumou J, Schuijt TJ, Boder E, Hovius JW, Fikrig E - PLoS Pathog. (2014)

Bottom Line: 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 Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Borrelia burgdorferi 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 Borrelia 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 Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.

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In vitro analysis of Ixofin3D-Borrelia burgdorferi interaction.A. Imunofluorescence microscopy of PFA-fixed in vitro grown B. burgdorferi to assess binding to rIxofin3D-PF. B. burgdorferi was detected with FITC-conjugated B. burgdorferi 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. ELISA assessment of dose-dependent binding of rIxofin3D-PF to B. burgdorferi membrane protein extract-coated plates compared to rIxophilin, a tick protein that does not bind to Borrelia.
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ppat-1004278-g004: In vitro analysis of Ixofin3D-Borrelia burgdorferi interaction.A. Imunofluorescence microscopy of PFA-fixed in vitro grown B. burgdorferi to assess binding to rIxofin3D-PF. B. burgdorferi was detected with FITC-conjugated B. burgdorferi 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. ELISA assessment of dose-dependent binding of rIxofin3D-PF to B. burgdorferi membrane protein extract-coated plates compared to rIxophilin, a tick protein that does not bind to Borrelia.

Mentions: We were unable to express the full-length protein transcript of Ixofin3D in the Drosophila expression system (DES), but succeeded in expressing a partial fragment of Ixofin3D protein encompassing amino acids 104 to 319 (rIxofin3D-PF) that is also contained in the protein fragment expressed in YSD Clone 1 (Fig. 2A). The 37 kDa rIxofin3D-PF generated in Drosophila S2 cells was glycosylated as seen by Periodic-Acid Schiff's staining (Fig. 3A). Polyclonal rabbit antibodies against rIxofin3D-PF bound to uninfected and B. burgdorferi-infected fed guts (Fig. 3B) as seen by confocal microscopy, suggesting that Ixofin3D is expressed on the surface of the gut. Consistent with the qRT-PCR analysis, quantification of pixel intensity in the TRITC channel (representing anti-rIxofin3D-PF serum binding to native Ixofin3D) using the ImageJ software showed significantly increased binding of rIxofin3D-PF antibodies to the tick gut in 24 h and 72 h fed ticks upon B. burgdorferi infection compared to that in 24 h and 72 h fed uninfected guts (Fig. 3C), and in B. burgdorferi-infected 72 h fed tick guts when compared to B. burgdorferi-infected 24 h fed guts (Fig. 3C) suggesting that Ixofin3D expression was increased in B. burgdorferi infected guts during feeding. rIxofin3D-PF incubated with in vitro grown PFA-fixed non-permeabilized B. burgdorferi N40 showed binding of rIxofin3D-PF to spirochetes as seen by indirect immunofluorescence using rabbit polyclonal antibodies against purified rIxofin3D-PF (Fig. 4A) indicating a potential interaction between Ixofin3D and an exposed B. burgdorferi protein ligand. Under similar conditions, rIxophilin, a tick gut thrombin inhibitor protein [22], not known to engage directly with spirochetes, did not show binding to in vitro grown spirochetes (Fig. 4A). Furthermore, in an ELISA assay using microplates coated with B. burgdorferi membrane protein extract, a dose-dependent increase in rIxofin3D-PF binding to B. burgdorferi membrane protein extracts was observed, whereas dose-dependent binding of rIxophilin was not observed (Fig. 4B).


A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission.

Narasimhan S, Coumou J, Schuijt TJ, Boder E, Hovius JW, Fikrig E - PLoS Pathog. (2014)

In vitro analysis of Ixofin3D-Borrelia burgdorferi interaction.A. Imunofluorescence microscopy of PFA-fixed in vitro grown B. burgdorferi to assess binding to rIxofin3D-PF. B. burgdorferi was detected with FITC-conjugated B. burgdorferi 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. ELISA assessment of dose-dependent binding of rIxofin3D-PF to B. burgdorferi membrane protein extract-coated plates compared to rIxophilin, a tick protein that does not bind to Borrelia.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4125277&req=5

ppat-1004278-g004: In vitro analysis of Ixofin3D-Borrelia burgdorferi interaction.A. Imunofluorescence microscopy of PFA-fixed in vitro grown B. burgdorferi to assess binding to rIxofin3D-PF. B. burgdorferi was detected with FITC-conjugated B. burgdorferi 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. ELISA assessment of dose-dependent binding of rIxofin3D-PF to B. burgdorferi membrane protein extract-coated plates compared to rIxophilin, a tick protein that does not bind to Borrelia.
Mentions: We were unable to express the full-length protein transcript of Ixofin3D in the Drosophila expression system (DES), but succeeded in expressing a partial fragment of Ixofin3D protein encompassing amino acids 104 to 319 (rIxofin3D-PF) that is also contained in the protein fragment expressed in YSD Clone 1 (Fig. 2A). The 37 kDa rIxofin3D-PF generated in Drosophila S2 cells was glycosylated as seen by Periodic-Acid Schiff's staining (Fig. 3A). Polyclonal rabbit antibodies against rIxofin3D-PF bound to uninfected and B. burgdorferi-infected fed guts (Fig. 3B) as seen by confocal microscopy, suggesting that Ixofin3D is expressed on the surface of the gut. Consistent with the qRT-PCR analysis, quantification of pixel intensity in the TRITC channel (representing anti-rIxofin3D-PF serum binding to native Ixofin3D) using the ImageJ software showed significantly increased binding of rIxofin3D-PF antibodies to the tick gut in 24 h and 72 h fed ticks upon B. burgdorferi infection compared to that in 24 h and 72 h fed uninfected guts (Fig. 3C), and in B. burgdorferi-infected 72 h fed tick guts when compared to B. burgdorferi-infected 24 h fed guts (Fig. 3C) suggesting that Ixofin3D expression was increased in B. burgdorferi infected guts during feeding. rIxofin3D-PF incubated with in vitro grown PFA-fixed non-permeabilized B. burgdorferi N40 showed binding of rIxofin3D-PF to spirochetes as seen by indirect immunofluorescence using rabbit polyclonal antibodies against purified rIxofin3D-PF (Fig. 4A) indicating a potential interaction between Ixofin3D and an exposed B. burgdorferi protein ligand. Under similar conditions, rIxophilin, a tick gut thrombin inhibitor protein [22], not known to engage directly with spirochetes, did not show binding to in vitro grown spirochetes (Fig. 4A). Furthermore, in an ELISA assay using microplates coated with B. burgdorferi membrane protein extract, a dose-dependent increase in rIxofin3D-PF binding to B. burgdorferi membrane protein extracts was observed, whereas dose-dependent binding of rIxophilin was not observed (Fig. 4B).

Bottom Line: 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 Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Borrelia burgdorferi 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 Borrelia 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 Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.

Show MeSH
Related in: MedlinePlus