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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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Yeast Surface Display (YSD) approach to identify tick gut proteins that interact with B. burgdorferi membrane proteins.A. EBY-100 yeast cells transformed with an Ixodes scapularis 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. After each magnetic sort, binding of Alexa488-labeled B. burgdorferi membrane protein extract to EBY-100 yeast cells was analysed using flow cytometry. C. Flow cytometric analysis of individual yeast displayed clones from Sort 4. Borrelia-binding clones 1, 2 and 3 and one representative non-binding clones are shown. In panels B and C cell populations within the gate represent yeast-displayed clones bound to Alexa488-labeled B. burgdorferi membrane protein extract. D. 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 B. burgdorferi-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 (p<0.05) indicated by one asterisk (p<0.05) or two asterisks (p<0.01).
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ppat-1004278-g001: Yeast Surface Display (YSD) approach to identify tick gut proteins that interact with B. burgdorferi membrane proteins.A. EBY-100 yeast cells transformed with an Ixodes scapularis 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. After each magnetic sort, binding of Alexa488-labeled B. burgdorferi membrane protein extract to EBY-100 yeast cells was analysed using flow cytometry. C. Flow cytometric analysis of individual yeast displayed clones from Sort 4. Borrelia-binding clones 1, 2 and 3 and one representative non-binding clones are shown. In panels B and C cell populations within the gate represent yeast-displayed clones bound to Alexa488-labeled B. burgdorferi membrane protein extract. D. 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 B. burgdorferi-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 (p<0.05) indicated by one asterisk (p<0.05) or two asterisks (p<0.01).

Mentions: B. burgdorferi membrane protein extracts were prepared as described [18] from in vitro grown B. burgdorferi (N40) temperature-shifted to 37°C for 24 hours. A YSD expression library of I. scapularis gut cDNAs [14] was probed with biotin-labeled B. burgdorferi membrane protein extracts as described in Materials and Methods. Four rounds of magnetic-activated cell sorting (MACS) screens provided a 40-fold enrichment of YSD clones expressing gut proteins that interacted with B. burgdorferi membrane proteins (Fig. 1A–B). Cells from the 4th sort were plated and one hundred colonies were individually tested for their ability to bind to B. burgdorferi membrane protein extracts by fluorescence-activated cell sorting (FACS) analysis using Alexa488-labeled B. burgdorferi membrane protein extracts. Recombinant plasmids were isolated from colonies that showed greater than 15% binding (40 clones) (Fig. 1C) and insert sizes compared by restriction digestion analysis. Clones with identical insert sizes were grouped (four groups) and two representative clones from each group were sequenced. Four unique clones encoding partial fragments of tick gut proteins were identified and provided a unique identifier based on their in silico predicted function (Table 1).


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)

Yeast Surface Display (YSD) approach to identify tick gut proteins that interact with B. burgdorferi membrane proteins.A. EBY-100 yeast cells transformed with an Ixodes scapularis 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. After each magnetic sort, binding of Alexa488-labeled B. burgdorferi membrane protein extract to EBY-100 yeast cells was analysed using flow cytometry. C. Flow cytometric analysis of individual yeast displayed clones from Sort 4. Borrelia-binding clones 1, 2 and 3 and one representative non-binding clones are shown. In panels B and C cell populations within the gate represent yeast-displayed clones bound to Alexa488-labeled B. burgdorferi membrane protein extract. D. 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 B. burgdorferi-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 (p<0.05) indicated by one asterisk (p<0.05) or two asterisks (p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004278-g001: Yeast Surface Display (YSD) approach to identify tick gut proteins that interact with B. burgdorferi membrane proteins.A. EBY-100 yeast cells transformed with an Ixodes scapularis 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. After each magnetic sort, binding of Alexa488-labeled B. burgdorferi membrane protein extract to EBY-100 yeast cells was analysed using flow cytometry. C. Flow cytometric analysis of individual yeast displayed clones from Sort 4. Borrelia-binding clones 1, 2 and 3 and one representative non-binding clones are shown. In panels B and C cell populations within the gate represent yeast-displayed clones bound to Alexa488-labeled B. burgdorferi membrane protein extract. D. 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 B. burgdorferi-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 (p<0.05) indicated by one asterisk (p<0.05) or two asterisks (p<0.01).
Mentions: B. burgdorferi membrane protein extracts were prepared as described [18] from in vitro grown B. burgdorferi (N40) temperature-shifted to 37°C for 24 hours. A YSD expression library of I. scapularis gut cDNAs [14] was probed with biotin-labeled B. burgdorferi membrane protein extracts as described in Materials and Methods. Four rounds of magnetic-activated cell sorting (MACS) screens provided a 40-fold enrichment of YSD clones expressing gut proteins that interacted with B. burgdorferi membrane proteins (Fig. 1A–B). Cells from the 4th sort were plated and one hundred colonies were individually tested for their ability to bind to B. burgdorferi membrane protein extracts by fluorescence-activated cell sorting (FACS) analysis using Alexa488-labeled B. burgdorferi membrane protein extracts. Recombinant plasmids were isolated from colonies that showed greater than 15% binding (40 clones) (Fig. 1C) and insert sizes compared by restriction digestion analysis. Clones with identical insert sizes were grouped (four groups) and two representative clones from each group were sequenced. Four unique clones encoding partial fragments of tick gut proteins were identified and provided a unique identifier based on their in silico predicted function (Table 1).

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