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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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RNA-mediated knockdown of ixofin3D expression results in decreased aggregation of Borrelia burgdorferi on the gut.A. Confocal microscopy of PFA-fixed guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. B. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding to spirochetes) of the confocal images obtained in A. Each data point represents one region of interest. C. Confocal microscopy of PFA-fixed salivary glands from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. In A and C magnification ×40. D. Spirochetes in each salivary gland pair counted manually. Each data point represents one salivary gland pair. E. Confocal microscopy of guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA washed to remove unbound Borrelia, and PFA fixed prior to staining. Nuclei were stained with propidium iodide (red), and spirochetes with anti-B. burgdorferi (N40) IgG (FITC-green). Magnification ×40. F. qRT-PCR assessment of Borrelia burden in washed tick guts. Each data point in B represents a pool of 3 guts. G. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding) of the confocal images obtained in E. Each data point represents one region of interest. Error bars in B, D, F and G represent mean ± SEM. Mean values significantly different in a two-tailed non-parametric Mann-Whitney test indicated by one asterisk (p<0.05) or indicated by two asterisks (p<0.01).
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ppat-1004278-g007: RNA-mediated knockdown of ixofin3D expression results in decreased aggregation of Borrelia burgdorferi on the gut.A. Confocal microscopy of PFA-fixed guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. B. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding to spirochetes) of the confocal images obtained in A. Each data point represents one region of interest. C. Confocal microscopy of PFA-fixed salivary glands from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. In A and C magnification ×40. D. Spirochetes in each salivary gland pair counted manually. Each data point represents one salivary gland pair. E. Confocal microscopy of guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA washed to remove unbound Borrelia, and PFA fixed prior to staining. Nuclei were stained with propidium iodide (red), and spirochetes with anti-B. burgdorferi (N40) IgG (FITC-green). Magnification ×40. F. qRT-PCR assessment of Borrelia burden in washed tick guts. Each data point in B represents a pool of 3 guts. G. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding) of the confocal images obtained in E. Each data point represents one region of interest. Error bars in B, D, F and G represent mean ± SEM. Mean values significantly different in a two-tailed non-parametric Mann-Whitney test indicated by one asterisk (p<0.05) or indicated by two asterisks (p<0.01).

Mentions: Borrelia replicates in the gut in preparation for transmission, and adheres tightly to the gut epithelium in order to migrate towards the basal lamina of the gut epithelium, moving away from the lumen [13]. RNAi-mediated decrease in ixofin3D expression did not demonstrate alteration in Borrelia burden in the tick gut as seen by qRT-PCR (Fig. 5B). However, this assessment cannot distinguish gut epithelium-bound Borrelia from those that are not bound to the gut epithelium. Therefore, we assessed by confocal microscopy, if Ixofin3D might enhance Borrelia adherence to the gut epithelium. RNAi-mediated decrease in ixofin3D expression resulted in significantly decreased Borrelia clustering to the gut epithelium as seen by confocal microscopy (Fig. 7A) and quantification of the pixel intensity in the FITC channel (representing binding of anti-B. burgdorferi serum to spirochetes) using the ImajeJ software (Fig. 7B). Consistent with the qRT-PCR observations, the numbers of spirochetes was also significantly reduced in the salivary glands of ds ixofin3D RNA-injected nymphs (Fig. 7C–D). To further assess if Ixofin3D might facilitate spirochete aggregation to the tick gut, fed tick guts were washed to remove luminal blood-meal contents and spirochetes loosely adhering to the gut epithelium. Borrelia burden assessed in the washed gut epithelium by confocal microscopy and quantification of the pixel intensity in the FITC channel using the ImageJ software and by qRT-PCR showed decreased gut-bound Borrelia burden in ds ixofin3D RNA-injected nymphal guts when compared to that in ds gfp RNA-injected nymphal guts (Fig. 7E–G).


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)

RNA-mediated knockdown of ixofin3D expression results in decreased aggregation of Borrelia burgdorferi on the gut.A. Confocal microscopy of PFA-fixed guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. B. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding to spirochetes) of the confocal images obtained in A. Each data point represents one region of interest. C. Confocal microscopy of PFA-fixed salivary glands from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. In A and C magnification ×40. D. Spirochetes in each salivary gland pair counted manually. Each data point represents one salivary gland pair. E. Confocal microscopy of guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA washed to remove unbound Borrelia, and PFA fixed prior to staining. Nuclei were stained with propidium iodide (red), and spirochetes with anti-B. burgdorferi (N40) IgG (FITC-green). Magnification ×40. F. qRT-PCR assessment of Borrelia burden in washed tick guts. Each data point in B represents a pool of 3 guts. G. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding) of the confocal images obtained in E. Each data point represents one region of interest. Error bars in B, D, F and G represent mean ± SEM. Mean values significantly different in a two-tailed non-parametric Mann-Whitney test indicated by one asterisk (p<0.05) or indicated by two asterisks (p<0.01).
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ppat-1004278-g007: RNA-mediated knockdown of ixofin3D expression results in decreased aggregation of Borrelia burgdorferi on the gut.A. Confocal microscopy of PFA-fixed guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. B. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding to spirochetes) of the confocal images obtained in A. Each data point represents one region of interest. C. Confocal microscopy of PFA-fixed salivary glands from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA. Nuclei, and spirochetes stained with propidium iodide (red), and anti-B. burgdorferi (N40) IgG (FITC-green) respectively. In A and C magnification ×40. D. Spirochetes in each salivary gland pair counted manually. Each data point represents one salivary gland pair. E. Confocal microscopy of guts from 72 h fed B. burgdorferi-infected nymphs injected with ds ixofin3D or ds gfp RNA washed to remove unbound Borrelia, and PFA fixed prior to staining. Nuclei were stained with propidium iodide (red), and spirochetes with anti-B. burgdorferi (N40) IgG (FITC-green). Magnification ×40. F. qRT-PCR assessment of Borrelia burden in washed tick guts. Each data point in B represents a pool of 3 guts. G. Mean pixel intensities of regions of interest in the FITC channel (representing anti-B. burgdorferi serum binding) of the confocal images obtained in E. Each data point represents one region of interest. Error bars in B, D, F and G represent mean ± SEM. Mean values significantly different in a two-tailed non-parametric Mann-Whitney test indicated by one asterisk (p<0.05) or indicated by two asterisks (p<0.01).
Mentions: Borrelia replicates in the gut in preparation for transmission, and adheres tightly to the gut epithelium in order to migrate towards the basal lamina of the gut epithelium, moving away from the lumen [13]. RNAi-mediated decrease in ixofin3D expression did not demonstrate alteration in Borrelia burden in the tick gut as seen by qRT-PCR (Fig. 5B). However, this assessment cannot distinguish gut epithelium-bound Borrelia from those that are not bound to the gut epithelium. Therefore, we assessed by confocal microscopy, if Ixofin3D might enhance Borrelia adherence to the gut epithelium. RNAi-mediated decrease in ixofin3D expression resulted in significantly decreased Borrelia clustering to the gut epithelium as seen by confocal microscopy (Fig. 7A) and quantification of the pixel intensity in the FITC channel (representing binding of anti-B. burgdorferi serum to spirochetes) using the ImajeJ software (Fig. 7B). Consistent with the qRT-PCR observations, the numbers of spirochetes was also significantly reduced in the salivary glands of ds ixofin3D RNA-injected nymphs (Fig. 7C–D). To further assess if Ixofin3D might facilitate spirochete aggregation to the tick gut, fed tick guts were washed to remove luminal blood-meal contents and spirochetes loosely adhering to the gut epithelium. Borrelia burden assessed in the washed gut epithelium by confocal microscopy and quantification of the pixel intensity in the FITC channel using the ImageJ software and by qRT-PCR showed decreased gut-bound Borrelia burden in ds ixofin3D RNA-injected nymphal guts when compared to that in ds gfp RNA-injected nymphal guts (Fig. 7E–G).

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