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EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin.

Mohan Kumar D, Lin M, Xiong Q, Webber MJ, Kural C, Rikihisa Y - MBio (2015)

Bottom Line: Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci.Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA.

No MeSH data available.


Related in: MedlinePlus

N-WASP is recruited to rEtpE-C-coated-bead entry foci, and it interacts with rEtpE-C and native EtpE. (A) Spinning-disk confocal microscopy images at four different time points from the three-dimensional time-lapse video of RF/6A cells transfected with GFP–N-WASP (cyan) and then incubated with rEtpE-C-coated beads (red). xy planes show the upper surface of an RF/6A cell that had internalized rEtpE-C-coated beads. Each white arrow indicates a cluster of N-WASP. Cell surface extensions surrounding the cluster of beads are highlighted with yellow arrows in the side views (xz and yz planes). Scale bar, 10 µm. (B) Affinity pulldown of THP-1 cell lysate incubated with rEtpE-C or rECH0825 bound to Ni-silica matrix. Bound proteins were eluted with imidazole and subjected to immunoblot analysis with anti-N-WASP and anti-His antibodies. One lane between rEtpE-C and rECH0825 was deleted to save the space without altering molecular mass or exposure time. (C) THP-1 cells were incubated with E. chaffeensis for 30 min, lysed, and immunoprecipitated (IP) with anti-EtpE-C antibody- or control IgG-bound protein A-agarose. The immunoprecipitates were subjected to immunoblotting with anti-N-WASP antibody. (B and C) Numbers to the left indicate kDa.
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fig5: N-WASP is recruited to rEtpE-C-coated-bead entry foci, and it interacts with rEtpE-C and native EtpE. (A) Spinning-disk confocal microscopy images at four different time points from the three-dimensional time-lapse video of RF/6A cells transfected with GFP–N-WASP (cyan) and then incubated with rEtpE-C-coated beads (red). xy planes show the upper surface of an RF/6A cell that had internalized rEtpE-C-coated beads. Each white arrow indicates a cluster of N-WASP. Cell surface extensions surrounding the cluster of beads are highlighted with yellow arrows in the side views (xz and yz planes). Scale bar, 10 µm. (B) Affinity pulldown of THP-1 cell lysate incubated with rEtpE-C or rECH0825 bound to Ni-silica matrix. Bound proteins were eluted with imidazole and subjected to immunoblot analysis with anti-N-WASP and anti-His antibodies. One lane between rEtpE-C and rECH0825 was deleted to save the space without altering molecular mass or exposure time. (C) THP-1 cells were incubated with E. chaffeensis for 30 min, lysed, and immunoprecipitated (IP) with anti-EtpE-C antibody- or control IgG-bound protein A-agarose. The immunoprecipitates were subjected to immunoblotting with anti-N-WASP antibody. (B and C) Numbers to the left indicate kDa.

Mentions: rEtpE-C-coated beads enter nonphagocytic and phagocytic cells in a DNase X-dependent manner (11). To further study whether N-WASP is specifically recruited to entry foci in the absence of other bacterial proteins, we incubated GFP–N-WASP-transfected RF/6A cells with rEtpE-C-coated beads. Live-cell imaging with spinning-disk confocal microscopy revealed that GFP–N-WASP was recruited to entry foci within 1 min of incubation (Fig. 5A, cluster of beads denoted by white arrows; see also Movie S1 in the supplemental material); it appeared to be concentrated at the base of a filopodiumlike cell surface extension embracing the beads and was associated with the beads until the beads were ultimately internalized (Fig. 5A, yellow arrowheads in the xz and yz side views).


EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin.

Mohan Kumar D, Lin M, Xiong Q, Webber MJ, Kural C, Rikihisa Y - MBio (2015)

N-WASP is recruited to rEtpE-C-coated-bead entry foci, and it interacts with rEtpE-C and native EtpE. (A) Spinning-disk confocal microscopy images at four different time points from the three-dimensional time-lapse video of RF/6A cells transfected with GFP–N-WASP (cyan) and then incubated with rEtpE-C-coated beads (red). xy planes show the upper surface of an RF/6A cell that had internalized rEtpE-C-coated beads. Each white arrow indicates a cluster of N-WASP. Cell surface extensions surrounding the cluster of beads are highlighted with yellow arrows in the side views (xz and yz planes). Scale bar, 10 µm. (B) Affinity pulldown of THP-1 cell lysate incubated with rEtpE-C or rECH0825 bound to Ni-silica matrix. Bound proteins were eluted with imidazole and subjected to immunoblot analysis with anti-N-WASP and anti-His antibodies. One lane between rEtpE-C and rECH0825 was deleted to save the space without altering molecular mass or exposure time. (C) THP-1 cells were incubated with E. chaffeensis for 30 min, lysed, and immunoprecipitated (IP) with anti-EtpE-C antibody- or control IgG-bound protein A-agarose. The immunoprecipitates were subjected to immunoblotting with anti-N-WASP antibody. (B and C) Numbers to the left indicate kDa.
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Related In: Results  -  Collection

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fig5: N-WASP is recruited to rEtpE-C-coated-bead entry foci, and it interacts with rEtpE-C and native EtpE. (A) Spinning-disk confocal microscopy images at four different time points from the three-dimensional time-lapse video of RF/6A cells transfected with GFP–N-WASP (cyan) and then incubated with rEtpE-C-coated beads (red). xy planes show the upper surface of an RF/6A cell that had internalized rEtpE-C-coated beads. Each white arrow indicates a cluster of N-WASP. Cell surface extensions surrounding the cluster of beads are highlighted with yellow arrows in the side views (xz and yz planes). Scale bar, 10 µm. (B) Affinity pulldown of THP-1 cell lysate incubated with rEtpE-C or rECH0825 bound to Ni-silica matrix. Bound proteins were eluted with imidazole and subjected to immunoblot analysis with anti-N-WASP and anti-His antibodies. One lane between rEtpE-C and rECH0825 was deleted to save the space without altering molecular mass or exposure time. (C) THP-1 cells were incubated with E. chaffeensis for 30 min, lysed, and immunoprecipitated (IP) with anti-EtpE-C antibody- or control IgG-bound protein A-agarose. The immunoprecipitates were subjected to immunoblotting with anti-N-WASP antibody. (B and C) Numbers to the left indicate kDa.
Mentions: rEtpE-C-coated beads enter nonphagocytic and phagocytic cells in a DNase X-dependent manner (11). To further study whether N-WASP is specifically recruited to entry foci in the absence of other bacterial proteins, we incubated GFP–N-WASP-transfected RF/6A cells with rEtpE-C-coated beads. Live-cell imaging with spinning-disk confocal microscopy revealed that GFP–N-WASP was recruited to entry foci within 1 min of incubation (Fig. 5A, cluster of beads denoted by white arrows; see also Movie S1 in the supplemental material); it appeared to be concentrated at the base of a filopodiumlike cell surface extension embracing the beads and was associated with the beads until the beads were ultimately internalized (Fig. 5A, yellow arrowheads in the xz and yz side views).

Bottom Line: Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci.Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization.Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA.

No MeSH data available.


Related in: MedlinePlus