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Shigella Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation.

Lu R, Herrera BB, Eshleman HD, Fu Y, Bloom A, Li Z, Sacks DB, Goldberg MB - PLoS Pathog. (2015)

Bottom Line: We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread.OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1.Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts, United States of America; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.

No MeSH data available.


Related in: MedlinePlus

OspB and IQGAP1 reciprocally co-precipitate.(A) GST-IQGAP1 precipitates OspB-FLAG, but not IpgD-FLAG or IpaC-FLAG from S. flexneri culture supernatants. Western using FLAG antibody. P, precipitated proteins; S, supernatant. (B) GST-IQGAP1, but not GST alone precipitates His-OspB. Each protein was purified from E. coli (see Materials and Methods). The top two panels are from the same western blot, probed with antibody to His. The bottom panel is a Coomassie stain of the top portion of the same SDS-PAGE gel; GST can be seen as a white band migrating at 25 kD. (C) GST-OspB, but not GST alone precipitates IQGAP1. Each protein was purified from E. coli (see Materials and Methods). All five panels are from the same gel. After transfer, the top three panels were probed with antibody to IQGAP1, and the bottom two panels were probed with antibody to GST. (D) GST-OspB, but not GST precipitates endogenous IQGAP1 from lysates of MCF-7. GST-OspB and GST were purified from E. coli. Top and bottom panels are from the same gel. Top panel, western probed with antibody to IQGAP1; bottom panel, Coomassie stained gel. (E) Diagram of IQGAP1 fragments used for mapping site of OspB interaction. CHD, calponin homology domain; WW, polyproline binding region; IQ, IQ region; GRD, Ras GTPase-activating protein-related domain; aa, amino acid. (F) IQGAP1 full length protein and fragments precipitated by GST-OspB, but not by GST alone. GST-OspB and GST were generated in E. coli, and IQGAP1 constructs were generated as biotinylated proteins by in vitro transcription and translation. Biotinylated proteins were detected with HRP-labeled streptavidin (see Materials and Methods). Data represent three or more independent experiments. Apparent MWs are indicated in Kd.
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ppat.1005200.g002: OspB and IQGAP1 reciprocally co-precipitate.(A) GST-IQGAP1 precipitates OspB-FLAG, but not IpgD-FLAG or IpaC-FLAG from S. flexneri culture supernatants. Western using FLAG antibody. P, precipitated proteins; S, supernatant. (B) GST-IQGAP1, but not GST alone precipitates His-OspB. Each protein was purified from E. coli (see Materials and Methods). The top two panels are from the same western blot, probed with antibody to His. The bottom panel is a Coomassie stain of the top portion of the same SDS-PAGE gel; GST can be seen as a white band migrating at 25 kD. (C) GST-OspB, but not GST alone precipitates IQGAP1. Each protein was purified from E. coli (see Materials and Methods). All five panels are from the same gel. After transfer, the top three panels were probed with antibody to IQGAP1, and the bottom two panels were probed with antibody to GST. (D) GST-OspB, but not GST precipitates endogenous IQGAP1 from lysates of MCF-7. GST-OspB and GST were purified from E. coli. Top and bottom panels are from the same gel. Top panel, western probed with antibody to IQGAP1; bottom panel, Coomassie stained gel. (E) Diagram of IQGAP1 fragments used for mapping site of OspB interaction. CHD, calponin homology domain; WW, polyproline binding region; IQ, IQ region; GRD, Ras GTPase-activating protein-related domain; aa, amino acid. (F) IQGAP1 full length protein and fragments precipitated by GST-OspB, but not by GST alone. GST-OspB and GST were generated in E. coli, and IQGAP1 constructs were generated as biotinylated proteins by in vitro transcription and translation. Biotinylated proteins were detected with HRP-labeled streptavidin (see Materials and Methods). Data represent three or more independent experiments. Apparent MWs are indicated in Kd.

Mentions: We interrogated whether IQGAP1 might interact with any of 26 effector proteins translocated by the Shigella type 3 secretion system. GST-IQGAP1 pulled down OspB-FLAG from the culture supernatant of S. flexneri that expressed and secreted it (Fig 2A). Pull down by GST-IQGAP1 was specific, as other secreted proteins, including the effector IpgD and the secreted translocon protein IpaC, were not pulled down (Fig 2A), and was independent of other mammalian proteins, as GST-IQGAP1 used in these experiments had been purified from E. coli.


Shigella Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation.

Lu R, Herrera BB, Eshleman HD, Fu Y, Bloom A, Li Z, Sacks DB, Goldberg MB - PLoS Pathog. (2015)

OspB and IQGAP1 reciprocally co-precipitate.(A) GST-IQGAP1 precipitates OspB-FLAG, but not IpgD-FLAG or IpaC-FLAG from S. flexneri culture supernatants. Western using FLAG antibody. P, precipitated proteins; S, supernatant. (B) GST-IQGAP1, but not GST alone precipitates His-OspB. Each protein was purified from E. coli (see Materials and Methods). The top two panels are from the same western blot, probed with antibody to His. The bottom panel is a Coomassie stain of the top portion of the same SDS-PAGE gel; GST can be seen as a white band migrating at 25 kD. (C) GST-OspB, but not GST alone precipitates IQGAP1. Each protein was purified from E. coli (see Materials and Methods). All five panels are from the same gel. After transfer, the top three panels were probed with antibody to IQGAP1, and the bottom two panels were probed with antibody to GST. (D) GST-OspB, but not GST precipitates endogenous IQGAP1 from lysates of MCF-7. GST-OspB and GST were purified from E. coli. Top and bottom panels are from the same gel. Top panel, western probed with antibody to IQGAP1; bottom panel, Coomassie stained gel. (E) Diagram of IQGAP1 fragments used for mapping site of OspB interaction. CHD, calponin homology domain; WW, polyproline binding region; IQ, IQ region; GRD, Ras GTPase-activating protein-related domain; aa, amino acid. (F) IQGAP1 full length protein and fragments precipitated by GST-OspB, but not by GST alone. GST-OspB and GST were generated in E. coli, and IQGAP1 constructs were generated as biotinylated proteins by in vitro transcription and translation. Biotinylated proteins were detected with HRP-labeled streptavidin (see Materials and Methods). Data represent three or more independent experiments. Apparent MWs are indicated in Kd.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608727&req=5

ppat.1005200.g002: OspB and IQGAP1 reciprocally co-precipitate.(A) GST-IQGAP1 precipitates OspB-FLAG, but not IpgD-FLAG or IpaC-FLAG from S. flexneri culture supernatants. Western using FLAG antibody. P, precipitated proteins; S, supernatant. (B) GST-IQGAP1, but not GST alone precipitates His-OspB. Each protein was purified from E. coli (see Materials and Methods). The top two panels are from the same western blot, probed with antibody to His. The bottom panel is a Coomassie stain of the top portion of the same SDS-PAGE gel; GST can be seen as a white band migrating at 25 kD. (C) GST-OspB, but not GST alone precipitates IQGAP1. Each protein was purified from E. coli (see Materials and Methods). All five panels are from the same gel. After transfer, the top three panels were probed with antibody to IQGAP1, and the bottom two panels were probed with antibody to GST. (D) GST-OspB, but not GST precipitates endogenous IQGAP1 from lysates of MCF-7. GST-OspB and GST were purified from E. coli. Top and bottom panels are from the same gel. Top panel, western probed with antibody to IQGAP1; bottom panel, Coomassie stained gel. (E) Diagram of IQGAP1 fragments used for mapping site of OspB interaction. CHD, calponin homology domain; WW, polyproline binding region; IQ, IQ region; GRD, Ras GTPase-activating protein-related domain; aa, amino acid. (F) IQGAP1 full length protein and fragments precipitated by GST-OspB, but not by GST alone. GST-OspB and GST were generated in E. coli, and IQGAP1 constructs were generated as biotinylated proteins by in vitro transcription and translation. Biotinylated proteins were detected with HRP-labeled streptavidin (see Materials and Methods). Data represent three or more independent experiments. Apparent MWs are indicated in Kd.
Mentions: We interrogated whether IQGAP1 might interact with any of 26 effector proteins translocated by the Shigella type 3 secretion system. GST-IQGAP1 pulled down OspB-FLAG from the culture supernatant of S. flexneri that expressed and secreted it (Fig 2A). Pull down by GST-IQGAP1 was specific, as other secreted proteins, including the effector IpgD and the secreted translocon protein IpaC, were not pulled down (Fig 2A), and was independent of other mammalian proteins, as GST-IQGAP1 used in these experiments had been purified from E. coli.

Bottom Line: We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread.OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1.Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts, United States of America; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.

No MeSH data available.


Related in: MedlinePlus