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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 limits S. flexneri spread dependent on IQGAP1.(A) Area of spread of GFP-producing wild type S. flexneri in IQGAP1-/- and IQGAP1+/+ MEFs, transfected or not with Myc-IQGAP1. a.u., arbitrary units. (B) Area of spread of GFP-producing wild type (WT) or ospB S. flexneri complemented or not with ospB in IQGAP1-/- or IQGAP1+/+ cells. (C) Area of spread of GFP-producing WT or ospB S. flexneri in IQGAP1-/- or IQGAP1+/+ cells transfected with a plasmid carrying either ospB gfp or gfp alone. Mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.
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ppat.1005200.g001: OspB limits S. flexneri spread dependent on IQGAP1.(A) Area of spread of GFP-producing wild type S. flexneri in IQGAP1-/- and IQGAP1+/+ MEFs, transfected or not with Myc-IQGAP1. a.u., arbitrary units. (B) Area of spread of GFP-producing wild type (WT) or ospB S. flexneri complemented or not with ospB in IQGAP1-/- or IQGAP1+/+ cells. (C) Area of spread of GFP-producing WT or ospB S. flexneri in IQGAP1-/- or IQGAP1+/+ cells transfected with a plasmid carrying either ospB gfp or gfp alone. Mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.

Mentions: IQGAP1 was selected from a pilot siRNA screen designed to identify human proteins that modulate Shigella spread. In this screen, siRNA to IQGAP1 was associated with an increase in the area of wild type S. flexneri strain 2457T spread through HeLa cell monolayers (IQGAP1 siRNA, 1200 ± 182 a.u. versus control siRNA, 596 ± 42 a.u., p = 0.04, Student’s two-tailed t test), determined by measuring the area of GFP-producing bacteria at individual infectious foci within the monolayer in 384-well format. The impact of IQGAP1 siRNA on area of bacterial spread was validated in 6-well format, where siRNA knock-down of IQGAP1 led to a 1.8-fold increase in area of spread of S. flexneri (IQGAP1 siRNA, 12 ± 1.4 versus control siRNA, 6 ± 0.4 a.u., p = 0.03, S1 Fig). Upon independently examining the role of IQGAP1 in S. flexneri spread using monolayers of mouse embryonic fibroblasts (MEFs) that lack or contain IQGAP1, we observed a similar 1.7-fold increase in area of spread in the absence of IQGAP1 (Fig 1A), together suggesting that IQGAP1 might restrict the extent of bacterial spread. Complementation with Myc-IQGAP1 in trans significantly reduced the area of spread for IQGAP1-/- MEFs (Fig 1A), indicating that the observed increase in spread in the IQGAP1-/- MEFs was due to the absence of IQGAP1 per se.


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 limits S. flexneri spread dependent on IQGAP1.(A) Area of spread of GFP-producing wild type S. flexneri in IQGAP1-/- and IQGAP1+/+ MEFs, transfected or not with Myc-IQGAP1. a.u., arbitrary units. (B) Area of spread of GFP-producing wild type (WT) or ospB S. flexneri complemented or not with ospB in IQGAP1-/- or IQGAP1+/+ cells. (C) Area of spread of GFP-producing WT or ospB S. flexneri in IQGAP1-/- or IQGAP1+/+ cells transfected with a plasmid carrying either ospB gfp or gfp alone. Mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005200.g001: OspB limits S. flexneri spread dependent on IQGAP1.(A) Area of spread of GFP-producing wild type S. flexneri in IQGAP1-/- and IQGAP1+/+ MEFs, transfected or not with Myc-IQGAP1. a.u., arbitrary units. (B) Area of spread of GFP-producing wild type (WT) or ospB S. flexneri complemented or not with ospB in IQGAP1-/- or IQGAP1+/+ cells. (C) Area of spread of GFP-producing WT or ospB S. flexneri in IQGAP1-/- or IQGAP1+/+ cells transfected with a plasmid carrying either ospB gfp or gfp alone. Mean ± S.D. Data represent three or more independent experiments. *, p < 0.05, Student’s two-tailed t test.
Mentions: IQGAP1 was selected from a pilot siRNA screen designed to identify human proteins that modulate Shigella spread. In this screen, siRNA to IQGAP1 was associated with an increase in the area of wild type S. flexneri strain 2457T spread through HeLa cell monolayers (IQGAP1 siRNA, 1200 ± 182 a.u. versus control siRNA, 596 ± 42 a.u., p = 0.04, Student’s two-tailed t test), determined by measuring the area of GFP-producing bacteria at individual infectious foci within the monolayer in 384-well format. The impact of IQGAP1 siRNA on area of bacterial spread was validated in 6-well format, where siRNA knock-down of IQGAP1 led to a 1.8-fold increase in area of spread of S. flexneri (IQGAP1 siRNA, 12 ± 1.4 versus control siRNA, 6 ± 0.4 a.u., p = 0.03, S1 Fig). Upon independently examining the role of IQGAP1 in S. flexneri spread using monolayers of mouse embryonic fibroblasts (MEFs) that lack or contain IQGAP1, we observed a similar 1.7-fold increase in area of spread in the absence of IQGAP1 (Fig 1A), together suggesting that IQGAP1 might restrict the extent of bacterial spread. Complementation with Myc-IQGAP1 in trans significantly reduced the area of spread for IQGAP1-/- MEFs (Fig 1A), indicating that the observed increase in spread in the IQGAP1-/- MEFs was due to the absence of IQGAP1 per se.

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