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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 enhances cell proliferation dependent on IQGAP1 and inhibited by rapamycin.(A) Impact of insulin-like growth factor on area of spread of S. flexneri strains producing or not producing OspB. IGF, insulin-like growth factor. *, p < 0.05, Student’s two-tailed t test. (B) Relative cell density at the edge of infectious foci (normalized to monolayer background) for IQGAP1+/+ cells infected with the ospB mutant complemented or not with a plasmid expressing OspB. *, p < 0.05, Student’s one-tailed t test. (C) Proliferation rate of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05 compared with all other conditions at the same time point, 2-way ANOVA. (D) Saturation density of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05, Student’s two-tailed t test. (E) Proliferation rate of IQGAP1+/+ MEFs transiently transfected with OspB GFP or GFP alone and treated with 10 nM rapamycin or DMSO carrier alone. *, **, p < 0.05 compared with cells transfected with GFP alone at the same time point; ***, p < 0.05 compared with cells treated with rapamycin at the same time point; 2-way ANOVA. (F) Representative images of cells on day 3 of experiment shown in panel E. Data represent mean ± S.D. of three or more independent experiments.
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ppat.1005200.g003: OspB enhances cell proliferation dependent on IQGAP1 and inhibited by rapamycin.(A) Impact of insulin-like growth factor on area of spread of S. flexneri strains producing or not producing OspB. IGF, insulin-like growth factor. *, p < 0.05, Student’s two-tailed t test. (B) Relative cell density at the edge of infectious foci (normalized to monolayer background) for IQGAP1+/+ cells infected with the ospB mutant complemented or not with a plasmid expressing OspB. *, p < 0.05, Student’s one-tailed t test. (C) Proliferation rate of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05 compared with all other conditions at the same time point, 2-way ANOVA. (D) Saturation density of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05, Student’s two-tailed t test. (E) Proliferation rate of IQGAP1+/+ MEFs transiently transfected with OspB GFP or GFP alone and treated with 10 nM rapamycin or DMSO carrier alone. *, **, p < 0.05 compared with cells transfected with GFP alone at the same time point; ***, p < 0.05 compared with cells treated with rapamycin at the same time point; 2-way ANOVA. (F) Representative images of cells on day 3 of experiment shown in panel E. Data represent mean ± S.D. of three or more independent experiments.

Mentions: We considered whether the effect of OspB on the area of S. flexneri spread could be due to effects on cell proliferation. Infection of cells by Shigella species results in cell death, which is seen as cellular debris at the center of infectious foci. We reasoned that if cells containing IQGAP1 and infected with S. flexneri expressing OspB proliferated at an increased rate, then these cells might proliferate sufficiently quickly to replace some of the dying cells within foci of infection, enabling bacteria to spread into these new cells and causing the net area of bacterial spread to be smaller. To assess whether increased cell proliferation per se would lead to smaller net area of bacterial spread, we tested the effect of insulin-like growth factor on the area of bacterial spread. The addition of insulin-like growth factor to IQGAP1+/+ MEFs led to a 1.3 ± 0.1-fold increase (p = 0.03) in the number of cells over 24 hr. Insulin-like growth factor-induced increase in cell proliferation was associated with a 21% decrease in spread of the ospB mutant and an 18% decrease in spread of the ospB mutant complemented with OspB (Fig 3A), indicating that increased cell proliferation in the monolayer is associated with decreased area of S. flexneri spread.


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 enhances cell proliferation dependent on IQGAP1 and inhibited by rapamycin.(A) Impact of insulin-like growth factor on area of spread of S. flexneri strains producing or not producing OspB. IGF, insulin-like growth factor. *, p < 0.05, Student’s two-tailed t test. (B) Relative cell density at the edge of infectious foci (normalized to monolayer background) for IQGAP1+/+ cells infected with the ospB mutant complemented or not with a plasmid expressing OspB. *, p < 0.05, Student’s one-tailed t test. (C) Proliferation rate of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05 compared with all other conditions at the same time point, 2-way ANOVA. (D) Saturation density of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05, Student’s two-tailed t test. (E) Proliferation rate of IQGAP1+/+ MEFs transiently transfected with OspB GFP or GFP alone and treated with 10 nM rapamycin or DMSO carrier alone. *, **, p < 0.05 compared with cells transfected with GFP alone at the same time point; ***, p < 0.05 compared with cells treated with rapamycin at the same time point; 2-way ANOVA. (F) Representative images of cells on day 3 of experiment shown in panel E. Data represent mean ± S.D. of three or more independent experiments.
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ppat.1005200.g003: OspB enhances cell proliferation dependent on IQGAP1 and inhibited by rapamycin.(A) Impact of insulin-like growth factor on area of spread of S. flexneri strains producing or not producing OspB. IGF, insulin-like growth factor. *, p < 0.05, Student’s two-tailed t test. (B) Relative cell density at the edge of infectious foci (normalized to monolayer background) for IQGAP1+/+ cells infected with the ospB mutant complemented or not with a plasmid expressing OspB. *, p < 0.05, Student’s one-tailed t test. (C) Proliferation rate of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05 compared with all other conditions at the same time point, 2-way ANOVA. (D) Saturation density of IQGAP1+/+ and IQGAP1-/- MEFs transiently transfected with OspB GFP or GFP alone. *, p < 0.05, Student’s two-tailed t test. (E) Proliferation rate of IQGAP1+/+ MEFs transiently transfected with OspB GFP or GFP alone and treated with 10 nM rapamycin or DMSO carrier alone. *, **, p < 0.05 compared with cells transfected with GFP alone at the same time point; ***, p < 0.05 compared with cells treated with rapamycin at the same time point; 2-way ANOVA. (F) Representative images of cells on day 3 of experiment shown in panel E. Data represent mean ± S.D. of three or more independent experiments.
Mentions: We considered whether the effect of OspB on the area of S. flexneri spread could be due to effects on cell proliferation. Infection of cells by Shigella species results in cell death, which is seen as cellular debris at the center of infectious foci. We reasoned that if cells containing IQGAP1 and infected with S. flexneri expressing OspB proliferated at an increased rate, then these cells might proliferate sufficiently quickly to replace some of the dying cells within foci of infection, enabling bacteria to spread into these new cells and causing the net area of bacterial spread to be smaller. To assess whether increased cell proliferation per se would lead to smaller net area of bacterial spread, we tested the effect of insulin-like growth factor on the area of bacterial spread. The addition of insulin-like growth factor to IQGAP1+/+ MEFs led to a 1.3 ± 0.1-fold increase (p = 0.03) in the number of cells over 24 hr. Insulin-like growth factor-induced increase in cell proliferation was associated with a 21% decrease in spread of the ospB mutant and an 18% decrease in spread of the ospB mutant complemented with OspB (Fig 3A), indicating that increased cell proliferation in the monolayer is associated with decreased area of S. flexneri spread.

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