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.
References
[1]
Bergounioux J, Elisee R, Prunier AL, Donnadieu F, Sperandio B, Sansonetti P, et al. Calpain activation by the Shigella flexneri effector VirA regulates key steps in the formation and life of the bacterium's epithelial niche. Cell Host Microbe. 2012;11(3):240–52. doi: 10.1016/j.chom.2012.01.013 pmid:22423964.
[2]
Carneiro LA, Travassos LH, Soares F, Tattoli I, Magalhaes JG, Bozza MT, et al. Shigella induces mitochondrial dysfunction and cell death in nonmyleoid cells. Cell Host Microbe. 2009;5(2):123–36. doi: 10.1016/j.chom.2008.12.011 pmid:19218084.
[3]
Lembo-Fazio L, Nigro G, Noel G, Rossi G, Chiara F, Tsilingiri K, et al. Gadd45alpha activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo. Cell death & disease. 2011;2:e122. doi: 10.1038/cddis.2011.4 pmid:21368893; PubMed Central PMCID: PMC3101704.
[4]
Miao EA, Mao DP, Yudkovsky N, Bonneau R, Lorang CG, Warren SE, et al. Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A. 2010;107(7):3076–80. doi: 10.1073/pnas.0913087107 pmid:20133635; PubMed Central PMCID: PMC2840275.
[5]
Suzuki T, Franchi L, Toma C, Ashida H, Ogawa M, Yoshikawa Y, et al. Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages. PLoS Pathog. 2007;3(8):e111. doi: 10.1371/journal.ppat.0030111 pmid:17696608; PubMed Central PMCID: PMC1941748.
[6]
Willingham SB, Bergstralh DT, O'Connor W, Morrison AC, Taxman DJ, Duncan JA, et al. Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC. Cell Host Microbe. 2007;2(3):147–59. doi: 10.1016/j.chom.2007.07.009 pmid:18005730; PubMed Central PMCID: PMC2083260.
[7]
Zychlinsky A, Prevost MC, Sansonetti PJ. Shigella flexneri induces apoptosis in infected macrophages. Nature. 1992;358(6382):167–9. doi: 10.1038/358167a0 pmid:1614548.
[8]
Pendaries C, Tronchere H, Arbibe L, Mounier J, Gozani O, Cantley L, et al. PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection. EMBO J. 2006;25(5):1024–34. doi: 10.1038/sj.emboj.7601001 pmid:16482216; PubMed Central PMCID: PMC1409730.
[9]
Kobayashi T, Ogawa M, Sanada T, Mimuro H, Kim M, Ashida H, et al. The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection. Cell Host Microbe. 2013;13(5):570–83. doi: 10.1016/j.chom.2013.04.012 pmid:23684308.
[10]
Dong N, Zhu Y, Lu Q, Hu L, Zheng Y, Shao F. Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses. Cell. 2012;150(5):1029–41. doi: 10.1016/j.cell.2012.06.050 pmid:22939626.
[11]
Brown MD, Bry L, Li Z, Sacks DB. Actin pedestal formation by enteropathogenic Escherichia coli is regulated by IQGAP1, calcium, and calmodulin. J Biol Chem. 2008;283(50):35212–22. doi: 10.1074/jbc.M803477200 pmid:18809683; PubMed Central PMCID: PMC2596374.
[12]
Kim H, White CD, Li Z, Sacks DB. Salmonella enterica serotype Typhimurium usurps the scaffold protein IQGAP1 to manipulate Rac1 and MAPK signalling. The Biochemical journal. 2011;440(3):309–18. doi: 10.1042/BJ20110419 pmid:21851337.
[13]
McLaughlin LM, Govoni GR, Gerke C, Gopinath S, Peng K, Laidlaw G, et al. The Salmonella SPI2 effector SseI mediates long-term systemic infection by modulating host cell migration. PLoS Pathog. 2009;5(11):e1000671. doi: 10.1371/journal.ppat.1000671 pmid:19956712; PubMed Central PMCID: PMC2777311.
[14]
Zurawski DV, Mumy KL, Faherty CS, McCormick BA, Maurelli AT. Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein. Molecular microbiology. 2009;71(2):350–68. doi: 10.1111/j.1365-2958.2008.06524.x pmid:19017275; PubMed Central PMCID: PMC2783611.
[15]
Ambrosi C, Pompili M, Scribano D, Limongi D, Petrucca A, Cannavacciuolo S, et al. The Shigella flexneri OspB effector: an early immunomodulator. International journal of medical microbiology: IJMM. 2015;305(1):75–84. doi: 10.1016/j.ijmm.2014.11.004 pmid:25434600.
[16]
Fukazawa A, Alonso C, Kurachi K, Gupta S, Lesser CF, McCormick BA, et al. GEF-H1 mediated control of NOD1 dependent NF-kappaB activation by Shigella effectors. PLoS Pathog. 2008;4(11):e1000228. doi: 10.1371/journal.ppat.1000228 pmid:19043560; PubMed Central PMCID: PMC2583055.
[17]
Wang JB, Sonn R, Tekletsadik YK, Samorodnitsky D, Osman MA. IQGAP1 regulates cell proliferation through a novel CDC42-mTOR pathway. Journal of cell science. 2009;122(Pt 12):2024–33. doi: 10.1242/jcs.044644 pmid:19454477; PubMed Central PMCID: PMC2723156.
[18]
Chen F, Zhu HH, Zhou LF, Wu SS, Wang J, Chen Z. IQGAP1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation by Akt activation. Experimental & molecular medicine. 2010;42(7):477–83. doi: 10.3858/emm.2010.42.7.049 pmid:20530982; PubMed Central PMCID: PMC2912475.
[19]
Hedman AC, Smith JM, Sacks DB. The biology of IQGAP proteins: beyond the cytoskeleton. EMBO reports. 2015. doi: 10.15252/embr.201439834 pmid:25722290.
Sansonetti PJ, Mounier J, Prevost MC, Mege RM. Cadherin expression is required for the spread of Shigella flexneri between epithelial cells. Cell. 1994;76(5):829–39. pmid:8124719. doi: 10.1016/0092-8674(94)90358-1
[22]
Noritake J, Fukata M, Sato K, Nakagawa M, Watanabe T, Izumi N, et al. Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact. Molecular biology of the cell. 2004;15(3):1065–76. doi: 10.1091/mbc.E03-08-0582 pmid:14699063; PubMed Central PMCID: PMC363077.
[23]
Van Hoorde L, Braet K, Mareel M. The N-cadherin/catenin complex in colon fibroblasts and myofibroblasts. Cell adhesion and communication. 1999;7(2):139–50. pmid:10427966. doi: 10.3109/15419069909034397
[24]
Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004;6(11):1122–8. doi: 10.1038/ncb1183 pmid:15467718.
[25]
Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol. 2004;14(14):1296–302. doi: 10.1016/j.cub.2004.06.054 pmid:15268862.
[26]
Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307(5712):1098–101. doi: 10.1126/science.1106148 pmid:15718470.
[27]
Tekletsadik YK, Sonn R, Osman MA. A conserved role of IQGAP1 in regulating TOR complex 1. J Cell Sci. 2012;125(Pt 8):2041–52. doi: 10.1242/jcs.098947 pmid:22328503; PubMed Central PMCID: PMC3360921.
[28]
Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008;320(5882):1496–501. doi: 10.1126/science.1157535 pmid:18497260; PubMed Central PMCID: PMC2475333.
[29]
Tattoli I, Sorbara MT, Vuckovic D, Ling A, Soares F, Carneiro LA, et al. Amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program. Cell host & microbe. 2012;11(6):563–75. Epub 2012/06/19. doi: 10.1016/j.chom.2012.04.012 pmid:22704617.
[30]
Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274–93. doi: 10.1016/j.cell.2012.03.017 pmid:22500797; PubMed Central PMCID: PMC3331679.
[31]
Laplante M, Sabatini DM. Regulation of mTORC1 and its impact on gene expression at a glance. Journal of cell science. 2013;126(Pt 8):1713–9. doi: 10.1242/jcs.125773 pmid:23641065; PubMed Central PMCID: PMC3678406.
[32]
Owen KA, Meyer CB, Bouton AH, Casanova JE. Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages. PLoS Pathog. 2014;10(6):e1004159. doi: 10.1371/journal.ppat.1004159 pmid:24901456.
[33]
Dragoi AM, Agaisse H. The class II phosphatidylinositol 3-phosphate kinase PIK3C2A promotes Shigella flexneri dissemination through formation of vacuole-like protrusions. Infection and immunity. 2015;83(4):1695–704. doi: 10.1128/IAI.03138-14 pmid:25667265; PubMed Central PMCID: PMC4363405.
[34]
Smith JM, Hedman AC, Sacks DB. IQGAPs choreograph cellular signaling from the membrane to the nucleus. Trends Cell Biol. 2015;25(3):171–84. doi: 10.1016/j.tcb.2014.12.005 pmid:25618329; PubMed Central PMCID: PMC4344846.
[35]
Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121(2):179–93. doi: 10.1016/j.cell.2005.02.031 pmid:15851026.
[36]
Roy M, Li Z, Sacks DB. IQGAP1 binds ERK2 and modulates its activity. The Journal of biological chemistry. 2004;279(17):17329–37. doi: 10.1074/jbc.M308405200 pmid:14970219.
[37]
Roy M, Li Z, Sacks DB. IQGAP1 is a scaffold for mitogen-activated protein kinase signaling. Molecular and cellular biology. 2005;25(18):7940–52. doi: 10.1128/MCB.25.18.7940–7952.2005 pmid:16135787; PubMed Central PMCID: PMC1234344.
[38]
Ren JG, Li Z, Sacks DB. IQGAP1 modulates activation of B-Raf. Proc Natl Acad Sci U S A. 2007;104(25):10465–9. doi: 10.1073/pnas.0611308104 pmid:17563371; PubMed Central PMCID: PMC1965536.
[39]
Li H, Xu H, Zhou Y, Zhang J, Long C, Li S, et al. The phosphothreonine lyase activity of a bacterial type III effector family. Science. 2007;315(5814):1000–3. doi: 10.1126/science.1138960 pmid:17303758.
[40]
Chung LK, Philip NH, Schmidt VA, Koller A, Strowig T, Flavell RA, et al. IQGAP1 Is Important for Activation of Caspase-1 in Macrophages and Is Targeted by Yersinia pestis Type III Effector YopM. MBio. 2014;5(4):e01402–14. doi: 10.1128/mBio.01402-14 pmid:24987096.
[41]
Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr Opin Cell Biol. 2005;17(6):596–603. doi: 10.1016/j.ceb.2005.09.009 pmid:16226444.
[42]
Yang H, Rudge DG, Koos JD, Vaidialingam B, Yang HJ, Pavletich NP. mTOR kinase structure, mechanism and regulation. Nature. 2013;497(7448):217–23. doi: 10.1038/nature12122 pmid:23636326.
[43]
Labrec EH, Schneider H, Magnani TJ, Formal SB. Epithelial Cell Penetration as an Essential Step in the Pathogenesis of Bacillary Dysentery. J Bacteriol. 1964;88(5):1503–18. Epub 1964/11/01. pmid:16562000; PubMed Central PMCID: PMC277436.
[44]
Maurelli AT, Blackmon B, Curtiss R 3rd. Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun. 1984;43(1):397–401. pmid:6360906; PubMed Central PMCID: PMC263440.
[45]
Ho YD, Joyal JL, Li Z, Sacks DB. IQGAP1 integrates Ca2+/calmodulin and Cdc42 signaling. The Journal of biological chemistry. 1999;274(1):464–70. pmid:9867866. doi: 10.1074/jbc.274.1.464
[46]
Weiss DS, Chen JC, Ghigo JM, Boyd D, Beckwith J. Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL. J Bacteriol. 1999;181(2):508–20. pmid:9882665; PubMed Central PMCID: PMC93405.
[47]
Costa SC, Schmitz AM, Jahufar FF, Boyd JD, Cho MY, Glicksman MA, et al. A new means to identify type 3 secreted effectors: functionally interchangeable class IB chaperones recognize a conserved sequence. MBio. 2012;3(1). doi: 10.1128/mBio.00243-11 pmid:22334517; PubMed Central PMCID: PMC3280449.
[48]
Ren JG, Li Z, Crimmins DL, Sacks DB. Self-association of IQGAP1: characterization and functional sequelae. J Biol Chem. 2005;280(41):34548–57. doi: 10.1074/jbc.M507321200 pmid:16105843.
[49]
Erdemir HH, Li Z, Sacks DB. IQGAP1 binds to estrogen receptor-alpha and modulates its function. The Journal of biological chemistry. 2014;289(13):9100–12. doi: 10.1074/jbc.M114.553511 pmid:24550401; PubMed Central PMCID: PMC3979404.
