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In bacteria, the targeting of proteins to specific subcellular sites is essential for many functions, including pathogenesis and cell division, but how localization occurs is poorly understood. We are investigating the molecular mechanisms by which bacteria establish polarity. The Shigella outer membrane protein IcsA, a member of the autotransporter family of proteins, is present at one pole of the bacterium, where it mediates actin tail assembly. IcsA and all other autotransporters tested are directly secreted at the bacterial pole. We are using IcsA as a model protein to study factors that are required for proper localization of polar proteins, as well as the broader role of this targeting pathway.

References:

Janakiraman A, Fixen KR, Gray AN, Niki H, Goldberg MB. A genome-scale screen identifies a role for DnaK in chaperoning of polar autotransporters in Shigella. J Bacteriol. 2009; 191(20): 6300-6311.link

Wagner JK, Heindl JE, Gray AN, Jain S, Goldberg MB. Contribution of the periplasmic chaperone Skp to efficient presentation of the autotransporter IcsA on the surface of Shigella flexneri. J Bacteriol. 2009 Feb;191(3):815-21. link

Edgar R, Rodkey A, Feeney M, Semsey S, Kessel M, Goldberg MB, Adhya S, Oppenheim AB. Bacteriophage infection is targeted to cellular poles. Mol Microbiol. 2008; 68:1107. link

Jain S, van Ulsen P, Benz I, Schmidt MA, Fernandez R, Tommassen J, Goldberg MB. Polar localization of the autotransporter family of large bacterial virulence proteins. J Bacteriol. 2006 Jul;188(13):4841-50. link

Janakiraman A, Goldberg MB. Recent advances on the development of bacterial poles. Trends Microbiol. 2004 Nov;12(11):518-25. Review.link

Janakiraman A, Goldberg MB. Evidence for polar positional information independent of cell division and nucleoid occlusion. Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):835-40. link

Nilsen T, Ghosh AS, Goldberg MB, Young KD. Branching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coli. Mol Microbiol. 2004 May;52(4):1045-54. link

Charles M, Perez M, Kobil JH, Goldberg MB. Polar targeting of Shigella virulence factor IcsA in Enterobacteriacae and Vibrio. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9871-6. link

Steinhauer J, Agha R, Pham T, Varga AW, Goldberg MB. The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface. Mol Microbiol. 1999 Apr;32(2):367-77. link

Shigella sp. cause disease by invading and spreading through the colonic epithelium. Following entry into the cytosol of the host cell, Shigella spread by inducing the assembly of an actin tail at the bacterial pole. The Shigella protein IcsA is required for this process and for virulence. IcsA utilizes the cellular Cdc42 signal transduction pathway for actin rearrangement. This cellular pathway involves Cdc42 activation of N-WASP, a member of the Wiskott-Aldrich syndrome protein (WASP) family. Shigella IcsA participates in actin tail assembly by binding N-WASP. We have recently shown that a second cellular protein, Toca-1, is required for activation of N-WASP and is recruited to Shigella independently of IcsA and N-WASP. This project focuses on the molecular mechanisms by which Shigella activates and regulates this actin assembly pathway.

References:

Germane KL, Ohi R, Goldberg MB, Spiller BW. Structural and functional studies indicate that VirA is not a protease and doesn't directly destabilize microtubules. Biochemistry. 2008. 47:10241-3. link

Leung Y, Ally S, Goldberg MB. Bacterial actin assembly requires toca-1 to relieve N-wasp autoinhibition. Cell Host Microbe. 2008 Jan 17;3(4):39-47. link

Ally S, Sauer NJ, Loureiro JJ, Snapper SB, Gertler FB, Goldberg MB. Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins. Cell Microbiol. 2004 Apr;6(4):355-66. link

Magdalena J, Goldberg MB. Quantification of Shigella IcsA required for bacterial actin polymerization. Cell Motil Cytoskeleton. 2002 Apr;51(4):187-96. link

Goldberg MB. Actin-based motility of intracellular microbial pathogens. Microbiol Mol Biol Rev. 2001 Dec;65(4):595-626. Review. link

Snapper SB, Takeshima F, Anton I, Liu CH, Thomas SM, Nguyen D, Dudley D, Fraser H, Purich D, Lopez-Ilasaca M, Klein C, Davidson L, Bronson R, Mulligan RC, Southwick F, Geha R, Goldberg MB, Rosen FS, Hartwig JH, Alt FW. N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol. 2001 Oct;3(10):897-904. link

Critical to the disease process is the ability of intracellular Shigella to spread into adjacent cells in the infected tissue. Using actin-based motility, Shigella move to the cell periphery, where they form filopodia-like protrusions - extensions of the host plasma membrane that contain a bacterium at the tip. We have identified and are currently characterizing novel host proteins and bacterial effectors that mediate protrusion formation and intercellular spread. In addition, we are taking unbiased genetic and chemical approaches to identify novel host molecules and pathways that are complicit in these processes.

References:

Heindl JE, Saran I, Yi CR, Lesser CF, Goldberg MB. Requirement for formin-induced actin polymerization during spread of Shigella. Infect Immun. 2009: in press. link

The Shigella actin assembly protein IcsA is an outer membrane protein that belongs to the autotransporter family of secreted proteins. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and include many proteins involved in virulence. Most autotransporters are unusually large, presenting special challenges for translocation. We have shown that autotransporters are secreted at the bacterial pole, and that they require the outer membrane YaeT complex for proper assembly in the outer membrane. We are investigating the mechanisms by which members of this important family of proteins are translocated across the bacterial membranes and periplasm and characterizing those specialized aspects of the secretion machinery that participate in secretion of proteins at the bacterial pole.

References:

Janakiraman A, Fixen KR, Gray AN, Niki H, Goldberg MB. A genome-scale screen identifies a role for DnaK in chaperoning of polar autotransporters in Shigella. J Bacteriol. 2009; 191(20): 6300-6311.link

Wagner JK, Heindl JE, Gray AN, Jain S, Goldberg MB. Contribution of the periplasmic chaperone Skp to efficient presentation of the autotransporter IcsA on the surface of Shigella flexneri. J Bacteriol. 2009 Feb;191(3):815-21. link

Jain S, Goldberg MB. Requirement for YaeT in the outer membrane assembly of autotransporter proteins. J Bacteriol. 2007 Jul;189(14):4393-8. link

Jain S, van Ulsen P, Benz I, Schmidt MA, Fernandez R, Tommassen J, Goldberg MB. Polar localization of the autotransporter family of large bacterial virulence proteins. J Bacteriol. 2006 Jul;188(13):4841-50. link

Brandon LD, Goehring N, Janakiraman A, Yan AW, Wu T, Beckwith J, Goldberg MB. IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed. Mol Microbiol. 2003 Oct;50(1):45-60. link

Brandon LD, Goldberg MB. Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA. J Bacteriol. 2001 Feb;183(3):951-8. link