bacterial protein translocation & pathogenesis

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Bacterial Protein Translocation & Pathogenesis. David R. Sherman HSB G-153 221-5381 dsherman@u.washington.edu. Lecture outline. Cellular addresses Getting stuck in the membrane: YidC Crossing the inner membrane: Sec-dependent SRP Sec B TAT-mediated - PowerPoint PPT Presentation

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  • Bacterial Protein Translocation & PathogenesisDavid R. ShermanHSB G-153221-5381dsherman@u.washington.edu

    Copyright (c) by W. H. Freeman and Company

  • Lecture outline Cellular addresses Getting stuck in the membrane: YidC Crossing the inner membrane:Sec-dependentSRPSec BTAT-mediated Crossing the outer barrier - specialized secretion systems

    An example in gram (+) bacteria (paper discussion)

    Copyright (c) by W. H. Freeman and Company

  • Protein destinationsApprox 10% of proteins cross at least the inner membrane.Approx 30% of proteins are membrane associated.

    Copyright (c) by W. H. Freeman and Company

  • Machinery of bacterial protein translocation

    Cytosolic membrane (Gram +/-): YidC. Sec machinery. Tat translocation.

    Cell wall (Gram +/-): very little known.

    Outer membrane (Gram -): several specialized systems.

    Much better studied in Gram-negatives.

    Copyright (c) by W. H. Freeman and Company

  • Membrane insertion via YidCMulti-pass membrane protein.Needed for insertion of some (all?) membrane proteins.Can act alone or w/ Sec YEG.Evolutionary origin of secretion?

    Copyright (c) by W. H. Freeman and Company

  • Across the cytoplasmic membrane -- the Sec machineryGeneral features:Sec YEG: heterotrimeric pore-forming membrane proteins.

    SecA: membrane-associated ATPase.

    Substrates are generally unfolded.

    Substrates have a signal peptide:usually N-terminal1(+) basic AAs followed by10-20 hydrophobic AAs

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  • SecYEG topologyHomologous to eukaryotic Sec61p complex.9:494-500, 2001

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  • SRP-mediated translocationSRP: homologous to eukaryotic SRPFfh (54 homolog) 48kDa GTPaseffs (4.5S RNA)essential for cell viability

    Recognizes ribosome-bound nascent membrane proteins.

    Substrate recognition is via signal sequence.

    SecA is NOT needed for membrane association, but IS needed for translocation.

    Copyright (c) by W. H. Freeman and Company

  • SRP targeting

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  • SecB-mediated translocationSecB:acidic, cytosolic chaperone.recognizes mature, unfolded proteins.destination -- periplasm, outer membrane or beyond.

    Substrate recognition is NOT via the signal sequence.

    Binding motif:~9 AAs long.hydrophobic and basic.acidic AAs strongly disfavored.

    Copyright (c) by W. H. Freeman and Company

  • SecB protein targetingNat Struct Biol. 2001 8(6):492-8.

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  • Sec interactions9:494-500, 2001

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  • Twin-arginine (Tat)-mediated protein translocationIndependent of the Sec system.

    TatA and TatC are essential.

    Transports folded proteins.

    Not found in eukaryotes; some bacteria.

    # of Tat substrates per organism varies very widely --None (Clostridium tetani, Fusobacterium)145 (Streptomyces coelicolour)

    Copyright (c) by W. H. Freeman and Company

  • Twin-arginine (Tat)-mediated protein translocation

    Targeting signal (in the first 35 AAs) has 3 regions:N-term is positively charged (S/T)-R-R-x-F-L-Khydrophobic a-helical domainC (cleavage) domain.

    TATFIND 1.2

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  • Specialized secretion pathways (Gram-negative bacteria)1. Type I pili.2. Type I secretion.3. Type II secretion/general secretory pathway/type IV pili.4. Type III secretion (TTSS).5. Type IV secretion.6. Type V/autotransporters.

    Classification is based on the sequence/structure of the transport machinery and their catalyzed reactions.

    These systems are usually associated with virulence.

    Copyright (c) by W. H. Freeman and Company

  • Assembly of type I piliAllow for attachment during the initial stages of infection.Assembled in two stages:Sec-dependentPap C/D-dependent

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  • Type I secretion of repeat toxins: HlyALipid-modified toxin.11-17 repeats of 9 AAs.Binds Ca++Punches holes.Sec-independent.Requires ABC-transporter (HlyB).C-term signal sequence.

    Copyright (c) by W. H. Freeman and Company

  • Type II secretion -- the general secretory pathwayOccurs in 2 steps -- 1st is Sec-dependent; 2nd requires 10 proteins and ATP. Secretion signal?Many examples:**cholera toxin**alkaline phosphataseproteaseselastaseType IV pili

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  • Type IV pili (a type II machine)

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  • Type III secretionTriggered by contact w/ host cells.Sec-independent, similar to flagellar assembly.Assembly of the needle occurs at the tip.Needle

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  • Type IV secretionVery versatile; Sec- and ATP-dependent.

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  • Autotransporters: Neisseria IgA proteaseSynthesized as a pre-proenzyme.C-term b-barrel inserts in OM, pulls N-term through.N-term auto-cleaves, promoting release.

    Copyright (c) by W. H. Freeman and Company

  • Cell wall proteins of Gram (+)sInitially Sec-dependent.N-term signal cleaved.C-term signal sorts to CW.L-P-x-T-G.Amide linkage to peptidoglycan.

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  • So whats in common?All secretion systems must: assemble themselves. recognize the appropriate substrates. maintain proper folding state. determine their final locations.

    Copyright (c) by W. H. Freeman and Company

  • Additional reading (not assigned)The structural basis of protein targeting and translocation in bacteria.Driessen AJ, Manting EH, van der Does C. Nat Struct Biol. 2001 8(6):492-8.

    The Tat protein export pathway.Berks BC, Sargent F, Palmer T. Mol Microbiol. 2000 Jan;35(2):260-74.

    Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey.Dilks K, Rose RW, Hartmann E, Pohlschroder M. J Bacteriol. 2003 Feb;185(4):1478-83.

    Protein secretion and the pathogenesis of bacterial infections.Lee VT, Schneewind O. Genes Dev. 2001 Jul 15;15(14):1725-52.

    Getting out: protein traffic in prokaryotes.Pugsley AP, Francetic O, Driessen AJ, de Lorenzo V. Mol Microbiol. 2004 Apr;52(1):3-11.

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  • Fig 1.Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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  • Fig. 2Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

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  • Fig. 3Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

    Copyright (c) by W. H. Freeman and Company

  • Fig. 4Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

    Copyright (c) by W. H. Freeman and Company

  • Fig. 5Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

    Copyright (c) by W. H. Freeman and Company

  • Fig. 6Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

    Copyright (c) by W. H. Freeman and Company

  • Fig. 7Guinn et al, Mol Microbiol, 2004, 51(2):359-370.

    Copyright (c) by W. H. Freeman and Company

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