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SBM 2044: Lecture 4

SBM 2044: Lecture 4. Weapons delivery & deployment (Part II). Secretion & targeting of protein virulence factors in Gram-positive bacteria. Sec-dependant General secretion pathway (GSP) . Gram-positive bacteria. Gram-negative bacteria. Sufficient to get protein out. In this case, other

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SBM 2044: Lecture 4

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  1. SBM 2044: Lecture 4 Weapons delivery & deployment (Part II) Secretion & targeting of protein virulence factors in Gram-positive bacteria

  2. Sec-dependant General secretion pathway (GSP) Gram-positive bacteria Gram-negative bacteria Sufficient to get protein out. In this case, other mechanisms needed to retain wall - associated proteins Proteins reach periplasm, but OM is additional barrier - need other mechanisms to get protein out thro’ OM. (Types I - V secretion) OM IM sec sec Type II secretion Signal-peptide

  3. Targeting secreted proteins to Gram-positive cell walls Four distinct mechanisms identified to date: Rare: • Binding to wall teichoic acid • Binding to membrane anchored LTA • Lipoprotein ‘anchors’ • C-terminal wall-associating signals More widespread:

  4. 1. Binding to cell-wall teichoic acid Streptococcus pneumoniae and Streptococcus suis Pneumococcal surface protein A (PspA) Pneumococcal autolysin (LytA) S. suis autolysin- [homologous to pneumococcal LytA] C-terminal ends share homologous choline-binding domains – enable binding to TA of these species

  5. O H H H H H O H O P O C C C C C O P O C O O H O OH O H H R R’ n The structure of teichoic acid: Polymer of either Glycerol phosphate or Ribitol phosphate, with various substituents (R) poly-ribitol phosphate In most species studied to date R = D-alanine R’ = N-acetylglucosamine In S. pneumoniae and S. suis R = phosphodiester linked choline - chemically more stable than ester-linked D-Ala

  6. 2. Binding to membrane anchored LTA • Single example recognised only recently • InlB of Listeria monocytogenes – has C-terminal • domain that ‘targets’ LTA – mechanism??

  7. 3. Lipoproteins • attached at outer surface of cytoplasmic membrane by a • lipid anchor Examples include penicillinase in S. aureus • Similar mechanisms used in both Gram + & Gram . Distinctive N-terminal signal peptides distinct Sec apparatus with specialized signal peptidase (called signal peptidase II) recognized by

  8. N- Short hydrophobic sequence Signal peptidase II cleavage site 1-3 positively charged a.a. -Leu-x-y- Cys- x and y usually small, uncharged residues A diglyceride is attached to the N-terminal Cys of the mature protein Diglyceride Lipoprotein signal peptides Contrast with ‘typical’ GSP secretion signal-peptide ( Lecture 3 )

  9. 4. ‘Sorting’ via C-terminal wall-associating signals Vast majority of Gram + wall-associated proteins share structurally similar C-terminal wall-associating signals Hydrophobic /Charged ‘tail’ membrane ‘anchor’ -C 15 - 20 hydrophobic residues Pro-rich region 5 - 10 mostly charged LPxTG motif

  10. C-terminal wall-associating signals Studies of S. aureus Protein A, showed that membrane ‘anchor’ plays a transient role in a more complex wall-associating pathway Pro-rich ‘flexible’ wall-spanning Hydrophobic Membrane ‘anchor’ Charged ‘tail’ + + Care: do not be misled by some textbooks/reviews which say proteins anchored in membrane.

  11. C N Wall-associating signal Signal peptidase wall-associated ‘Sortase’ N-terminal signal peptide -L-P-x-T Cleavage at LPxTG Cross-linked to cell-wall G Some, but not necessarily all, covalently linked to wall (e.g. InaA, Prot. A) N C Majority ‘cleaved’ at LPxTG Minority simply ‘anchored’? (e.g. ActA in Listeria) mRNA

  12. Retaining secreted proteins in Gram-positive cell walls 1. Binding to wall teichoic acid Limited to a very few species (e.g. S. pneumoniae, S. suis) 2. Binding to membrane anchored LTA Single example recognised only recently (InlB of Listeria monocytogenes) 3. Lipoprotein ‘anchors’ A minorityof wall-associated proteins in many speciesanchored to outer surface of cell membrane via an N-terminal lipid anchor 4. C-terminal wall-associating signals Vast majority of wall-associated proteins studied to date share structurally similar C-terminal wall-associating signals

  13. Retaining proteins at Gram-negative cell-surfaces First step: Sec-dependent secretion to periplasm (GSP) Then: • Targeting of integral OM proteins - OM-interacting • ‘surfaces’ result from folding in periplasm • (may involve periplasmic Dsb and Ppi enzymes) • OR • Individual biogenesis pathways – e.g. fimbriae

  14. References • Navarre and Schneewind. Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope (1999). Microbiology and Molecular Biology Reviews, 63, 174-229. • Ton-That et al. Protein sorting to the cell wall envelope of Gram-positive bacteria (2004). Biochimica et Biophysica Acta, 1694, 269-278.

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