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

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:

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 PspA and LytA – Very distinctive proteins – only common feature is conserved C-terminal choline-binding domain S. suis – a pig pathogen

n The structure of teichoic acid: poly-ribitol phosphate R R’ Polymer of either Glycerol phosphate or Ribitol phosphate, with various substituents (R) poly-ribitol phosphate O P O C C C C C O P O C O H H H H H O H H O OH O H H R R’ n 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

2. Binding to membrane anchored LTA Single example recognised only recently InlB of Listeria monocytogenes – has C-terminal domain that ‘targets’ LTA – mechanism?? Exogenous addition of InlB allows it to associate with wall and mediate invasion – hence wall ‘targeting’ sequence, analagous to the C-terminal wall targeting sequence of lysostaphin (secreted by Staph. simulans and targets cell walls of other staph species)

attached at outer surface of cytoplasmic membrane by a lipid anchor 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

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

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 First recognized by comparing C-terminal sequences of S. aureus Protein A & streptococcal M proteins Pro-rich region 15 - 20 hydrophobic residues 5 - 10 mostly charged LPxTG motif

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 Originally suggested that wall-association due simply to ‘anchoring’ of C-terminal ends of proteins in membrane BUT this was vast oversimplification Membrane ‘anchor’ Charged ‘tail’ + + Care: do not be misled by some textbooks/reviews which say proteins anchored in membrane.

wall-associated ‘Sortase’ N C Wall-associating signal Signal peptidase N-terminal signal peptide -L-P-x-T Cleavage at LPxTG Cross-linked to cell-wall G In S. aureus, the Thr X-linked to the terminal glycine of the pentaglycine x-bridge, probably prior to incorporation of the peptidoglycan precursor into the wall N Some, but not necessarily all, covalently linked to wall (e.g. InaA, Prot. A) C Minority simply ‘anchored’? (e.g. ActA in Listeria) Majority ‘cleaved’ at LPxTG mRNA

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 minority of wall-associated proteins in many species anchored 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

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

Major types of surface proteins in Gram-positive bacteria Major types of surface proteins in Gram-positive bacteria. Protein A, an immunoglobulin-binding protein, is covalently linked to the cell wall and exposed on the cell surface. The amidase LytA is loosely attached to choline (CH) residues decorating teichoic acid (TA) and lipoteichoic acid (LTA) in Streptococcus pneumoniae. The actin-polymerizing protein ActA of Listeria monocytogenes is membrane-anchored and exposed to the medium. The β-lactamase BlaZ encoded by the resistance plasmid PI258 of Staphylococcus aureus is associated to the membrane and partially released into the medium. InlB of L. monocytogenes is loosely attached to LTA. It is weakly exposed on the cell surface. Proc Natl Acad Sci U S A. 2000 May 9; 97(10): 5013–5015. Copyright © 2000, The National Academy of Sciences

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.