1. Weapons delivery & deployment
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-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

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

5. 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

6. 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)

7. 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

8. 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 )

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
First recognized by comparing C-terminal sequences of S. aureus Protein A & streptococcal M proteins
Pro-rich region
hydrophobic
residues
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
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.

11. 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

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 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

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. 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 May 9; 97(10): 5013–5015.
Copyright © 2000, The National Academy of Sciences

15. 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,
Ton-That et al. Protein sorting to the cell wall envelope of Gram-positive bacteria (2004). Biochimica et Biophysica Acta, 1694,