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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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Presentation Transcript
slide1

SBM 2044: Lecture 4

Weapons delivery & deployment

(Part II)

Secretion & targeting

of protein virulence factors in Gram-positive bacteria

slide2

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

slide3

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:

slide4

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

slide5

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

slide6

2. Binding to membrane anchored LTA

  • Single example recognised only recently
  • InlB of Listeria monocytogenes – has C-terminal
  • domain that ‘targets’ LTA – mechanism??
slide7

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

slide8

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 )

slide9

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

slide10

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.

slide11

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

slide12

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

slide13

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
references
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.