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Bacterial Protein Translocation &
Pathogenesis
David R. Sherman
HSB G-153
221-5381
[email protected]
Lecture outline
• Cellular addresses
• Getting stuck in the membrane: YidC
• Crossing the inner membrane:
Sec-dependent
SRP
Sec B
TAT-mediated
• Crossing the outer barrier - specialized secretion systems
• An example in gram (+) bacteria (paper discussion)
Protein destinations
Approx 10% of proteins cross at least the inner membrane.
Approx 30% of proteins are membrane associated.
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.
Membrane insertion via YidC

Multi-pass membrane protein.

Needed for insertion of some (all?) membrane proteins.

Can act alone or w/ Sec YEG.

Evolutionary origin of secretion?
Across the cytoplasmic membrane -the Sec machinery
General features:
Sec YEG: heterotrimeric pore-forming membrane proteins.
SecA: membrane-associated ATPase.
Substrates are generally unfolded.
Substrates have a signal peptide:
usually N-terminal
1(+) basic AAs followed by10-20 hydrophobic AAs
SecYEG topology
9:494-500, 2001
Homologous to eukaryotic Sec61p complex.
SRP-mediated translocation
SRP: homologous to eukaryotic SRP
Ffh (54 homolog) 48kDa GTPase
ffs (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.
SRP targeting
SecB-mediated translocation
SecB: 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.
SecB protein targeting
Nat Struct Biol. 2001 8(6):492-8.
Sec interactions
9:494-500, 2001
Twin-arginine (Tat)-mediated protein
translocation
Independent 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)
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-K
hydrophobic a-helical domain
C (cleavage) domain.
TATFIND 1.2
Specialized secretion pathways
(Gram-negative bacteria)
1.
2.
3.
4.
5.
6.
Type I pili.
Type I secretion.
Type II secretion/general secretory pathway/type IV pili.
Type III secretion (TTSS).
Type IV secretion.
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.
Assembly of type I pili
Allow for attachment during the
initial stages of infection.
Assembled in two stages:
Sec-dependent
Pap C/D-dependent
Type I secretion of repeat toxins: HlyA
Lipid-modified toxin.
11-17 repeats of 9 AAs.
Binds Ca++
Punches holes.
Sec-independent.
Requires ABC-transporter (HlyB).
C-term signal sequence.
Type II secretion -- the “general” secretory
pathway
Many examples:
**cholera toxin**
alkaline phosphatase
proteases
elastase
Type IV pili
Occurs in 2 steps -- 1st is Sec-dependent; 2nd requires 10
proteins and ATP. Secretion signal?
Type IV pili (a type II machine)
Type III secretion
Needle
Triggered by contact w/ host cells.
Sec-independent, similar to flagellar assembly.
Assembly of the needle occurs at the tip.
Type IV secretion
Very versatile; Sec- and ATP-dependent.
Autotransporters: Neisseria IgA protease
Synthesized as a pre-proenzyme.
C-term b-barrel inserts in OM, pulls N-term through.
N-term auto-cleaves, promoting release.
Cell wall proteins of Gram (+)s
Initially Sec-dependent.
N-term signal cleaved.
C-term signal sorts to CW.
L-P-x-T-G.
Amide linkage to
peptidoglycan.
So what’s in common?
All secretion systems must:
• assemble themselves.
• recognize the appropriate substrates.
• maintain proper folding state.
• determine their final locations.
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):147883.
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.
Fig 1.
B
A
2.0×10
07
1.5×10
07
125
day 0
5.0×10 06
100
% metabolism
day 7
1.0×10 07
+





75
RD1
tn3870
tn3871
tn3874
3875
tn3876
50
25
x H37Rv
- tn0982
-- tn3864
0
2
4
6
8
days
H
37
R
H
3
v: 7R
37 tn v
R 09
H v:tn 82
37 3
H Rv 86
37 : 4
R R
H v: D1
37 tn
R 3
H v: 870
37 tn
R 3
H v:t 871
37 n
3
H Rv: 874
37 3
R 87
v:
tn 5
38
76
0
H
bacteria / well
day 4
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 2
A
B
120
120
100
% metabolism
% metabolism
100
80
60
40
20
80
60
40
20
0
0
0
2
4
days
6
8
0
2
4
6
8
days
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 3
% of cells infected
75
H37Rv
***
H37Rv: RD1
50
***
25
0
% of infected cells with
>10 bacilli
B
A
100
***
75
50
***
25
0
day 0
day 4
day 7
day 0
day 4
day 7
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 4
bacteria / lung
1.0E+08
H37Rv
1.0E+07
H37Rv:  RD1
1.0E+06
H37Rv:tn3870
1.0E+05
H37Rv:tn3876
H37Rv:  3875
1.0E+04
1.0E+03
1.0E+02
0
4
weeks
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 5
A
C
B
D
E
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 6
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 7
A
B
300
spot forming units
(IFN-)
spot forming units
(IFN-)
300
200
100
0
0
25
50
ng/ml CFP-10
75
filtrate
pellet
200
100
0
v
R D1 870 G18 871 G18 874 408 875 406 876 429
7
R 3
3
3
3
3
3
H  tn 0:K tn 1:K tn :MH  :MH tn :MH
7
7
4
8
8
7
76
75
3
3
8
8
8
3
3
tn
tn
3
tn
tn
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.