bacterial protein translocation & pathogenesis
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
Bacterial Protein Translocation & Pathogenesis
David R. ShermanHSB [email protected]
Lecture outline
• Cellular addresses
• Getting stuck in the membrane: YidC
• Crossing the inner membrane:Sec-dependent
SRPSec 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.
Signal sequence at N-terminus.
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-terminal1(+) basic AAs followed by10-20 hydrophobic AAs
SecYEG topology
Homologous to eukaryotic Sec61p complex.9:494-500, 2001
SRP-mediated translocation
SRP: homologous to eukaryotic SRPFfh (54 homolog) 48kDa GTPaseffs (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-Khydrophobic -helical domainC (cleavage) domain.
TATFIND 1.2
Inner membrane hierarchy Co-translational:
SecYEG; Sec A, SRP Very hydrophobic N-terminal signal
Newly synthesized, unfolded: SecYEG, SecA, SecB Hydrophobic N-terminal signal
Fully folded: Tat, Hydrophobic N-terminal signal w/ RR
Very little cross-talk!
Specialized secretion pathways (Gram-negative bacteria)
1. Type I pili.2. Type I secretion.3. Type II secretion/general secretory pathway/type IV pili.4. Type III secretion (TTSS).5. Type IV secretion.6. 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-dependentPap C/D-dependent
Uropathogenic E. coli
Type I secretion of repeat toxins: E. coli 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
Occurs in 2 steps -- 1st is Sec-dependent; 2nd requires 10 proteins and ATP. Secretion signal?
Many examples:**cholera toxin**alkaline phosphataseproteaseselastaseType IV pili
Type IV pili (a type II machine)
Type III secretion
Triggered by contact w/ host cells.Sec-independent, similar to flagellar assembly.Assembly of the needle occurs at the tip.
Needle
The type III needle complex
Nature 435, 702-707 (2 June 2005)
Type IV secretion
Very versatile; Sec- and ATP-dependent.
Pertussistoxin
Agarobacterium
Autotransporters: Neisseria IgA protease
Synthesized as a pre-proenzyme.C-term -barrel inserts in OM, pulls N-term through.N-term auto-cleaves, promoting release.
Also calledtype V secretion
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):1478-83.
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.
0
25
50
75
100
125
0 2 4 6 8
days
% m
etab
olis
m
A B
+ RD1 tn3870 tn3871 tn3874 3875 tn3876
x H37Rv- tn0982-- tn3864
H37Rv
H37Rv:t
n098
2
H37Rv:t
n386
4
H37Rv:
RD1
H37Rv:t
n387
0
H37Rv:t
n387
1
H37Rv:t
n387
4
H37Rv:
3875
H37Rv:t
n387
6
5.0×1006
1.0×1007
1.5×1007
2.0×1007day 0
day 4
day 7
bact
eria
/ w
ell
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 2
0
20
40
60
80
100
120
0 2 4 6 8days
% m
etab
olis
m
A
0204060
80100120
0 2 4 6 8days
% m
etab
olis
m
B
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 3
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
day 0 day 4 day 70
25
50
75 H37Rv
H37Rv: RD1
***
***
% o
f cel
ls in
fect
ed
A B
day 0 day 4 day 70
25
50
75
100
***
***
% o
f inf
ecte
d ce
lls w
ith>1
0 ba
cilli
Fig. 4
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
0 4
weeks
bact
eria
/ lu
ng
H37RvH37Rv: RD1H37Rv:tn3870H37Rv: 3875H37Rv:tn3876
Fig. 5
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
A B
C ED
Fig. 6
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
Fig. 7
Guinn et al, Mol Microbiol, 2004, 51(2):359-370.
A B
0 25 50 750
100
200
300
ng/ml CFP-10
spot
form
ing
units
(IFN-
)
H37RvRD1
tn387
0
tn387
0:KG18
tn387
1
tn387
1:KG18
tn387
4
tn387
4:MH40
8 38
75
3875
:MH40
6
tn387
6
tn387
6:MH42
90
100
200
300 filtratepellet
spot
form
ing
units
(IFN-
)