Molecular Microbiology

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Molecular Microbiology Pathogenesis BIOL 5392
(March 4, 2009) Dr. Joseph Vogel MPRB Room
10220 jvogel_at_borcim.wustl.edu 314-747-1029
"Facts are meaningless you can use facts to
prove anything that's even remotely true!
Facts, schmacks. Homer Simpson
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• Additional papers you can read if you are
  interested/confused
• Type I
• Delepelaire. Type I secretion in gram-negative
  bacteria. Biochim Biophys Acta. 2004.
  1694149-61.
• Type II
• Johnson et al. Type II secretion from structure
  to function. FEMS Microbiol Lett. 2006.
  255175-86.
• Type III
• Cornelis GR. The type III secretion
  injectisome. Nat Rev Microbiol. 2006. 4811-25.
  Review.

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• Additional papers you can read if you are
  interested/confused
• Type IV
• Christie PJ. Type IV secretion the
  Agrobacterium VirB/D4 and related conjugation
  systems. Biochim Biophys Acta. 2004. 1694219-34.
• Type V
• Desvaux et al. The autotransporter secretion
  system. Res Microbiol. 2004. 15553-60.
• Henderson et al. Type V protein secretion
  pathway the autotransporter story. Microbiol
  Mol Biol Rev. 2004. 68692-744.
• Bonus paper
• Buttner D, Bonas U. Common infection strategies
  of plant and animal pathogenic bacteria. Curr
  Opin Plant Biol. 2003. 6312-9.
• TAT
• Robinson Bolhuis. Tat-dependent protein
  targeting in prokaryotes and chloroplasts.
  Biochim Biophys Acta. 2004. 1694135-47.

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Some people believe there is only one truly
important secretion system
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Primitive view of the first three types of
secretion systems
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More complicated view of secretion
Buttner Bonas. Trends Microbiol. 2002
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Bacterial protein secretion systems (historical
landmarks)

• 1981 First secA mutant isolated
• 1985 First T1SS identified (E. coli
  a-hemolysin)
• 1987 First characterization of a T2SS
  (Klebsiella)
• (Dr. Vogel started graduate school in 1987)
• 1987 First T5SS proteins identified (IgA1
  proteases)
• 1990 Discovery of T3SSs (Yersinia)
• 1996 Existence of TAT (twin-arginine pathway)
  proposed
• 1997 T4SS proposed based on Agrobacterium
  T-DNA transfer

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Why is it difficult for Gram-negative bacterium
to secrete proteins?
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• Type I Secretion
• Type I secretions are generally known as the
  ATP-binding cassette (ABC) protein type
• Best examples are HylA (hemolysin A) secretion
  by E. coli and RTX toxins (repeats in toxins)
• Type I secretion systems contain only three
  different transport components (2 in the inner
  memb. and one that forms a general pore in the
  outer memb.)
• Proteins are secreted directly through the pore
  across both membranes in one step
• Signal sequence is at the c-terminus of the
  substrate (generally not cleaved)

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Type I secretion (topological model of hemolysin
secretion)
Gentschev I, Dietrich G, Goebel W. Trends
Microbiol. 2002
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• Type II Secretion
• Type II secretion is exemplified by Klebsiella
  oxytoca pullulanase (PulA) secretion and Vibrio
  cholerae cholera toxin secretion
• Type II secretion is a two step process
• Protein is first exported into the periplasm by
  the sec apparatus (can also use the Tat pathway)
• Protein is then transported across the outer
  membrane by a dedicated secretion apparatus
  composed of 12-16 proteins
• Type II pathway is also called the
  secreton-dependent pathway or the terminal path
  of the general secretion pathway (NOT the general
  secretion path or sec)

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Type II secretion systems (terminal path of the
general secretion pathway)
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Cholera toxin secretion (type II) piston?
EpsD (secretin or pore)
Cholera toxin A and B subunits
Sec machinery
Sandkvist. Biology of type II secretion. Mol
Microbiol. 2001
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Three different versions of Type II secretion
Mattick et al 1993. Mol. Micro.
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• Type V Secretion
• Type V (autotransporters or two-partner system)
• Simple system consisting of only 1 or 2 proteins
• Amino-terminal signal sequence
• Export occurs in two steps
• First step occurs by the sec machinery
• Second step occurs by transport across from the
  periplasm and across the outer membrane via a
  b-barrel
• Examples include Neisseria gonorrhoeae IgA1
  protease

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Type V Secretion
Desvaux et al. 2004. Research in Microbiology
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• Type III secretion systems
• First described in Yersinia species in mid-1950s
• Y. pestis strains are unable to grow at 37
  degrees in media lacking calcium
• Mutants that are calcium-independent are
  avirulent
• Virulence (and calcium dependence) was due to a
  70 kb plasmid (e.g. pCD1)
• Virulence plasmid directs massive secretion of
  10 proteins into the culture supernatant under
  inducing conditions

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Yersinia Ysc injectisome
Guy R. Cornelis. Nature Reviews Molecular Cell
Biology 3, 742-754 (2002).
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Salmonella type 3 secretion needles
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Structural comparison between a flagellum and a
type III secretion apparatus
Cellular Microbiology 2000 Fig. 13.3 page 247
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Evolutionary proposal Bacterial type III
protein secretion systems (TTSS) may have evolved
from a flagellum
Milt Saier Trends in Micro 2004. 12 113-115
Homologous structures in the basal regions of
both structures are colored the same.
Non-homologous structures are colored
dissimilarly. IM, inner (cytoplasmic) membrane
PG, peptidoglycan cell wall layer OM, outer
(lipopolysaccharide-containing) membrane.
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Type III secretion systems (widespread) Species
T3SS Diseases Yersinia spp Ysc (Yops)
Bubonic plague Ysa in Y. enterocolitica
gastrointenstinal disorders Shigella spp Mxi
(Spa) Bacillary dysenteria Salmonella
spp SPI-1/Inv (invasion) Mild food poisoning to
life-threatening systemic infections
depending on serovar SPI-2/Ssa (intracellular
survival) Pseudomonas Psc Pneumonia chronic
bronchopneumonia aeruginosa in patients with
cystic fibrosis EPEC EHEC Esc Diarrhea,
attachment/effacement lesion Chlamydiaceae Sct
(internalization) Sexually transmitted disease,
trachoma, pneumonia, atheroschlerosis? Bord
etella Bsc (establish long term
infection) Whooping cough
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Components of type III secretion
T3S translocation pore

• Hollow, elongated, stiff needle-like structure
• 45-80 nm in length
• Length controlled by a ruler

Cylindrical base

• Cytoplasmic secretion chaperones
• small, acidic proteins with ampiphathic
  c-terminal helix
• bind substrates
• stabilize the protein and/or assist in export

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• Type III secretion systems
• Secretion is sec-independent
• Secretion occurs in one step (no periplasmic
  intermediate)
• Signal sequence is not cleaved at the
  amino-terminus
• (Big fight over whether it is protein and/or RNA
  based)

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The Yersinia T3SS effectors -- used to inhibit
phagocytosis
Juris, Shao Dixon Cellular Microbiology 2002
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• YopH
• yopH mutant is avirulent in mice
• Has a 262 aa domain with tyrosine phosphatase
  activity
• Active site is a cysteine
• Dephosphorylates p130Cas, FAK and paxillin
  (causes disruption of focal adhesion complexes
  thereby inhibiting phagocytosis)
• Down-regulates signalling cascades required for
  the activation of Fc-mediated respiratory burst
  in macrophages and neutrophils
• Suppresses both T-cell cytokine production and
  expression of the B-cell co-stimulatory
  receptor B7.2

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• YopE
• Acts with YopH to block phagocytosis
• Disrupts actin stress fibers
• GAP (GTPase-activating protein) for Rho family
  members
• Has homology to ExoS (Pseudomonas) and SptP
  (Salmonella)

Conserved Arg finger
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• YopT
• Strong depolymerizing effect on actin
• Modifies Rho-family proteins and displaces them
  from the membrane to the cytosol
• Appears to be a cysteine protease
• Cleaves RhoA, Rac, and Cdc42 close to their
  carboxyl terminus, releasing them into the
  cytosol

Surface antigen p76 (Haemophilus
somnus) Filamentous haemagglutinin-like
protein Lsp2 (Haemophilus ducreyi) PfhB2
(Pasteurella multocida) All of unknown
function Conserved residues shown in blue
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• YopO/YpkA
• Looks like a eukaryotic Ser/Thr kinase
• Specific target has not yet been identified
• Cells infected with multiple yop mutant
  expressing YopO, round-up, but do not detach
  (different from YopE)
• Requires a eukaryotic kinase activator (actin)
• YpkA induces disruption of actin fibers in
  transient transfection experiments of HeLa
  cells

Yellow shows invariant residues in kinases Blue
lysine is specific for Ser/Thr kinases
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• YopM
• 41 kDa protein necessary virulence determinant
• Composed of leucine-rich repeats (LRRs)
• LRRs are found in diverse group of proteins
  (Rnase inhibitor, Toll receptors, proteoglycans,
  platlet glycoproteins)
• Protein-protein interaction motif (signaling
  pathways)
• Initially thought to interact with thrombin and
  inhibit thrombin-induced platelet aggregation
  (extracellular)
• Later shown to be translocated and localizes to
  the nucleus
• Function not clear

Examples of proteins with leucine-rich
repeats (LRR is the blue box) IpaH
(Shigella) SspH SlrP (Salmonella) YF4R
(Rhizobium) ID431 (Bradyrhizobium)
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• YopJ
• Induces apoptosis in macrophages, not epithelial
  cells
• Homologues to AvrRxv from Xanthomonas which
  activates programmed cell death in plants
• Blocks TNF release in macrophages
• Blocks MAP kinase activity
• yopJ mutant is not attenuated in an animal model

YopJ (Yersinia) AVP (Adenovirus protease) Ulp1
(yeast ubiquitin-like protein protease) Catalytic
triad in yellow, green red
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• Type IV secretion systems
• Plasmid transfer systems (e.g. F plasmid)
• Adapted conjugation systems (e.g. pathogens)

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Crown gall
Tzfira and Citovsky 2000 Molecular Plant Pathology
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Crown gall (Agrobacterium tumefaciens)
Tzfira and Citovsky 2000 Molecular Plant Pathology
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• Agrobacterium tumefaciens
• Gram negative bacterium found in the soil
• Able to genetically transform plants (crown gall)
• Transfers a single-stranded segment (the
  T-strand) of the bacteriums tumor-inducing (Ti)
  plasmid into the host cell

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• Agrobacterium tumefaciens
• T-DNA integrates in the plant genome
• T-strand products direct the synthesis of plant
  growth regulators results in tumorous
  proliferation of plant cells
• T-strand products also cause the tumor to produce
  opines, compounds that represent a unique carbon
  and nitrogen source metabolized by the bacterium
• Researchers can replace T-strand sequences with
  recombinant DNA to engineer plants for
  agricultural and research purposes

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• Steps of host transformation by Agrobacterium
• plant cell recognition
• (2) plant-wound-signal-mediated induction of
  virulence genes
• (3) T-strand generation
• (4) T-complex and virulence factor export to the
  host cytoplasm
• (5) T-strand nuclear import
• (6) T-strand integration into the host genome

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Cellular Micro. Fig 14.1
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virB operon

• VirB1 trans-glycosylase
• non-essential virulence factor- may play role
  in mediating specific contacts with plants
• VirB2 pilin
• VirB4, VirB11 and VirD4 ATPases (Walker boxes)
• (VirD4 is probably the receptor for substrates)
• VirB6 and VirB10 inner membrane pore
• VirB7 and VirB9 outer membrane pore

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Assembly of the VirB complex
Cellular Micro. Fig 14.3
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Pretty model of the Agrobacterium tumefaciens T4SS
Cascales Christie, Science 2004
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• Type IV secretion systems
• Plasmid transfer systems (e.g. F plasmid)
• Adapted conjugation systems (e.g. pathogens)

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1. Plasmid transfer systems
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2. Adapted conjugation systems
Pertussis Toxin
T-DNA
Bordetella
A
Nucleus
Agrobacterium
A
Oncogenesis Opine production
ADP-ribosylation of GTP-binding proteins
Eukaryotic Target
Cytoskeletal Rearrangements (IL-8 induction
tyrosine phosphorylation)
Intracellular survival (e.g. prevent
phagosome- lysosome fusion)
CagA
Helicobacter
Legionella, Coxiella, Rickettsia, Brucella,
Bartonella
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Two things you need to know about conjugation
that your mother never told you 1. Most, if
not all, conjugation systems work via a
membrane fusion event 2. DNA is not the real
substrate of conjugation systems
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1. Plasmid transfer systems
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Bacterial conjugation
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• Plasmid transfer steps
• 1. Adherence via pilus

2. Pilus retraction
3. Fusion of the membranes

• 4. DNA processing
• 5. Transfer of ssDNA to the recipient

6. Synthesis of the complementary DNA strands
7. Cell separation
Brock et al. 1994
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• Plasmid transfer steps
• 1. Adherence via pilus

2. Pilus retraction
3. Fusion of the membranes

• 4. DNA processing
• 5. Transfer of ssDNA to the recipient

6. Synthesis of the complementary DNA strands
7. Cell separation
Brock et al. 1994
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RP4 plasmid conjugation - membrane fusion
Samuels AL, Lanka E, Davies JE. J. Bact. 2000
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• Plasmid transfer steps
• 1. Adherence via pilus

2. Pilus retraction
3. Fusion of the membranes

• 4. DNA processing
• 5. Transfer of ssDNA to the recipient

6. Synthesis of the complementary DNA strands
7. Cell separation
Brock et al. 1994
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• DNA processing and transfer
• Nicking at the oriT (origin of transfer) by a
  relaxase
• Covalent attachment of the relaxase to the 5
  prime end of one strand
• Unwinding of the two strands (helicase activity)
• Transfer of the relaxasome into the recipient
  (protein ssDNA is transferred)

ssDNA intermediate
oriT
Relaxase
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Plasmid transfer
relaxase/nickase
Plasmid transfer is mediated by transfer of a
protein (DNA can be viewed as cargo!!!)
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• Type IV secretion systems
• Plasmid transfer systems (e.g. F plasmid)
• Adapted conjugation systems (e.g. pathogens)
• Two flavors of type IV secretion systems
• Type IVA VirB-like (e.g. Agrobacterium VirBs)
• Type IVB IncI-like (e.g. Legionella Dot/Icms)

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Type IVA secretion systems
T-DNA
D4
B8
B1
B4
B6
B9
B10
B11
B5
B3
B2
B7
pKM101
L
B
C
M
D
N
E
O
F
G
A
J
R388
B
M
K
J
I
H
G
F
E
D
L
(Rh1)
Ti
E
C
D
I
F
B
RP4
C
D
E
I
F
G
N
B
F
169
E
A
L
D
C
B
L. pneum.
B3
B2
D4
B1
B4
B5
B6
B8
B9
B10
B11
B7
B. pertussis
C
D
B
A
I
F
G
H
E
H. pylori
E
528
527
525
524
T
B. suis
B3
B2
B7
B4
B5
B6
B9
B10
B11
B8
B. abortus
B3
B2
B7
B4
B5
B6
B9
B10
B11
B8
B1
B. henselae
B3
B2
B7
B4
B5
B6
B9
B10
B11
B8
Wolbachia
VirB
D4
B9
B10
B11
B8
R. etli
B9
B10
B11
B8
R. prowazekii
457
103 / 784
286 / 290
291
292
293
287
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Type IVB secretion systems
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• Type IVB secretion systems
• (not virB-like)
• IncI plasmids
• ColIb-P9 plasmid
• R64 plasmid
• P. syringae pPT23A-like plasmid
• Legionella pneumophila dot/icm genes
• Coxiella burnetii dot/icm homologues

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2. Adapted conjugation systems
Pertussis Toxin
T-DNA
Bordetella
A
Nucleus
Agrobacterium
A
Oncogenesis Opine production
ADP-ribosylation of GTP-binding proteins
Eukaryotic Target
Cytoskeletal Rearrangements (IL-8 induction
tyrosine phosphorylation)
Intracellular survival (e.g. prevent
phagosome- lysosome fusion)
CagA
Helicobacter
Legionella, Coxiella, Rickettsia, Brucella,
Bartonella
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Pertussis toxin secretion uses an oddball type IV
system
Target Cell
A
Pilus
OM
PT Holotoxin
A
IM
Coupling protein
GSP
Chaperones
CagA
S
S
VirE2, VirF
PT subunits
S
RecA, Sog
N-terminal signal sequence
T-strand/relaxase
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How would one identify the secretion apparatus
components that directly contact substrate?
Protein being secreted
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TrIP (transfer immunoprecipitation)
-gt modified ChIP (chromosome immunoprecipitation)
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Definition of a bacterial type IV secretion
pathway for a DNA substrate. Cascales and
Christie. 2004. Science. 3041170-3.
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(No Transcript)
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End of day 1 (go back to your labs and do some
real work)