Transcript Design and Function of Bacterial Toxins

Bacterial secretion
Disease
function of susceptibility
of host
immune
competent/compromised
immunizations
age
trauma
genetics
antimicrobial therapy
relates to mechanism of
bacterial pathogenesis
secretion of factors (toxins)
direct host cell manipulation
(type III / type IV
secretion systems)
Bacterial secretion
Function - protection (secretion of toxins / enzymes - virulence factors)
- transport of cell surface, cell wall, cell membrane proteins
- communication
Mechanisms - differ between Gram-negative and Gram-positive bacteria
Experimental approaches to study bacterial secretion
Describe bacterial secretory mechanisms
Role of secretory processes in pathogenesis Type III (Type IV)
Bacterial protein secretion
Gram-negative - translocation past the cytoplasmic / periplasm / outer membrane
Gram-positive - translocation cytoplasmic membrane / cell wall
Studying bacterial secretion processes
• Identify / develop a secretory mutant phenotype
• Identify secretory components
• Clone / sequence - examine data banks for sequence / motif similarities pathogencity islands
• Examine function based on:
- sequence homologies (enzyme / adherence / channel)
- biochemical analyses
- site-directed mutagenesis
• Determine crystal structure of protein components
• Use biochemical / two-hybrid analyses to determine protein-protein interaction
components of apparatus
• Electron microscopy to visualize secretory structure
Bacterial transport mechanisms serve as models for studying
eukaryotic membrane-transport mechanisms
Gram-negative secretion
Type I - ATP-binding cassette (ABC) transporter
Type II - general pathway (Sec-dependent) - major secretory pathway
Type III - contact-dependent translocation into eukaryotic cells
Type IV - (Sec-like dependent) - translocation of DNA / protein complex
Type V - auto-transporter (Sec-dependent) - includes b-pore forming domain
~
Tat - (twin arginine transport) - moves folded proteins across CM
SRP- (signal recognition particle) (Sec-dependent) - used for CM proteins
Gram-negative - Type I secretion (ABC secretion)
Properties:
- ATP-binding cassette transporter (also in eukaryotes)
- Single step traversal across CM and OM
- Signal sequence at C-terminus - is not removed
- ABC channel - 6-12 transmembrane helices
- Accessory factor - bridges periplasmic space
- Post-translationally coordinated synthesis-translocation
OM
P
accessory
factor
CM
Genes fused or coordinately expressed on operon:
protein - GGXGSD
ATP
ADP
ABC transporter
accessory factor (MFP)
Outer membrane transport may not be linked
N
C
Type I secreted proteins:
RTX toxin (repeat in toxin)
E. coli hemolysin
bacteriocins
metalloproteases
Gram-negative - Type II secretion
Sec-dependent secretory pathway
signal peptidase
leader peptide
N
++
hydrophobic
C
1-5
7-15
3-7
mature protein
Two step process:
Step 1 - Transfer across cytoplasmic membrane
- Leader (signal) peptide (18-26 aa)
- SecA - binds leader (L), inserts in CM channel
(requires ATP)
- SecB - cytosolic chaperone (keeps unfolded)
- SecYEG - CM channel complex
SecY,E,G
N L
SecB
C
(www.genome.ad.jp/kegg/ pathway/map/map03090.html)
post-translational translocation
Gram-negative - Type II secretion
Step 2 - Transfer through periplasm /
outer membrane transfer
Periplasm:
Protein folded into final structure & complex
- signal peptide removal
- chaperone-mediated protein folding
- disulfide bond formation
- oligomerization
- proline isomerization
OM
P
CM
Sec
ATP
ADP
Outer membrane translocation:
Protein - bacteria specific mechanisms
- Secreton - homology to pilus components
- Secretins - homology to phage OM proteins
- driven by PMF (?ATP)
Type II secreted proteins:
Majority of virulence factors
- pullulanase
- AB toxins
- proteases
Type III secretion
Host-cell contact induced secretion process
Gram-negative
bacterium
induction /
translocation
of type III
effectors
(intracellular
enzyme activity)
direct
manipulation of
host cell actin
/ function
Gram-negative - Type III secretion regulon
Type III secretion components
pscU
pscT pscS pscR pscQ pscP pscO
pscL pscK pscJ pscI pscH pscG pscF pscE
pscD
pscN
pscC
popN
pscB
exsD
pcr1 pcr2 pcr3 pcr4
exsA exsB exsC
pcrD
popD popB pcrH
*
pcrR
pcrV
*
pcrG
* *
Type III effectors
spcU
exoU
orf1
exoS
exoT
*
exoY
* *
*
*
(Pseudomonas aeruginosa regulon, Frank, Yahr 1997; Figure courtesy of Dara Frank)
Properties:
- Induced by contact with host cell
- Coordinately induces - regulatory, structural and effector genes - encoded on a
pathogenicity island (chromosomal / plasmid / phage)
- No Sec-dependent signal sequence
- Provides a conduit for the direct translocation of bacterial proteins into host cells
- Evolutionary relationship with flagella
Gram-negative - Type III secretory apparatus
Salmonella
Shigella
S. typhimurium
flagellum
(Kubori, 1998; Blocker, 2001; Plano, 2001)
Comparison of type III secretion structures
Flagellum
Yersinia
E. coli
P. syringae
2 µM
10-15 µM
~90 nM
58 nM
(Tampakaki et al., Cellular Microbiology, 2004)
Shigella type III secretion needle structure
7 nN
2 nN
(Deane et al., PNAS USA, 2006)
Structure / function of type III effectors
A-B toxin
A-subunit
S
S
L enzyme activity /
intracellular trafficking
B-subunit
receptor binding
internalization
Type III effectors
A-subunit
YopE
SopE
YopH
YopO
T enzyme activity
GAP - Rho, Rac, Cdc42
GEF - Rho
phosphatase
kinase
A-subunit
SptP
ExoS
ExoT
A-subunit
GAP - Rho, Rac, Cdc42 - phosphatase
GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferase
GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferase
type III effectors function in a coordinated manner within the host cell
Gram-negative - Type IV secretion
Properties
- Used in export of protein complexes / DNA
- Can translocate directly into host cell
- Show homology to pilus-mediated conjugal
transfer systems
- Sec-like dependent translocation into periplasm
- B11 - related to ATP-ases of type II system
- D4 - DNA binding - may function in DNA transfer
- B6, B7, B8 B9, B10 - core periplasmic components
- B2, B5 - pilus components
Bacteria that use type IV secretion:
(H-J. Yeo, G. Waksman, J. Bacteriol. 2004)
Agrobacterium tumefaciens - VirB-VirD
Bordetella pertussis - pertussis toxin
Helicobacter pylori - CagA
Legionella pneumophila
Gene organization of Type IV secretion
A. tumefaciens
Type V secretion - autotransporter
(Desvaux et al., Res in Microbiol. 2004)
Properties:
- Insertion of b-domain - formation b-barrel pore in outer membrane
- Signal sequence - directs protein membrane translocation
- Linker region - leads protein secretion through pore
- Auto-chaperone - triggers protein folding
- Folded protein - released (or not) from membrane
Bacteria that use type V secretion:
Neisseria gonorrhoeae - IgA1 protease
Helicobacter pylori - VacA
Haemophilus influenzae - Hsf fibrillar protein
(Wilson, McNab, Henderson
Bacterial Disease Mechanisms, 2002)
Gram-negative secretion
Sec-(or Sec-like) dependent
Sec-independent
Type I
Type II
Type III
Type IV
Type V
host
cell
host
cell
OM
P
Sec B11
CM
ATP
ADP
ATP
ADP
N
ATP
ATP
ADP
N
ADP
N
C
Sec
C
C
(Adapted from Stathopoulus et al. (2000); provided by E Rucks)
Gram-positive secretion
Type I - ATP-binding cassette (ABC) transporter
Type II - general pathway (Sec-dependent) - major secretory pathway
Type III - oligolysin-dependent translocation
No type IV secretion Type V - auto-transporter (Sec-dependent) - includes b-pore forming domain
~
Tat - (twin arginine transport) - moves folded proteins across CM
SRP - (signal recognition particle) (Sec-dependent) - used for CM proteins
Gram-positive - secretion
Type I - ATP-binding (ABC)
CW
CM
ATP
ADP
CW
accessory
factor
trans
memb
pore
N
Type II - Sec-dependent
Type V - autotransporter
b-barrel
pore
CW
CM
CM
Sec
Sec
ATP
ADP
Protein - C-terminal
signal
C
bacteriocins
N
Protein - N-terminal
signal
Protein translocation unit
C
majority of proteins
Staphylococcus alpha toxin
Gram-positive - Type III secretion
Cytolysin-Mediated Translocation
Gram-negative
Streptococcus pyogenes
Gram-positive
Properties:
- spn (NAD glycohydrolase) - slo (streptolysin O)
genes linked and co-transcribed
Cytotoxic lymphocyte
- SPN and SLO exported by Sec-dependent
secretory process
- SLO - pore forming cytolysin - binds cholesterol
in membrane - oligomerizes to form pore allows translocation of SPN
(Madden, Ruiz, Caparon, Cell, 2001)
Role of secretory processes in bacterial pathogenesis
Gram-negative - type III secretion
Pseudomonas aeruginosa - extracellular pathogen
Salmonella spp - intracellular pathogen
Pseudomonas aeruginosa
Bacteriology - Gram-negative rod,
motile / aerobe
ubiquitous, highly adaptable bacterium
Disease - opportunistic pathogen
(www.bact.wisc.edu/ Bact330/lecturepseudomonas)
Pathogenesis - complex / multi-factorial
related to regulated secretion of multiple
virulence factors
primarily an extracellular pathogen
Pyocyanin production by P. aeruginosa
Identification / diagnosis - culture / isolate
forms smooth, fluorescent green colonies at 42oC
characteristic sweet (grape-like) odor
(students.washington.edu/ chenamos/Pseudomonas)
Pseudomonas aeruginosa
Disease
opportunistic pathogen
nosocomial infections
indwelling catheters, urinary tract, lung, bloodstream
complicated by antibiotic / disinfectant resistance
infects compromised individuals
burns, wounds, immuno-compromised
cystic fibrosis
disease manifestations
chronic and acute lung infection
nosocomial pneumonia
corneal ulcers
urinary tract infections
wound infections
chronic lung infections in CF patients
P. aeruginosa - infections
Nosocomial pneumonia
Contact lens associated corneal ulcer
Ear piercing infections
( www.opt.pacificu.edu/.../ 13036-AS/Fig%2017.jpg)
Greenish pigment-associated infection
(www.uni-mainz.de/.../ tag/heussel/aj97_p1c.jpg)
(www.dadlnet.dk/ufl/ 0244/VP-html/VP38141-3.jpg)
P. aeruginosa - Green nail syndrome
Hot tub dermatitis
(www.rsdfoundation.org/ images/image16.gif)
Folliculitis
(www.skinatlas.com/ greenailopt.jpg)
(www.dermnet.com/ thumbnailIndex)
Pseudomonas aeruginosa
Cystic fibrosis:
lethal autosomal recessive disease
characterized by pulmonary obstruction
pancreatic exocrine deficiency
high sodium and chloride in sweat
male infertility
most common, serious inherited disease
among Caucasians
Mutation in CFTR gene - cause of cystic fibrosis
(CF transmembrane conductance regulator)
90% of morbidity and mortality of CF patients relates to chronic lung
infection (by Pseudomonas aeruginosa)
CF transmembrane conductance regulator
( www.cfgenetherapy.org.uk/ CFTR.htm)
DF508 - most frequent mutation
recognized as non-functional protein
not modified in ER - degraded
(wsrv.clas.virginia.edu/ ~rjh9u/gif/cfmap3.gif)
Pseudomonas aeruginosa virulence factors
Type I secretion:
Planktonic P. aeruginosa
hemolysin
Type II secretion:
proteases
elastase (LasB) - zinc metalloprotease
LasA - serine protease
alkaline protease
exotoxin A - ADP-ribosylating toxin
(textbookofbacteriology.net/ P.aeruginosa.jpeg)
Type III secretion:
ExoS - GAP / ADP-ribosylating enzyme
ExoT - GAP / ADP-ribosylating enzyme
ExoU - PLA2
ExoY - adenylate cyclase
Bacterial biofilm magnified 7,000x
No Type IV secretion
Biofilm formation
alginate - mucopolysaccharide
quorum sensing
(www.math.utah.edu/.../ quorum_talk.html)
Studying the role of type III
secretion in pathogenesis
Pseudomonas type III secretion effectors
Effect on eukaryotic cell
ExoS
GAP
ADP-ribosylates LMWG-proteins
Rho, Rac, Cdc42
GAP
Ras, Ral, Rabs, Rac
ADP-ribosylates Crk
ExoT
Rho, Rac, Cdc42
ExoU
cell inactivation
anti-phagocytic
CF ( 85%), wound,
UT, soil isolates
anti-phagocytic
alters cytoskeletal structure
100% isolates
PLA2 - cytotoxic
CF (15%)
corneal isolates
adenylate cyclase
ExoY
cyclic AMP
CF (97%)
(Feltman, et al, 2001, Fleiszig, 1997)
Effects of ExoS on human epithelial cells
388 DExoS (1 hour)
Strain 388 (1 hour)
Strain 388
(Fraylick et al, Infect. Immun. 1999)
Effects of ExoS on eukaryotic cell function
• Inhibition of DNA synthesis
• Cell rounding (altered cytoskeleton)
• Anti-phagocytic / anti-invasive
• Loss of cell surface microvilli
• Loss of adhesion or re-adhesion
• Loss of cell viability
ExoS is a bi-functional toxin
R146
1
E379 E381
99
233
453
ADP-ribosyltransferase
Rho-GAP
GDP-inactive Rho, Rac, Cdc42
CONH2
GTP
PI
Cellular Targets
N
ExoS
GAP
GEF
CH2
O
CH
O Adenine
2
focal adhesions / stress fibers
filopodia / lamellopodia
(Goehring et al.)
CH
ExoS
P
P
GDP
GTP active Rho, Rac, Cdc42
[Ras-family LMWG-proteins]
2
CONH2
P
P
CH
2
NAD
O
+
N
O Adeninenicotinamid
e
ADP-ribosylated protein
(Iglewski, Coburn, Barbieri)
Effects of ExoS GAP and ADPRT activity
on macrophages
0
ExoS
GAP-mutant
(Rocha et al, Infect. Immun. 2002)
ADPRT mutant
Bi-functional effects of ExoS on cell function
GAP-ADPRT
GAP-ADPRT
*
*
Rac
Ras
Ral
GTP
GTP
GTP
anti-phagocytic
****
* Cdc42
*
Rac1,
Rabs 5, 8, 11, 7
inhibits DNA synthesis
affects adherence
alters morphology
affects cell viability
Eukaryotic cell
(E. McGuffie, J. Fraylick, E. Rucks, J. LaRoche, C. Rocha, J. Barbieri)
Salmonella
Salmonella enteritidis - gastroenterititis
Salmonella typhimurium - gastroenterititis
Salmonella typhi - typhoid fever
Bacteriology - Gram-negative
facultative, motile rod
non-lactose fermentor / H2S production
Pathogenesis -
(www.ipsiaponti.it/.../ bacilli/salmonella.htm)
intracellular pathogen
Virulence factors Two type III secretion processes
- SPI-1 - (Salmonella pathogenicity island-1)
involved in initial invasion
- SPI-2 - (Salmonella pathogenicity island-2)
involved in intracellular survival
(microvet.arizona.edu/.../ salmonella/sem.html)
Salmonella invasion
- Salmonella can directly invade epithelial cells
- Or can cross intestinal epithelium via M cells - likely main portal of entry
- Also invades macrophages
Fimbriae-mediated contact with
epithelial cells induces bacterial
appendages - invasomes
Entry of bacteria into cells / and
presence or loss of invasomes
Invasomes disappear upon entry into
cell
Salmonella - SPI-1 type III secretory process
(www.niaid.nih.gov/biodefense/images/SALMON_1.jpg)
Salmonella invasion:
SipB
SipC
SipD
(E. Stebbins, J. Galan, Nature, 2001)
Mimicry of type III effectors - eukaryotic proteins
R
SptP - GAP / tyrosine phosphatase activity
SopE - GEF for Rho / Rac / Cdc42
SopB - inositol phosphatase - PI(1,3,4,5,6)P5
to PI(1,4,5,6)P4
SipA - binds actin, inhibits depolymerization
SipB - binds activates caspase-1, induction
of apoptosis in macrophages
R
(E. Stebbins, J. Galan, Nature, 2001)
Salmonella - SPI-2 type III secretory process
SPI-2 - Salmonella survival/ growth in Salmonella containing vacuoles (SCV)
(identified using signature tagged mutagenesis - 40 kb island)
SPI-2 - includes 13 effector
proteins affecting:
(SR Waterman, DW Holden, Cell. Microbiol. 2003)
- Actin rearrangement
- Inhibits endocytic trafficking
- Avoidance NADPH-oxidase
killing
- Delayed apoptosis
- SCV membrane dynamics
- Assembly of F-actin mesh
around SCV membrane
- Accumulation of cholesterol
around SCV
- Interference nitric oxide
synthesis
Alternative uses of bacterial secretion processes
- Type I (ABC) secretion signals can be fused to heterologous proteins
which are efficiently secreted from bacteria - use in biotechnology
- Type III secretion used to deliver proteins directly into eukaryotic cell cytosol
- Type IV secretion used to deliver complex proteins directly into host cells
Type IV translocation
domain
Protein sequence of choice
- Type IV secretion used to deliver DNA (contributes to spread of
antibiotic resistance genes)
Protection against secretion-linked virulence factors
• Anti-bacterial agents - antibodies / vaccines / antibiotics
• Innate immune response
• Cellular immune response effective against intracellular bacteria
• Humoral immune response not effective against type III effectors
Concepts - bacterial secretion
• Mechanisms of bacterial secretion
differences between Gram-positive / Gram-negative bacteria
• Methods used to study secretory processes identification and function of secretory components and effectors
• How bacteria use type III secretion to manipulate host cell function
• Functional mimicry between bacterial and eukaryotic cell proteins