Bacterial secretion
Disease
function of susceptibilityof host
relates to mechanism ofbacterial pathogenesis
immune competent/compromised
immunizationsage
trauma genetics
antimicrobial therapy
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 secretionDescribe bacterial secretory mechanismsRole of secretory processes in pathogenesis - Type III (Type IV)
Gram-negative - translocation past the cytoplasmic / periplasm / outer membrane
Gram-positive - translocation cytoplasmic membrane / cell wall
Bacterial protein secretion
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 -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)
Type I secreted proteins: RTX toxin (repeat in toxin) E. coli hemolysin bacteriocins metalloproteases
ATPADP
N
C
OM
P
CM
accessory factor
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
protein - GGXGSD ABC transporter accessory factor (MFP)
Genes fused or coordinately expressed on operon:
Outer membrane transport may not be linked
Gram-negative - Type II secretion
N
C
SecBL
SecY,E,G
(www.genome.ad.jp/kegg/ pathway/map/map03090.html)
Sec-dependent secretory pathway
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
post-translational translocation
signal peptidaseleader peptide
1-5 7-15 3-7
N ++ hydrophobic C mature protein
Sec
ATPADP
Periplasm:Protein folded into final structure & complex - signal peptide removal- chaperone-mediated protein folding- disulfide bond formation- oligomerization- proline isomerization
Outer membrane translocation:Protein - bacteria specific mechanisms - Secreton - homology to pilus components- Secretins - homology to phage OM proteins- driven by PMF (?ATP)
OM
P
CM
Type II secreted proteins: Majority of virulence factors - pullulanase - AB toxins - proteases
Gram-negative - Type II secretion
Step 2 - Transfer through periplasm / outer membrane transfer
induction /translocation
of type IIIeffectors
Gram-negativebacterium
(intracellularenzyme activity)
directmanipulation ofhost cell actin
/ function
Type III secretion
Host-cell contact induced secretion process
spcU exoU orf1 exoS exoT exoY* *
* **
(Pseudomonas aeruginosa regulon, Frank, Yahr 1997; Figure courtesy of Dara Frank)
Type III effectors
pscU pscT pscS pscR pscQ pscP pscO pscN popN pcr1 pcr2 pcr3 pcr4 pcrD pcrR
pscL pscK pscJ pscI pscH pscG pscF pscE pscD pscC pscB exsD exsA exsB exsC popD popB pcrH pcrV pcrG**
*
*
Type III secretion components
Gram-negative - Type III secretion regulon
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
(Kubori, 1998; Blocker, 2001; Plano, 2001)
S. typhimurium flagellum
Comparison of type III secretion structures
Flagellum Yersinia E. coli P. syringae
(Tampakaki et al., Cellular Microbiology, 2004)
10-15 µM
58 nM
~90 nM
2 µM
2 nN
7 nN
Shigella type III secretion needle structure
(Deane et al., PNAS USA, 2006)
Structure / function of type III effectors
A-B toxinS SA-subunit B-subunit
L enzyme activity / receptor binding internalization intracellular trafficking
type III effectors function in a coordinated manner within the host cell
A-subunit
T enzyme activityYopE GAP - Rho, Rac, Cdc42 SopE GEF - RhoYopH phosphataseYopO kinase
SptP GAP - Rho, Rac, Cdc42 - phosphatase ExoS GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferaseExoT GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferase
A-subunit A-subunit
Type III effectors
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: Agrobacterium tumefaciens - VirB-VirD Bordetella pertussis - pertussis toxin Helicobacter pylori - CagA Legionella pneumophila
A. tumefaciens
Gene organization of Type IV secretion
(H-J. Yeo, G. Waksman, J. Bacteriol. 2004)
Type V secretion - autotransporter
Bacteria that use type V secretion: Neisseria gonorrhoeae - IgA1 protease Helicobacter pylori - VacA Haemophilus influenzae - Hsf fibrillar protein
(Wilson, McNab, HendersonBacterial Disease Mechanisms, 2002)
Properties:- Insertion of -domain - formation -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
(Desvaux et al., Res in Microbiol. 2004)
OM
P
CM
Sec-(or Sec-like) dependentSec-independent
Type I
ATPADP
N
C
N
C
Type III Type II Type IV
Sec
(Adapted from Stathopoulus et al. (2000); provided by E Rucks)
ATPADPADP
ATP ATP
ADP
host cell
host cell
Type V
SecB11
Gram-negative secretion
N
C
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 -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
ATPADP
N
C
CM
CW
Type I - ATP-binding (ABC)
transmembpore
Protein - C-terminal signal
accessory factor
bacteriocins
Sec
-barrel pore
Type V - autotransporter
CM
CW
Staphylococcus alpha toxin
Protein - translocation unit
ATPADP
CM
CW
N
C
Sec
Protein - N-terminal signal
Type II - Sec-dependent
majority of proteins
Gram-positive - Type III secretion
Cytolysin-Mediated Translocation
Properties:
- spn (NAD glycohydrolase) - slo (streptolysin O) genes linked and co-transcribed
- 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
Streptococcus pyogenes
(Madden, Ruiz, Caparon, Cell, 2001)
Gram-negative
Gram-positive
Cytotoxic lymphocyte
Role of secretory processes in bacterial pathogenesis
Gram-negative - type III secretion
Pseudomonas aeruginosa - extracellular pathogenSalmonella spp - intracellular pathogen
Pathogenesis - complex / multi-factorial related to regulated secretion of multiple virulence factors primarily an extracellular pathogen
Identification / diagnosis - culture / isolate forms smooth, fluorescent green colonies at 42oC characteristic sweet (grape-like) odor
(students.washington.edu/ chenamos/Pseudomonas)
Pyocyanin production by P. aeruginosa
Pseudomonas aeruginosa
Bacteriology - Gram-negative rod, motile / aerobe ubiquitous, highly adaptable bacterium
(www.bact.wisc.edu/ Bact330/lecturepseudomonas)
Disease - opportunistic pathogen
opportunistic pathogen
nosocomial infectionsindwelling catheters, urinary tract, lung, bloodstream
complicated by antibiotic / disinfectant resistance
infects compromised individualsburns, wounds, immuno-compromised
cystic fibrosis
disease manifestations chronic and acute lung infection
nosocomial pneumoniacorneal ulcers
urinary tract infectionswound infections
chronic lung infections in CF patients
Disease
Pseudomonas aeruginosa
( www.opt.pacificu.edu/.../ 13036-AS/Fig%2017.jpg)
Contact lens associated corneal ulcer
(www.skinatlas.com/ greenailopt.jpg)
P. aeruginosa - Green nail syndromeHot tub dermatitis (www.rsdfoundation.org/ images/image16.gif)
Greenish pigment-associated infection
Ear piercing infections
(www.dadlnet.dk/ufl/ 0244/VP-html/VP38141-3.jpg)
Nosocomial pneumonia
(www.uni-mainz.de/.../ tag/heussel/aj97_p1c.jpg)
P. aeruginosa - infections
Folliculitis
(www.dermnet.com/ thumbnailIndex)
Pseudomonas aeruginosa
90% of morbidity and mortality of CF patients relates to chronic lung infection (by Pseudomonas aeruginosa)
Cystic fibrosis:
lethal autosomal recessive diseasecharacterized by pulmonary obstruction pancreatic exocrine deficiencyhigh sodium and chloride in sweatmale infertility
most common, serious inherited disease among Caucasians
Mutation in CFTR gene - cause of cystic fibrosis(CF transmembrane conductance regulator)
( www.cfgenetherapy.org.uk/ CFTR.htm)
CF transmembrane conductance regulator
(wsrv.clas.virginia.edu/ ~rjh9u/gif/cfmap3.gif)
F508 - most frequent mutationrecognized as non-functional protein
not modified in ER - degraded
Pseudomonas aeruginosa virulence factors
Type I secretion: hemolysin
Type II secretion: proteases
elastase (LasB) - zinc metalloprotease LasA - serine protease alkaline protease
exotoxin A - ADP-ribosylating toxin
Type III secretion: ExoS - GAP / ADP-ribosylating enzyme ExoT - GAP / ADP-ribosylating enzyme ExoU - PLA2
ExoY - adenylate cyclase
No Type IV secretion
Planktonic P. aeruginosa
(textbookofbacteriology.net/ P.aeruginosa.jpeg)
Biofilm formation alginate - mucopolysaccharide quorum sensing (www.math.utah.edu/.../ quorum_talk.html)
Bacterial biofilm magnified 7,000x
Studying the role of type III secretion in pathogenesis
Pseudomonas type III secretion effectors
Rho, Rac, Cdc42
GAP ADP-ribosylates Crk anti-phagocytic alters cytoskeletal structure 100% isolates
ExoT
PLA2 - cytotoxic CF (15%)
corneal isolates
ExoU
Rho, Rac, Cdc42 Ras, Ral, Rabs, Rac
GAP ADP-ribosylates LMWG-proteins cell inactivation anti-phagocytic CF ( 85%), wound,
UT, soil isolates
ExoS
Effect on eukaryotic cell
adenylate cyclase cyclic AMP CF (97%)
ExoY
(Feltman, et al, 2001, Fleiszig, 1997)
(Fraylick et al, Infect. Immun. 1999)
Strain 388
388 ExoS (1 hour) Strain 388 (1 hour)
Effects of ExoS on human epithelial cells
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
1 99 233 453
Rho-GAP
focal adhesions / stress fibersfilopodia / lamellopodia
R146
GTP
GDP
PI
GTP active Rho, Rac, Cdc42
GDP-inactive Rho, Rac, Cdc42
ExoS GAP GEF
(Goehring et al.)
E379 E381
(Iglewski, Coburn, Barbieri)
O
N
CH2
CH2
PP
Adenine
CONH2
ExoS
Cellular Targets[Ras-family LMWG-proteins]
+
ADP-ribosylated protein
nicotinamide
O
OCH2
CH2
PP
AdenineON
CONH2
NAD
ExoS is a bi-functional toxin
ADP-ribosyltransferase
Effects of ExoS GAP and ADPRT activity on macrophages
0 ExoS GAP-mutant ADPRT mutant
(Rocha et al, Infect. Immun. 2002)
Bi-functional effects of ExoS on cell function
RacGTP
GAP-ADPRT
GAP-ADPRT
anti-phagocytic
RalGTP
*
inhibits DNA synthesisaffects adherencealters morphologyaffects cell viability
Rabs 5, 8, 11, 7
Rac1, Cdc42
* * * *RasGTP
** *
Eukaryotic cell
(E. McGuffie, J. Fraylick, E. Rucks, J. LaRoche, C. Rocha, J. Barbieri)
Pathogenesis - intracellular pathogen
Salmonella enteritidis - gastroenterititis
Salmonella typhimurium - gastroenterititis Salmonella typhi - typhoid fever
Salmonella
Bacteriology - Gram-negative facultative, motile rod non-lactose fermentor / H2S production
(www.ipsiaponti.it/.../ bacilli/salmonella.htm)
(microvet.arizona.edu/.../ salmonella/sem.html)
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
Salmonella invasion
Fimbriae-mediated contact with epithelial cells induces bacterial appendages - invasomes
- Salmonella can directly invade epithelial cells - Or can cross intestinal epithelium via M cells - likely main portal of entry- Also invades macrophages
Invasomes disappear upon entry into cellEntry of bacteria into cells / and
presence or loss of invasomes
Salmonella - SPI-1 type III secretory process
(E. Stebbins, J. Galan, Nature, 2001)
Salmonella invasion:
SipBSipCSipD
(www.niaid.nih.gov/biodefense/images/SALMON_1.jpg)
Mimicry of type III effectors - eukaryotic proteins
R
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
(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:
- 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
(SR Waterman, DW Holden, Cell. Microbiol. 2003)
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 secretion used to deliver DNA (contributes to spread of antibiotic resistance genes)
Type IV translocation Protein sequence of choice domain
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