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)
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 Genes fused or coordinately expressed on operon: protein GGXGSD ABC transporter accessory factor (MFP) ATP ADP N Outer membrane transport may not be linked C Type I secreted proteins: RTX toxin (repeat in toxin) E. coli hemolysin bacteriocins metalloproteases
Gram-negative - Type II secretion
Sec-
dependent secretory pathway SecY,E,G SecB N L C (www.genome.ad.jp/kegg/ pathway/map/map03090.html) signal peptidase leader peptide N ++ hydrophobic C mature protein 1-5 7-15 3-7 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
OM P CM
Gram-negative - Type II secretion
ATP ADP Sec 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 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 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 effectors
spcU exoU orf1 exoS * exoT * * *
(
Pseudomonas aeruginosa
regulon, Frank, Yahr 1997; Figure courtesy of Dara Frank )
exoY
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 10 15 µM 2 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-subunit S S A-B toxin B-subunit L enzyme activity / intracellular trafficking receptor binding internalization Type III effectors A-subunit T enzyme activity YopE GAP - Rho, Rac, Cdc42 SopE GEF - Rho YopH phosphatase YopO kinase A-subunit A-subunit SptP ExoS GAP - Rho, Rac, Cdc42 phosphatase GAP - Rho, Rac, Cdc42 ADP-ribosyltransferase ExoT 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 (H-J. Yeo, G. Waksman, J. Bacteriol. 2004) Bacteria that use type IV secretion: 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)
ATP ADP N
Gram-negative secretion
Sec-independent Sec-(or Sec-like) dependent Type I Type III Type II Type IV Type V C host cell ATP ADP host cell OM P CM Sec B11 N ATP ADP N C C (Adapted from Stathopoulus et al. (2000); provided by E Rucks) ATP ADP Sec
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) Type II - Sec-dependent Type V - autotransporter CW CM ATP ADP accessory factor trans memb pore N Protein - C-terminal signal CW CM C bacteriocins CW
b
-barrel pore ATP ADP N Sec CM Sec Protein - N-terminal signal Protein translocation unit C majority of proteins Staphylococcus alpha toxin
Gram-positive - Type III secretion
Cytolysin-Mediated Translocation
Streptococcus pyogenes
Gram-negative Gram-positive Properties :
spn
(NAD glycohydrolase) -
slo
genes linked and co-transcribed (streptolysin O) 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 Cytotoxic lymphocyte (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 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 42 o C characteristic sweet (grape-like) odor
(
www.bact.wisc.edu/ Bact330/lecturepseudomonas) Pyocyanin production by P. aeruginosa (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 Ear piercing infections Contact lens associated corneal ulcer
( www.opt.pacificu.edu/.../ 13036-AS/Fig%2017.jpg)
Greenish pigment-associated infection
(www.uni-mainz.de/.../ tag/heussel/aj97_p1c.jpg)
P. aeruginosa - Green nail syndrome
(www.dadlnet.dk/ufl/ 0244/VP-html/VP38141-3.jpg)
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
(wsrv.clas.virginia.edu/ ~rjh9u/gif/cfmap3.gif) ( www.cfgenetherapy.org.uk/ CFTR.htm) D
F508 - most frequent mutation recognized as non-functional protein not modified in ER - degraded
Pseudomonas aeruginosa virulence factors
Planktonic
P. aeruginosa
Type I secretion: hemolysin Type II secretion: proteases elastase (LasB) LasA zinc metalloprotease 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 ExoY PLA 2 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
ExoS ExoT ExoU
Pseudomonas type III secretion effectors
GAP ADP-ribosylates LMWG-proteins Effect on eukaryotic cell Rho, Rac, Cdc42 Ras, Ral, Rabs, Rac cell inactivation anti-phagocytic CF ( 85%), wound, UT, soil isolates GAP ADP-ribosylates Crk Rho, Rac, Cdc42 anti-phagocytic alters cytoskeletal structure 100% isolates PLA 2 - cytotoxic CF (15%) corneal isolates ExoY adenylate cyclase (Feltman, et al, 2001, Fleiszig, 1997) cyclic AMP CF (97%)
Effects of ExoS on human epithelial cells
388
D
ExoS (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
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Loss of cell surface microvilli
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Loss of adhesion or re-adhesion
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Loss of cell viability
ExoS is a bi-functional toxin R146 E379 E381 1 99 233 453 Rho-GAP ADP-ribosyltransferase ExoS GDP-inactive Rho, Rac, Cdc42 P I GTP GAP GEF GDP GTP active Rho, Rac, Cdc42 focal adhesions / stress fibers filopodia / lamellopodia CH 2 P P CH 2 CONH 2 O N ExoS CH 2 P P O Adenine Cellular Targets [Ras-family LMWG-proteins] O CH 2 + CONH 2 O N Adenine
nicotinamid e NAD ADP-ribosylated protein
(Goehring et al.) (Iglewski, Coburn, Barbieri)
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
Rac GTP GAP ADPRT GAP -ADPRT * Ras GTP * Ral GTP * * * * Rabs 5, 8, 11, 7 * * anti-phagocytic 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 Salmonella typhi -
gastroenterititis typhoid fever Bacteriology Gram-negative facultative, motile rod non-lactose fermentor / H 2 S production Pathogenesis intracellular pathogen Virulence factors Two type III secretion processes - SPI-1 (Salmonella pathogenicity island-1) - SPI-2 involved in initial invasion (Salmonella pathogenicity island-2) involved in intracellular survival
(
www.ipsiaponti.it/.../ bacilli/salmonella.htm) (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 R (E. Stebbins, J. Galan, Nature, 2001) 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
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 translocation Protein sequence of choice domain 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
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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