1. Bacterial secretion 1

2. Disease function of susceptibility of host relates to mechanism of
bacterial pathogenesis
immune competent/compromised
immunizations
age
trauma
genetics
antimicrobial therapy
secretion of factors (toxins)
direct host cell manipulation
(type III / type IV
secretion systems)

3. 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)

4. Bacterial protein secretion
Gram-negative - translocation past the cytoplasmic / periplasm / outer membrane
Gram-positive - translocation cytoplasmic membrane / cell wall

5. 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

6. 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

7. 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 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
ATP
ADP N C OM P CM
accessory
factor
Type I secreted proteins:
RTX toxin (repeat in toxin)
E. coli hemolysin
bacteriocins
metalloproteases

8. Gram-negative - Type II secretion
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 peptidase
leader peptide
N hydrophobic C mature protein N C
SecB L
SecY,E,G
( pathway/map/map03090.html)
Sec-dependent secretory pathway

9. 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
Sec
Outer membrane translocation:
Protein - bacteria specific mechanisms
- Secreton - homology to pilus components
- Secretins - homology to phage OM proteins
- driven by PMF (?ATP) CM
ATP
ADP
Type II secreted proteins:
Majority of virulence factors
- pullulanase
- AB toxins
- proteases

10. Type III secretion Host-cell contact induced secretion process
Gram-negative
bacterium
direct
manipulation of
host cell actin
/ function
induction /
translocation
of type III
effectors
(intracellular
enzyme activity)

11. Gram-negative - Type III secretion regulon
pscU pscT pscS pscR pscQ pscP pscO pscN popN pcr1 pcr2 pcr3 pcr 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
Type III effectors
spcU exoU orf 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

12. Gram-negative - Type III secretory apparatus
Salmonella Shigella
Type III apparatus resembles the basal body of flagella, with an injection needle at the tip replacing the hook and flagellar filament.
S. typhimurium flagellum
(Kubori, 1998; Blocker, 2001; Plano, 2001)

13. 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

14. Shigella type III secretion needle structure
(Deane et al., PNAS USA, 2006)

15. Structure / function of type III effectors
A-B toxin
S S
A-subunit
B-subunit
L enzyme activity / receptor binding internalization
intracellular trafficking
A-subunit
T enzyme activity
YopE GAP - Rho, Rac, Cdc42
SopE GEF - Rho
YopH phosphatase
YopO kinase
SptP GAP - Rho, Rac, Cdc42 - phosphatase ExoS GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferase
ExoT GAP - Rho, Rac, Cdc42 - ADP-ribosyltransferase
Type III effectors
type III effectors function in a coordinated manner within the host cell

16. 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
A. tumefaciens
Gene organization of Type IV secretion
(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

17. (Wilson, McNab, Henderson Bacterial Disease Mechanisms, 2002)
Type V secretion - autotransporter
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
(Desvaux et al., Res in Microbiol. 2004)
(Wilson, McNab, Henderson
Bacterial Disease Mechanisms, 2002)
Bacteria that use type V secretion:
Neisseria gonorrhoeae - IgA1 protease
Helicobacter pylori - VacA
Haemophilus influenzae - Hsf fibrillar protein

18. Gram-negative secretion
Sec-independent
Sec-(or Sec-like) dependent
Type I
Type III
Type II
Type IV
Type V
host
cell
host
cell OM P
Sec
B11
Sec CM
ATP
ADP
ADP
ATP
ATP
ADP
ATP
ADP N C N C N C
(Adapted from Stathopoulus et al. (2000); provided by E Rucks)

19. 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

20. Gram-positive - secretion
ATP
ADP N C CM CW
Type I - ATP-binding (ABC)
trans
memb
pore
Protein - C-terminal
signal
accessory
factor
bacteriocins
ATP
ADP CM CW N C
Sec
Protein - N-terminal
signal
Type II - Sec-dependent
majority of proteins
Sec
b-barrel
pore
Type V - autotransporter CM CW
Staphylococcus alpha toxin
Protein -
translocation unit

21. 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

22. Role of secretory processes in bacterial pathogenesis
Gram-negative - type III secretion
Pseudomonas aeruginosa - extracellular pathogen
Salmonella spp - intracellular pathogen

23. Pseudomonas aeruginosa
Bacteriology - Gram-negative rod,
motile / aerobe
ubiquitous, highly adaptable bacterium
( Bact330/lecturepseudomonas)
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 42oC
characteristic sweet (grape-like) odor
(students.washington.edu/ chenamos/Pseudomonas)
Pyocyanin production by P. aeruginosa

24. 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

25. P. aeruginosa - infections
( AS/Fig%2017.jpg)
Contact lens associated corneal ulcer
Nosocomial pneumonia
( tag/heussel/aj97_p1c.jpg)
Ear piercing infections
( 0244/VP-html/VP jpg)
( images/image16.gif)
Greenish pigment-associated infection
( greenailopt.jpg)
P. aeruginosa - Green nail syndrome
Hot tub dermatitis
Folliculitis
( thumbnailIndex)

26. 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)

27. CF transmembrane conductance regulator
(wsrv.clas.virginia.edu/ ~rjh9u/gif/cfmap3.gif)
( CFTR.htm)
DF508 - most frequent mutation
recognized as non-functional protein
not modified in ER - degraded

28. Planktonic P. aeruginosa
Pseudomonas aeruginosa virulence factors
Planktonic P. aeruginosa
(textbookofbacteriology.net/ P.aeruginosa.jpeg)
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
Biofilm formation
alginate - mucopolysaccharide
quorum sensing
( quorum_talk.html)
Bacterial biofilm magnified 7,000x
phosphatidylcholine phosphorylcholine + diglyceride

29. Studying the role of type III secretion in pathogenesis

30. Pseudomonas type III secretion effectors
Rho, Rac, Cdc Ras, Ral, Rabs, Rac
GAP ADP-ribosylates LMWG-proteins
cell inactivation
anti-phagocytic
CF ( 85%), wound,
UT, soil isolates
ExoS
Effect on eukaryotic cell
Rho, Rac, Cdc42
GAP ADP-ribosylates Crk
anti-phagocytic
alters cytoskeletal structure
100% isolates
ExoT
PLA2 - cytotoxic CF (15%)
corneal isolates
ExoU
adenylate cyclase
cyclic AMP
CF (97%)
ExoY
(Feltman, et al, 2001, Fleiszig, 1997)

31. Effects of ExoS on human epithelial cells
388 DExoS (1 hour) Strain 388 (1 hour)
(Fraylick et al, Infect. Immun. 1999)
Strain 388

32. 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
Elisabet Fritz-Lindsten, Ake Forseberg - Hela cells, J774 macrophage

33. ExoS is a bi-functional toxin
Rho-GAP
focal adhesions / stress fibers
filopodia / 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 P
Adenine
CONH2
ExoS
Cellular Targets
[Ras-family LMWG-proteins] +
ADP-ribosylated protein
nicotinamide
NAD
ADP-ribosyltransferase

34. Effects of ExoS GAP and ADPRT activity on macrophages
ExoS GAP-mutant ADPRT mutant
(Rocha et al, Infect. Immun. 2002)

35. Bi-functional effects of ExoS on cell function
GAP-ADPRT
Eukaryotic cell
(E. McGuffie, J. Fraylick, E. Rucks, J. LaRoche, C. Rocha, J. Barbieri)
GAP-ADPRT
Ras
GTP *
Ral
GTP *
* * * *
Rac
GTP
Rabs 5, 8, 11, 7
Rac1, Cdc42
* *
anti-phagocytic
inhibits DNA synthesis
affects adherence
alters morphology
affects cell viability

36. Salmonella Bacteriology - Gram-negative Pathogenesis -
facultative, motile rod
non-lactose fermentor / H2S production
( bacilli/salmonella.htm)
Salmonella enteritidis - gastroenterititis
Salmonella typhimurium - gastroenterititis
Salmonella typhi - typhoid fever
Pathogenesis -
intracellular pathogen
(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

37. 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

38. Salmonella - SPI-1 type III secretory process(
(E. Stebbins, J. Galan, Nature, 2001)
Salmonella invasion:
SipB
SipC
SipD

39. Mimicry of type III effectors - eukaryotic proteins
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)

40. 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)

41. 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

42. 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

43. 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