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