OM

IM

RY ZSA

TUVWX

P

Q

1

3

Assembly

Secretion

ATP

Folding

2

TypIV piliation (Pil)

PilD

A

OM

IM

Q

CBATP

R

Y Z

TUVWX

S

ATP

XcpA

Q

Protein secretion (Xcp)

P

Bacteria overproducing XcpT are covered with extracellular pilus-like appendages.

Transmission Electron Microscopy, Negative Staining

Individual fibrils

« Bundled »

The pseudopilus hallmarks

XcpT is assembled in a multifibrillar structure calledtype II pseudopilus

TEM analysis, Immunogold labelling anti-XcpT

Type II pseudopilus

Type IV pilus

Flagella

Image FilteringOriginal Image

Fourier Transform

Filtered image

Enlargement

The Secreton-Dependent Pathway (Type II)

C

P

Xcp

IM

OM

XcpQ

XcpR

ATPADP + Pi

Secretin

Traffic ATPase

SÉCRÉTINE « TRAFFIC ATPASE »

SÉCRÉTION TYPE II YES YES

SÉCRÉTION TYPE III YES NO

SÉCÉRTION TYPE IV NO YES

DNA UPTAKE

GRAM+

NO YES

DNA UPTAKE

GRAM-

YES YES

TYPE IV PILI YES YES

PHAGE

FILAMENTEUX

YES NO

Molecular Mechanisms involving:

• Secretion

– Type II

– Type III

• Type IV Piliation

• Filamentous phage extrusion

• DNA uptake/Natural competence

• Assembly of S-layer proteins

• Secretion

– Type II

– Type IV

• Type IV Piliation

• Bacterial Conjugation

• DNA uptake/Natural competence

SECRETINS TRAFFIC ATPASES

BfpB SecretinE. coli EPEC

Scmidt et al., J Bacteriol 2001 183:4848-4859

LES SÉCRÉTINES TRANSPORTENT DE LARGES COMPLEXES

YscC PilQ XcpQ pIV

5 5-6 9-10 8

20 18 19-20 14

Type IIISecretion

Type IVpili

Type IISecretion

Filamentousphages

Inner diameter (nm)

Outer diameter (nm)

Process

Bitter & Tommassen, 1999, Trends in Microbiol., 7: 4-7

(12-14 subunits)

PAO1 GENOME SEQUENCE

Stover et al., 2000, Nature, 406: 959-964

Traffic ATPase

Secretin

hxcR

hplR

xcpR

pilT/pilU

hvbA

pilB

hxrA

0

1

2

3

4

5

66,3

Mb

Type IV piliation

Type IV piliation

Type II secretion

Traffic ATPases

W U P V T X Y Z Q R S

0

1

2

3

4

5

66,3

hxcQ

xqhC

xqhB

pscC Type III secretion

xqhA Type II secretion

xcpQ Type II secretion

pilQ Type IV piliation

Secretins

P. aeruginosa hxc gene cluster

WT xcp hxc94 kDa

67

43

30

20

(Supernatant fraction, Phosphate starvation)

W U P V T X Y Z Q R S

QPRSTUVWXYZ

hxc

xcp

ApR

Mass Spectrometry

0

50

100

150

200

250

300

100 200 300 400 500

growth time, min

PAO1 pMP220

PAO1 pMPTZ

PAO1 pMPVZ

phxcV-lacZ phxcT-lacZ

LapA (368) MFKRSLIAASLSVAALVSAQAMA-VTGGGASLPAELYK

lapB

LapB (370) MLKPMFGVVALTLLTSHTTLG-ATVTGGGASMPAKLYK

lapAhxc gene cluster

Tan & Worobec, 1993

Low molecular weight Alkaline Phosphatase

• Identification of a second xcp-like gene cluster involved in type II secretion

• Hxc system is specifically involved in secretion of low molecular weight alkaline phosphatases

• Expression of the hxc gene cluster appeared to be coregulated with lap genes expression

Which alternative systems can assemble the type II pseudopilus ?

SecretinATP

fixation motif

Pseudopilins

Xcp system(type II secreton)

W PSTXYZ QR V Uxcp

Q Pil system

(type IV pili)A B C

Pilin

pil

Prepilin peptidase

xcpA/pilD

W U P V T X Y Z Q R S

Hxc system(type II secreton) hxc

Requirement for the Xcp, Hxc or Pil system in the assembly of the type II pseudopilus

STRAINS MOLECULAR MACHINERIES PSEUDOPILUS ASSEMBLY

Xcp Hxc Pil

WT + + + Yes

xcpZ-Q - + + Yes

xcpA - - - No

pilQ/hxcR + - - Yes

xcpRS/pilQ - + - Yes

xcpRS/hxcR - - + Yes

xcpRS/hxcR/pilQ - - - No

hxcR

hplR

xcpR

pilT/pilU

hvbA

pilB

hxrA

0

1

2

3

4

5

66,3

Mb

Type IV piliation

Type IV piliation

Type II secretion

Traffic ATPases

0

1

2

3

4

5

66,3

hxcQ

xqhC

xqhB

pscC Type III secretion

xqhA Type II secretion

xcpQ Type II secretion

pilQ Type IV piliation

Secretins

A flp / tad gene cluster in the P. aeruginosa genome

Kachlany S.C. et al., (2000), J. Bact., 6169-6176Actinobacillus actynomycetemcomitans

Traffic ATPaseSecretin

Secretin Traffic ATPase

flp

A. actinomycetemcomitans adherence system:the « Flp pili »

A. actinomycetem-comitans

(negativ contrast)

Kachlany S.C. et al., (2001), Molecular microbiology, 40, 542-554

Bundled pili( electron microscopy)

- Gram-negative cocobacillus, agent of the juvenil periodontal disease

- Produces long fibrils of bundled pili responsible for non-specific adherence and autoaggregation

- Forms a tenacious biofilm on teeth

- Structural subunit of bundled pili is encoding by flp-1 gene. tad genes cluster encodes the assembly machinery of Flp pili

- Flp-1 is a member of the type IVB pilus sub-familly

Phylogeny of genes for secretion NTPases: Development of a diagnostickey for gene classification

Planet et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 2503-2508.

C

P

Xcp

IM

OM

ETA

SP

Sec Tat

XcpQ

The Secreton-Dependent Pathway (Type II)

XcpR

ATPADP + Pi

The P. aeruginosa Tat system

E. coli

P. aeruginosa

Amino acid identity (%)in corresponding proteins

39 30 4457

tatA

tatA

tatC

tatC

hisE

tatD tatE

tatD

tatB

PA5071 tatB

Is the system functional ?

KmR

The colicin A assay

OM

IM

OmpFBtuB

PALTol

pfColAColicin A (A B

 Viability of E. coli and P. aeruginosa strains upon pfColA production

 E. coli  P. aeruginosa

  WT WT (cai)a tatA-E WT tatC tatC (pTATC)

pMMB67EH + + + + + +

pRR-pfColA67

- + + - + -

Sec

SP

Tat

RR

The P. aeruginosa Tat substrates ?PAK-TC PAK

94

67

43

30

20

PAK PAK

PAK-TC PAKxcpRZ

MTENWKFRRRTFLKHGAQAATLAGLSGLFPETLRRALAVPlcH38 aa

MISKSRRSFIRLAAGTVGATVATSMLPSSIQAALAIPlcN35 aa

Phospholipases C

Mass spectrometry

Phospholipase C activity* in cell fractions

4.21

0.57

0.68

Cytoplasmic membrane

0.04<0.010.01PAK-TC (tatC)

0.164.740.03PAK-∆xcpRZ

0.100.502.80PAK

Wild-type

CytoplasmPeriplasmSupernatant Strain

* p-nitrophenyl-phosphatidylcholine as substrate

C

P

Xcp

IM

OM

ETA

SP

Sec

Plc’sRR

Tat

XcpQ

The Secreton-Dependent Pathway (Type II)

XcpR

ATPADP + Pi