dbg wuppertal 2011

P13, an atypical porin of Borrelia burgdorferi.

Dr. Iván Bárcena UribarriUniversity of Wuerzburg

Wuppertal, April 8-10, 2011

Introduction

The P13 porin in Borrelia burgdorferi

Results and conclusions

Cloning of P13 in E. coli and Nicotiana benthamiana

Contents

Borrelia P13 porin

Rosa PA. et al – 2005 – Nat. Rev. Microbiol.

Introduction

TMHMM server v 2.0computer prediction

P13

The general trimeric porin model versus P13.

Examples : OmpF (diffusion porin)LamB (specific porin)

16-18 β-strands

Introduction

P13 of Borrelia

The general trimeric porin model versus P13.

Examples : OmpF (diffusion porin)LamB (specific porin)

16-18 β-strands

Introduction

P13 of Borrelia

P13 periplasmic C-terminus processing

Sec

BAM

CtpA

P13 Signal seq.c-terminus

P13c-terminus

Introduction

OmpC Signal seq.

OmpC

IM

OM

?

n monomers?

P13 paralogues*

Gene Nucleotide position Location Function

bb0034 32089–31553 Chromosome Channel-forming protein P13

bba01 588–1070 lp54 Channel-forming protein BBA01**

bbg03 2104–2492 p28-2Conserved hypothetical protein, authentic frameshift

bbh41 28197–27628 lp28-3 Conserved hypothetical protein

bbi31 20127–19618 lp28-4 Conserved hypothetical protein

bbj02.1 1475–2367 lp38Conserved hypothetical protein, pseudogene

bbj03 1593–1742 lp38Hypothetical protein (located inside BBJ02.1)

bbq06 3623–4105 lp56 Conserved hypothetical protein

bbq81 49246–49047 lp56Conserved hypothetical protein, pseudogene

*Pinne M. et al (2004) Microbiology.**Pinne M. et al (2006) J Bacteriol.

Introduction

P13 paralogues*

Chaconas G. 2005 Mol. Microbiol.

Why are so many copies of the P13 gene in these genetically reduced bacteria?

Introduction

P13

P13 Homologues

P13 immunogenic potential

Introduction

Pinne M. et al (2004) Microbiology.

-Outer-surface-epitope regions are the most heterogeneous.-Design of three fragments (ABC) and recombinant expression.- Antibody 15G6 that recognizes the natural epitope only recognized fragment B.

Recombinant obtaining of P13 from B. burgdorferi in different

expression systems

Recombinant expression of P13:

Why P13?:

- Development of new targets for diagnosis/therapie strategies.

- Knowledge about structure and function of P13 and its function in Borrelia.

Why recombinant and no native?:

- Production of large amounts of P13

- Reduction of the costs

- Reduction of the time

- Reduction of the infection risk

Results

Potato virus X

pARAJS2vector

pICH31160vector

Agrobacterium tumefaciensEscherichia coli BL21 Omp8 rosetta

Results

DNA constructs

To transform E. coli: To transform A. tumefaciens:

Cytoplasm

Nucleus

Agroinfiltration of

Nicotiana benthamiana

Agrobacterium strain

carrying viral vector

RNA virus sequence

viral replication

Transcription

Export

translation

protein procesing

Cell-to-cell and/or

systemic movement

Results

DNA virus sequence

RNA virus sequence

Expression in Tobacco plants Plant cell

Adapted from Marillonnet, S. et al. (2004) Proc Natl Acad Sci U S A.

Results

rP13 expression in Nicotiana benthamiana

SDS-PAGE Western Blot

Conclusions

• P13 was produced successfully in two systems with a high yield expression.

• Development of better diagnosis/treatment strategies.

• Better understanding of the biological function of this fascinating protein

P13

Thank you for listening !