Identification of new Listeria monocytogenes LPXTG surface proteins involved in infection

Group of Molecular Microbiology

Mélanie Leroux

IBMC

Instituto de Biologia

Molecular e Celular

Listeria MonocytogenesGram positive

Hetrophobic, saprophyte and ubiquitar organism

Discovered by E.G.D Murray in 1926

First Human case in 1929

Food borne pathogen

Survives in extreme conditions encountered in the food chain (temperatures, pH, salts …etc)

Genome = 2 944 528 Bp

Rod-shaped (Bacillus) bacterium

Human listeriosis

• Maternofoetal listeriosis• Neonatal listeriosis• Highest hospitalization (90%) and mortality (30%) of food borne

infections• Meningitis• Meningoencephalitis• Septicemia• Abortion• Perinatal infections• Gastroenteritis • …etc.

Can be silently present in the gastro intestinal tract of healthy humans.

Dangerous for immunodefients individuals and pregnant women.

Listeria monocytogenes can induce :

Cycle of infection of the hostListeria monocytogenes is able to cross all body barriers :

Intestine barrier

Blood-brain barrier

Foeto-placental barrier

Listeria monocytogenes disseminates from the intestinal lumen to the central nervous system and to the fetoplacental unit .

Listeria contaminated food

Intestine

Intestine barrier

Lymph node

Bloodstream

Liver

Spleen

Bloodstream

Blood-brain barrier

Brain

Foeto-placental barrier Foetus

Mechanism of cell infection• Adhesion at the cellular surface:

recruit Internalin proteins like InlA and InlB, and others adhesion proteins.

• Entry into cell : bacteria are entrapped into a vacuole

→ Internalization mediated by the interaction with surface proteins and their receptors.

→ “zipper mechanism” : interaction of bacterial surface ligands with their respective cellular receptors.

• Escape from the vacuole :

vacuole lysis recruit pore forming proteins like LLO and PI-PLC.

• Multiplication.

• Actin polymerization: recruit ActA.

• Movement intracellular thanks to Actin tail.

• Cell to cell spread.

Genome of Listeria

The genome of Listeria monocytogenes EGDe can be compared with the genome of Listeria innocua wich is non pathogenic.

L. monocytogenes genome = 2 944 528 bp0 plasmid1 transposon90,3% coding

L. Innocuagenome = 3 011 209 bp1 plasmid0 transposon90,3% coding

LPXTG surface proteins & Virulence

Listeria monocytogenes surface proteins play an important role in the virulence, for example in the cross of barriers and entry into cell.

LPXTG proteins have an LPxTG motif involved in the linkage to peptidoglycan.

Why study LPXTG surface proteins? Listeria monocytogenes genome encodes 41 LPXTG surface proteins and 19 of those are absent from Listeria innocua.

-- Absent in L.innocua.

> Present in L.monocy-togenes cell wall fraction.

Study of two LPXTG poteins genes

Present in L.innocua and L.monocytogenes

Present in L.monocytogenes cell wall fraction

Present in L.innocua and L.monocytogenes

LPXTG motif

LRR domain

PKR repeats

Absent in L.monocytogenes cell wall fraction

Lmo1289

Lmo0842

1782 bp

6135 bp

ObjectivesConstruction of deletion mutants for this genes (lmo1289 and lmo0842) .

Mutagenesis by double recombination.

Creation of a plasmid vector containing the fragments up and down of the gene, in order to do recombination with L.monocytogenes genome.

Vector : pMAD

BamHISalIMluIEcoRISmaINcoIBglII

Multiplecloning

site

pMAD9666pb

Origine of replication for E.Coli.

Origin of replication for Listeria monocytogenes: thermosensible (30°C active, 42°C inactive) .

Gene of Ampicilline resistance

Gene bla (encoding for the β-galactosidase).Gene of Erythromycine

resistance

Gene Downstream fragmentUpstream fragment

Enzyme 1

Enzyme 2

Enzyme 2

Enzyme 3

PCR

Enzyme 1 Enzyme 2 Enzyme 2 Enzyme 3

Multiple site of clonage : Ezymes 1,2, 3

Enzyme 1

Enzyme 2

Enzyme 1 Enzyme 2

Enzyme 2

Enzyme 3

Enzyme 2 Enzyme 3

Primer A

Primer B

Primer C

Primer D

Mutagenisis by double recombinationGene Down fragmentUp fragment

Down fragmentUp fragment

• Extraction of pMAD from E.Coli.• Amplification PCR of Up and Down fragments.• Digestion of pMAD and fragments Up with enzymes 1 and 2.• Ligation pMAD with fragment Up.• Transfomation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline :

selection of bacteria with plasmid pMAD. • PCR on colonies E.Coli with primers Up : verification of the presence of the insert.• Extraction of pMAD/insert Up• Digestion of plasmid and fragments Down with enzymes 2 and 3.• Ligation pMAD/insert Up with fragment Down.• Transformation of bacteria E.Coli with pMAD/insert Up/insert Down, and selection with

Ampicilline.• PCR on colonies E.Coli with primer Down.• Extraction of pMAD/insert Up/insert Down.• Transformation of bacteria Listeria monocytogenes.• Mutagenesis by double recombination.

Experiments

Lmo 1289

First Results1. Extraction of pMAD from E.Coli.2. Amplification PCR of Up fragment with primers A and B.3. Digestion of pMAD and fragment Up with enzymes Sal 1 and Mlu1 (non-cutters in lmo1289 gene).4. Ligation pMAD with fragment Up.5. Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of

bacteria with plasmid pMAD. → 13 colonies6. PCR on colonies E.Coli with primers A and B: verification of the presence of the insert. → 2 positives

Control : PCR on Listeria monocytogenes EGDe genome with primers A and B

Positives: Colony 1 and 3

Primers

7. Culture of bacteria E.Coli (colony 1 and 3).8. Extraction of pMAD/insert Up of colony 1 and 3.9. Digestion of pMAD/insert Up and fragment Down with enzymes Mlu1 and Blg2.10. Ligation pMAD/insert Up with fragment Down. 11. Transformation of bacteria E.coli with products of ligation, and selection with Ampicilline.

Multiple site of clonage : Ezymes Sal1, Mlu1, Bgl2

Sal 1

Mlu 1

Sal1 Mlu 1

Fragment Up

Mlu 1

Bgl 2

Mlu 1 Bgl 2

??? ?

Work in progress

12. PCR on bacteria in order to see if they contain the insert.

13. Cultures of bacteria with pMAD/insert UP/insert Down.

14. Extraction of pMAD/insert Up/insert Down.

15. Transformation of bacteria Listeria monocytogenes.

16. Mutagenisis by double recombination.

Lmo 0842

First Results1. Extraction of pMAD from E.Coli.2. Amplification PCR of Up fragments with primers A and B.3. Digestion of pMAD and fragments Up with enzymes Sal 1 and EcoR 1 (non-cutters in lmo0842 gene).4. Ligation pMAD with fragment Up: 1µL pMAD for 4µL fragment5. Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of

bacteria with plasmid pMAD. → 1 colony6. PCR on colonies E.Coli with primers A and B: verification of the presence of the insert. → No results, 0 positives

Control : PCR on Listeria EGDe genome with primers A and B

Primers

Negative

4. Ligation pMAD with fragment Up: 2µL pMAD for 6µL fragment5. Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of

bacteria with plasmid pMAD. → 10 colonies6. PCR on colonies E.Coli with primers A and B: verification of the presence of the insert. → No results, 0 positives

4. Ligation pMAD with fragment Up: 1µL pMAD for 7µL fragment 5. Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of

bacteria with plasmid pMAD. → 3 colonies (11, 12, 13)6. PCR on colonies E.Coli with primers A and B: verification of the presence of the insert. → No results, 0 positives

Control

Primers

Work in progress

4. Others ligations pMAD with fragment Up.5. Transfomation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of

bacteria with plasmid pMAD. 6. PCR on colonies E.Coli with primers Up : verification of the presence of the insert.7. Culture of bacteria E.Coli with pMAD/insert Up8. Extraction of pMAD/insert Up

9. Digestion of plasmid and fragments Down with enzymes EcoR1 and Nco1.10. Ligation pMAD/insert Up with fragment Down.11. Transformation of bacteria E.Coli with pMAD/insert Up/insert Down, and selection with Ampicilline.12. PCR on colonies E.Coli with primers Down.13. Extraction of pMAD/insert Up/insert Down.

14. Transformation of bacteria Listeria monocytogenes.15. Mutagenesis by double recombination.

Mutagenesis Transformation of Listeria monocytogenese by the vector :

Vector : pMAD + fragments UP and DOWN of Lmo 1289

pMAD9666pb

Origine of replication for E.Coli.

Origin of replication for Listeria monocytogenes: thermosensible (30°C active, 42°C inactive) .

Gene of Ampicilline resistance

Gene for Erythromycine resistance

Gene bla (encoding for the β-galactosidase).

Fragment UP of Lmo 1289

Fragment DOWN of Lmo 1289

Gene Lmo1289

Fragment UP

Fragment DOWN

Listeria monocytogenes DNA genomic

Listeria monocytogenes EGDe

30°C = origin of replication active → Multiplication of Listeria

monocytogenes with the plasmid

Selection with Erythromycine: only bacteria with the plasmid pMAD will survive

42°C = origin of replication inactive

Selection with Erythromycin: only bacteria with the plasmid pMAD will survive.

Will survive only bacteria who have integrated the plasmid in DNA genomic (by recombination):

Gene for Erythromycine resistance

First recombination: integration of the vector in Listeria monocytogenes DNA genomic:

Perspectives

Study of bacteria multiplication.

Cells infection.

Mouse infection.

Study of different step of infection.

Study of adhesion property.

Thank You

for your attention !!