1. Group of Molecular Microbiology
Identification of new Listeria monocytogenes LPXTG surface proteins involved in infection
IBMC
Instituto de Biologia
Molecular e Celular
Group of Molecular Microbiology
Mélanie Leroux

2. Listeria Monocytogenes
Gram positive
Genome = Bp
Rod-shaped (Bacillus) bacterium
Hetrophobic, saprophyte and ubiquitar organism
Food borne pathogen
Bacillus are
Survives in extreme conditions encountered in the food chain (temperatures, pH, salts …etc)
Discovered by E.G.D Murray in 1926
First Human case in 1929

3. Human listeriosis
Can be silently present in the gastro intestinal tract of healthy humans.
Dangerous for immunodefients individuals and pregnant women.
Listeria monocytogenes can induce :
Maternofoetal listeriosis
Neonatal listeriosis
Highest hospitalization (90%) and mortality (30%) of food borne infections
Meningitis
Meningoencephalitis
Septicemia
Abortion
Perinatal infections
Gastroenteritis
…etc.

4. Cycle of infection of the host
Listeria 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
Liver
Blood-brain barrier
Brain
Bloodstream
Intestine
Bloodstream
Foeto-placental barrier
Spleen
Foetus
Intestine
barrier
Lymph node

5. 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.

6. 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 = bp
0 plasmid
1 transposon
90,3% coding
L. Innocua
genome = bp
1 plasmid
0 transposon
90,3% coding

7. 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.

8. 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.

9. Study of two LPXTG poteins genes
Lmo1289
LPXTG motif
LRR domain
1782 bp
PKR repeats
Present in L.innocua and L.monocytogenes
Absent in L.monocytogenes cell wall fraction
Lmo0842
6135 bp
Present in L.innocua and L.monocytogenes
Present in L.monocytogenes cell wall fraction

10. Objectives
Construction 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
Origine of replication for E.Coli.
Gene of Ampicilline resistance
BamHI
SalI
MluI
EcoRI
SmaI
NcoI
BglII
pMAD
9666pb
Multiplecloning site
Origin of replication for Listeria monocytogenes: thermosensible (30°C active, 42°C inactive) .
Gene bla (encoding for the
β-galactosidase).
Gene of Erythromycine resistance

11. PCR Primer A Primer C Enzyme 1 Enzyme 2 Enzyme 2 Enzyme 3 Primer B
Upstream fragment
Gene
Downstream fragment
Enzyme 2
Enzyme 3
Primer B
Primer D
PCR
Enzyme 1
Enzyme 2
Enzyme 2
Enzyme 3
Multiple site of clonage : Ezymes 1,2, 3
Enzyme 1
Enzyme 1
Enzyme 2
Enzyme 2
Enzyme 2
Enzyme 2
Enzyme 3
Enzyme 3

12. Mutagenisis by double recombination
Up fragment
Gene
Down fragment
Up fragment
Down fragment

13. Experiments 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.

14. Lmo 1289 First Results Extraction of pMAD from E.Coli.
Amplification PCR of Up fragment with primers A and B.
Digestion of pMAD and fragment Up with enzymes Sal 1 and Mlu1 (non-cutters in lmo1289 gene).
Ligation pMAD with fragment Up.
Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of bacteria with plasmid pMAD.
→ 13 colonies
PCR on colonies E.Coli with primers A and B: verification of the presence of the insert.
→ 2 positives
Positives:
Colony 1
and 3
Control : PCR on Listeria monocytogenes EGDe genome with primers A and B
Primers

15. ??? ? Culture of bacteria E.Coli (colony 1 and 3).
Multiple site of clonage : Ezymes Sal1, Mlu1, Bgl2
Sal 1
Fragment Up
Sal1
Mlu 1
Mlu 1
Culture of bacteria E.Coli (colony 1 and 3).
Extraction of pMAD/insert Up of colony 1 and 3.
Digestion of pMAD/insert Up and fragment Down with enzymes Mlu1 and Blg2.
Ligation pMAD/insert Up with fragment Down.
Transformation of bacteria E.coli with products of ligation, and selection with Ampicilline.
Mlu 1
Mlu 1
Bgl 2
Bgl 2
??? ?

16. Work in progress
PCR on bacteria in order to see if they contain the insert.
Cultures of bacteria with pMAD/insert UP/insert Down.
Extraction of pMAD/insert Up/insert Down.
Transformation of bacteria Listeria monocytogenes.
Mutagenisis by double recombination.

17. Lmo 0842 First Results Extraction of pMAD from E.Coli.
Amplification PCR of Up fragments with primers A and B.
Digestion of pMAD and fragments Up with enzymes Sal 1 and EcoR 1 (non-cutters in lmo0842 gene).
Ligation pMAD with fragment Up: 1µL pMAD for 4µL fragment
Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of bacteria with plasmid pMAD.
→ 1 colony
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
Negative
Primers

18. Ligation pMAD with fragment Up: 2µL pMAD for 6µL fragment
Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of bacteria with plasmid pMAD.
→ 10 colonies
PCR on colonies E.Coli with primers A and B: verification of the presence of the insert.
→ No results, 0 positives
Ligation pMAD with fragment Up: 1µL pMAD for 7µL fragment
Transformation of bacteria E.Coli with pMAD/insert Up, and selection with Ampicilline : selection of bacteria with plasmid pMAD.
→ 3 colonies (11, 12, 13)
PCR on colonies E.Coli with primers A and B: verification of the presence of the insert.
→ No results, 0 positives
Control
Primers

19. Work in progress Others ligations 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.
Culture of bacteria E.Coli with pMAD/insert Up
Extraction of pMAD/insert Up
Digestion of plasmid and fragments Down with enzymes EcoR1 and Nco1.
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 primers Down.
Extraction of pMAD/insert Up/insert Down.
Transformation of bacteria Listeria monocytogenes.
Mutagenesis by double recombination.

20. Mutagenesis Vector : pMAD + fragments UP and DOWN of Lmo 1289 pMAD
Transformation of Listeria monocytogenese by the vector :
Vector : pMAD + fragments UP and DOWN of Lmo 1289
Origine of replication for E.Coli.
Gene of Ampicilline resistance
Fragment UP of Lmo 1289
pMAD
9666pb
Origin of replication for Listeria monocytogenes: thermosensible (30°C active, 42°C inactive) .
Fragment DOWN of Lmo 1289
Gene for Erythromycine resistance
Gene bla (encoding for the β-galactosidase).

21. Listeria monocytogenes EGDe
Fragment UP
Gene Lmo1289
Fragment DOWN
Listeria monocytogenes DNA genomic
Selection with Erythromycine:
only bacteria with the plasmid pMAD will survive
30°C = origin of replication active
→ Multiplication of Listeria
monocytogenes with the plasmid

22. 42°C = origin of replication inactive
First recombination: integration of the vector in Listeria monocytogenes DNA genomic:
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):
42°C = origin of replication inactive
Gene for Erythromycine resistance

24. Perspectives Study of bacteria multiplication. Cells infection.
Mouse infection.
Study of different step of infection.
Study of adhesion property.

25. Thank You for your attention !!