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

Listeria Monocytogenes Gram positive Genome = 2 944 528 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

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.

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

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 bp 0 plasmid 1 transposon 90,3% coding L. Innocua genome = 3 011 209 bp 1 plasmid 0 transposon 90,3% 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 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

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

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

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

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.

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

??? ? 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 ??? ?

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.

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

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

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.

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).

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

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

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

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