Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

Hdl Handle:
http://hdl.handle.net/10147/125884
Title:
Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.
Authors:
Monk, Ian R; Casey, Pat G; Hill, Colin; Gahan, Cormac G M
Affiliation:
Alimentary Pharmabiotic Centre & Department of Microbiology, University College Cork, Western Road, Cork, Ireland.
Citation:
Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model. 2010, 10:318 BMC Microbiol.
Journal:
BMC microbiology
Issue Date:
2010
URI:
http://hdl.handle.net/10147/125884
DOI:
10.1186/1471-2180-10-318
PubMed ID:
21144051
Abstract:
Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.; We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlA(m) murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA(m)*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA(m)* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlA(m) strain.; We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. This murinized L. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.
Item Type:
Article
Language:
en
MeSH:
Amino Acid Substitution; Animals; Bacterial Proteins; Cell Line; Directed Molecular Evolution; Disease Models, Animal; Female; Humans; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; Mouth Diseases; Mutagenesis, Site-Directed; Virulence Factors
ISSN:
1471-2180

Full metadata record

DC FieldValue Language
dc.contributor.authorMonk, Ian Ren
dc.contributor.authorCasey, Pat Gen
dc.contributor.authorHill, Colinen
dc.contributor.authorGahan, Cormac G Men
dc.date.accessioned2011-03-28T14:51:05Z-
dc.date.available2011-03-28T14:51:05Z-
dc.date.issued2010-
dc.identifier.citationDirected evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model. 2010, 10:318 BMC Microbiol.en
dc.identifier.issn1471-2180-
dc.identifier.pmid21144051-
dc.identifier.doi10.1186/1471-2180-10-318-
dc.identifier.urihttp://hdl.handle.net/10147/125884-
dc.description.abstractInternalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.-
dc.description.abstractWe have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlA(m) murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA(m)*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA(m)* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlA(m) strain.-
dc.description.abstractWe have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. This murinized L. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.-
dc.language.isoenen
dc.subject.meshAmino Acid Substitution-
dc.subject.meshAnimals-
dc.subject.meshBacterial Proteins-
dc.subject.meshCell Line-
dc.subject.meshDirected Molecular Evolution-
dc.subject.meshDisease Models, Animal-
dc.subject.meshFemale-
dc.subject.meshHumans-
dc.subject.meshListeria monocytogenes-
dc.subject.meshListeriosis-
dc.subject.meshMice-
dc.subject.meshMice, Inbred BALB C-
dc.subject.meshMouth Diseases-
dc.subject.meshMutagenesis, Site-Directed-
dc.subject.meshVirulence Factors-
dc.titleDirected evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.en
dc.typeArticleen
dc.contributor.departmentAlimentary Pharmabiotic Centre & Department of Microbiology, University College Cork, Western Road, Cork, Ireland.en
dc.identifier.journalBMC microbiologyen

Related articles on PubMed

All Items in Lenus, The Irish Health Repository are protected by copyright, with all rights reserved, unless otherwise indicated.