Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2010-12-13

AUTHORS

Ian R Monk, Pat G Casey, Colin Hill, Cormac GM Gahan

ABSTRACT

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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. More... »

PAGES

318-318

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2180-10-318

DOI

http://dx.doi.org/10.1186/1471-2180-10-318

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1019245879

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/21144051


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