| Summary: | <p>Abstract</p> <p>Background</p> <p>Internalin A (InlA) is a critical virulence factor which mediates the initiation of <it>Listeria monocytogenes </it>infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates <it>L. monocytogenes </it>entry into human enterocytes, but has only a limited interaction with murine cells.</p> <p>Results</p> <p>We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host <it>Lactococcus lactis </it>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 <it>L. monocytogenes </it>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<sup>m </sup>murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA<it><sup>m</sup>*</it>. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates <it>Listeria</it>-optimized codons encoding the altered amino acids. <it>L. monocytogenes </it>EGD-e InlA<it><sup>m</sup>* </it>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<sup>m </sup>strain.</p> <p>Conclusions</p> <p>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 <it>L. monocytogenes </it>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 <it>L. monocytogenes </it>strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.</p>
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