The food-borne pathogenListeria monocytogenes causes severe infections in immunocompromised patients. At 30% the mortality rate of this pathogen far exceeds that of other foodborne pathogens. The first step of listerial infection involves the uptake of bacteria into epithelial cells of the intestine. This uptake is mediated by the interaction of themajor invasin Internalin (InlA) with its human receptor E-Cadherin. We have previously investigated the recognition complex between InlA and human E-cadherin by crystallizing functional fragments of both proteins (InlA’ and hEC1). The leucinerich-repeat (LRR) protein InlA is found to bind the N-terminal domain of E-cadherin by its 15 unit LRR-domain. Nevertheless, despite a large interaction surface, the complex is surprisingly weak. Presently, we have undertaken to investigate whether this low affinity is of biological relevance. For this purpose we have substituted individual amino acids in InlA’ to increase the affinity for hEC1. We have identified two residues that, when suitably mutated, improve the binding strength 2500-fold. We have furthermore generated strains of L. monocytogenes that incorporate the InlA-mutations within the genome. This allows the effect of the higher affinity InlA-variants on the uptake of L. monocytogenes into human epithelial cells to be analyzed. m14.p01