Yamazaki, S. sodium concentrations (2).L. monocytogenescan be transmitted by ingestion of contaminated food.L. monocytogenescan cross the intestinal, bloodbrain, and fetoplacental barriers (3). When the bacteria cross Chlorantraniliprole the bloodbrain barrier, it causes infection of the central nervous system and potentially results in fatal meningitis (4). T Chlorantraniliprole cell suppression makes pregnant women susceptible toL. monocytogenesinfection (5), and infection can result in the arrest of fetal development (6). Although infection and invasion mechanisms ofL. monocytogeneshave been well studied (7,8), no treatment has been developed to prevent infection by this bacterium. Two proteins, InlA and InlB, which are members of the internalin family of virulence factors, function in the first step in invadingL. monocytogenesinto host cells (9,10). The binding of InlA and InlB to host cell receptors activates a signaling cascade that triggers receptor-mediated endocytosis and bacterial internalization. Both InlA and InlB contain leucine-rich repeats (LRRs). Downstream of the LRRs, both Chlorantraniliprole proteins have Ig-like domains, which contain an LPxTG motif necessary for binding to the host cell (11). InlA binds to E-cadherin, a receptor expressed on epithelial cells, which allows the bacterium to invade the intestine (11). E-cadherin belongs to the family of classical cadherins of the superfamily of cadherin proteins responsible for Ca2+-dependent cell adhesion (12). E-cadherin has five extracellular cadherin (EC) domains, numbered consecutively from the N terminus. The cocrystal structure of the EC1 of E-cadherin (1105) in a complex with InlA (36496) has been reported (13). Single-domain antibodies (VHHs) that inhibit the interaction between InlB and its host cell receptor c-Met have recently been reported; these antibodies inhibitL. monocytogenescell invasion (14,15) Since it is known that synergistic action of InlA and InlB is required forL. monocytogenesto cross the placental barrier (16), a cocktail therapy of anti-InlA and anti-InlB antibodies could be effective. In addition, VHH antibodies, at about 15 kDa, are about 10-fold smaller in size than conventional immunoglobulin G antibodies. In this study, we isolated and purified two VHHs, vhh10, and vhh24 from an alpaca immune library utilizing a phage display method. The two VHHs bound to InlA with pM affinity and inhibited the binding of InlA to the two N terminal-most extracellular domains, EC12, of E-cadherinin vitro. Furthermore, vhh10 and vhh24 inhibitedL. monocytogenesinvasion into host cells in culture. High-resolution X-ray structural analysis of the complexes of InlA with anti-InlA VHH antibodies and with EC12 revealed that these VHHs competitively inhibit the interaction of InlA with E-cadherin. == Results == == Selection of anti-InlA VHH antibodies and the interaction analysis == To obtain anti-InlA VHH antibodies, an alpaca was immunized with recombinant InlA (36496). After immunization and confirmation of an increase in antibody titer against the antigen, RNA was extracted from lymphocytes to construct a VHH phage display library. Subsequently, panning was performed against InlA, and the enriched VHH clones were singly cloned and evaluated by phage ELISA. We obtained 28 clones that bound with high affinity (Fig. 1A). Analyses of complementarity-determining regions (CDRs) revealed the high convergence into two clones, vhh10 and vhh24 (Table 1). == Figure 1. == Selection of anti-InlA VHH antibodies.A, phage ELISA analysis of single clones from phage display library. ELISA assays were performed twice on each clone (first and second). Theasterisksmark clones selected for sequence analysis (total 28 clones).B, SPR analysis of the interaction between InlA and vhh10.C, SPR IL18BP antibody analysis of the interaction between InlA and vhh24.D, SPR analysis of the interaction between InlA and D3-L11. The color lines are the raw data and theblack linesare the fitting curves in SPR.E, ITC analysis of the interaction between InlA and vhh10.F, ITC analysis of the interaction between InlA and vhh24.G, ITC analysis of the interaction between InlA and D3-L11. ITC, isothermal titration calorimetry; SPR, surface plasmon resonance. == Table 1. == Amino acid sequences of vhh10 and vhh24 (IMGT numbering) These two VHHs were expressed and purified as recombinant proteins using anEscherichia coliexpression.