PURIFICATION OF THE INLB GENE-PRODUCT OF LISTERIA-MONOCYTOGENES AND DEMONSTRATION OF ITS BIOLOGICAL-ACTIVITY

Entry of Listeria monocytogenes into nonphagocytic cells requires the inlAB gene products. InlA and InlB are bacterial cell wall-associated polypeptides that can be released by sodium dodecyl sulfate treatment. By applying more gentle extraction methods, we have purified InlB in its native form. Treatment of bacteria with various nondenaturating ag ents including mutanolysin, thiol reagents, sodium chloride, and deter gents like Triton X-100 or 3-[(3-cholamidopropyl) dimethylammonio]-1-p ropanesulfonate did not release substantial amounts of InlB from the b acterial cell wall. Instead, InlB was nearly quantitatively extracted in a solubilized form by treatment of bacteria with 1 M Tris-Cl or oth er protonated amines at pH 7.5. However, the reduced solubility of the extracted InlB in low-salt buffers hampered further biochemical purif ication. A panel of monoclonal antibodies against listerial Tris-Cl ex tracts containing InlB was therefore produced to generate reagents for use in affinity chromatography. One of the monoclonal antibodies enab led purification of the InlB protein to homogeneity with relatively hi gh yields. When added externally, purified InlB associated with the su rface of noninvasive bacteria such as Listeria innocua or an L. monocy togenes inlB2 mutant, where it promoted entry of these strains into Ve ro cells >300- and 17-fold, respectively. This effect was even more dr amatic for HeLa cells, where the observed invasion was increased about 9,000- and 4,000-fold, respectively. The availability of purified nat ive, invasion-competent InlB will allow analysis of the molecular basi s of InlB mediated entry into tissue culture cell lines in greater det ail.