Figure 1.

Effect of human milk whey and human lactoferrin on H. influenzae IgA1 protease. (A) Western immunoblot showing that human milk whey removes the native IgA1 protease preprotein and the remnant Iga_β domain from H. influenzae strain Rd. The immunoblot was prepared with rabbit antiserum #331, which reacts with the IgA1 protease preprotein, Iga_p, and Iga_β. Samples were loaded as follows: lane 1, untreated Rd, whole cells; lane 2, untreated Rd, supernatant; lane 3, milk whey-treated Rd, whole cells; lane 4, milk whey-treated Rd, supernatant. Lane 1 shows the preprotein (P) and the remnant Iga_β domain (β) generated from processing of the preprotein. Lane 2 shows the two active IgA1 protease species released from the surface of the cell. Lanes 3 and 4 demonstrate that the preprotein and Iga_β were removed from whole cells, with the preprotein detectable in the supernatant (∗). The preprotein in the supernatant remained unprocessed, since milk contains antibody that inhibits autoproteolysis. The secondary antibody crossreacts with lactoferrin (Lf), which is indicated by the open arrow. (B) Western immunoblot showing removal of the IgA1 protease preprotein from H. influenzae strain Rd3–13 as a function of time. Bacteria were grown to mid-logarithmic phase and incubated with milk whey, and aliquots were then sampled at the indicated times. Whole cells (Upper) and the corresponding supernatants (Lower) were examined by using rabbit antiserum #331. The preprotein was progressively removed from cells and transferred to the supernatant. In control samples prepared from strain Rd3–13 after incubation in buffer alone (lane C), the preprotein remained associated with cells. Arrowheads point to the preprotein, and the bracket indicates a degradation product of the preprotein. ∗ marks the upward-shifted preprotein. (C) Western immunoblot showing that recombinant lactoferrin is sufficient to remove the IgA1 protease preprotein from H. influenzae strain Rd3–13. The immunoblot was prepared with rabbit antiserum #331. Lanes 2 and 3 contain samples that were treated with human milk lactoferrin, and lanes 6 and 7 contain samples that were treated with recombinant human lactoferrin prepared from BHK cells. Samples were loaded as follows: lanes 1 and 5, untreated Rd3–13, whole cells; lanes 2 and 6, milk lactoferrin or recombinant lactoferrin-treated Rd3–13, whole cells; lanes 3 and 7, milk lactoferrin or recombinant lactoferrin-treated Rd3–13, supernatants; lanes 4 and 8, milk lactoferrin or recombinant lactoferrin, without bacteria. Both milk lactoferrin and recombinant lactoferrin removed the preprotein (∗), which was then partially degraded (brackets). Both sources of lactoferrin resulted in an upward shift of the preprotein. The secondary antibody crossreacts with lactoferrin (Lf), which is indicated by the open arrow.