Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae

Abstract

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

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.

Figure 2

Western immunoblots demonstrating that treatment of H. influenzae strain DB117 with human milk lactoferrin or deglycosylated A. awamori recombinant human lactoferrin results in degradation of the Hap preprotein and Hap_β, with release of Hap_s into the culture supernatant. (A) Western immunoblot of whole-cell lysates of H. influenzae strain DB117 derivatives preincubated with either PBS alone (lanes 1–4) or PBS with 13 μM human milk lactoferrin (lanes 5–8). (B) Western immunoblot of whole-cell lysates of H. influenzae strain DB117 derivatives preincubated with either PBS alone (lanes 1–4) or PBS with 13 μM deglycosylated A. awamori recombinant human lactoferrin (lanes 5–8). (C) Western analysis of culture supernatants of H. influenzae strain DB117 derivatives preincubated with either PBS alone (lanes 1–4) or PBS with 13 μM deglycosylated A. awamori recombinant human lactoferrin (lanes 5–8). Western analysis was performed with antiserum Rab730, which reacts with the Hap preprotein, Hap_s, and Hap_β. The gels in all panels were loaded as follows: lane 1, PBS-treated DB117/vector; lane 2, PBS-treated DB117/wild-type Hap; lane 3, PBS-treated DB117/HapS243A; lane 4, PBS-treated DB117/Hap_β; lane 5, lactoferrin-treated DB117/vector; lane 6, lactoferrin-treated DB117/wild-type Hap; lane 7, lactoferrin-treated DB117/HapS243A; lane 8, lactoferrin-treated DB117/Hap_β. Arrowheads point to the Hap preprotein and Hap_β, arrows point to Hap degradation products, and asterisks indicate Hap_s.

Figure 3

Effect of human lactoferrin on Hap-mediated H. influenzae adherence to human epithelial cells. (A) Adherence to Chang conjunctival cells by DB117/vector and DB117/HapS243A after incubation in PBS, PBS with 13 μM human milk whey lactoferrin, or PBS with 13 μM deglycosylated A. awamori recombinant lactoferrin. Adherence was measured in a 30-min assay as previously described (33) and is reported relative to DB117/HapS243A after incubation in PBS, which was normalized to 100%. (B and C) Light micrographs of DB117/HapS243A associated with Chang conjunctival cells samples after staining with Giemsa (×2,250). The sample in B was incubated in PBS, and the sample in C was incubated with 13 μM deglycosylated A. awamori recombinant lactoferrin.

Figure 4

Effect of serine protease inhibitor PMSF on lactoferrin-associated proteolysis of H. influenzae Hap. DB117/HapS243A was incubated in PBS (lane 1), PBS with 433 nM deglycosylated A. awamori recombinant lactoferrin (lane 2), PBS with 433 nM recombinant lactoferrin and 7.5% 2-propanol (lane 3), or PBS with 433 nM recombinant lactoferrin and 7.5 mM PMSF in 2-propanol (lane 4). Whole-cell lysates were prepared and examined by Western analysis with antiserum Rab 730, which reacts with the Hap preprotein, Hap_s, and Hap_β. The arrowhead points to the Hap preprotein, and arrows point to Hap degradation products.

Figure 5

The H. influenzae P2, P5, and P6 outer-membrane proteins are not removed or degraded by exposure to human milk whey. Bacteria were grown to mid-logarithmic phase and then incubated with either buffer (lanes 1 and 2 in all panels) or human milk whey (lanes 3 and 4 in all panels). Whole-cell lysates (lanes 1 and 3 in all panels) and corresponding supernatants (lanes 2 and 4 in all panels) were examined by Western immunoblot analysis. A was probed with antiserum #331 against IgA1 protease, B was probed with mAb 6G3 against P2, C was probed with mAb 2C7 against P5, and D was probed with mAb 7F3 against P6. Strain Rd3–13 was used for the analysis of IgA1 protease, P5, and P6. Strain 1479 was used for the analysis of P2, as antibody 6G3 is specific for the strain 1479 P2 protein. The IgA1 protease preprotein was transferred to the supernatant, while P2, P5, and P6 were unaffected.