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. 2019 Apr 29;220(6):1049–1060. doi: 10.1093/infdis/jiz217

Figure 3.

Figure 3.

Haemophilus elongation factor thermal unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and lipoprotein P6 (P6) promote nontypeable Haemophilus influenzae (NTHi) adhesion to pulmonary epithelial cells by targeting laminin as a bridging molecule. (A–C) Direct interactions of recombinant NTHi laminin-binding proteins (Lbps) (EF-TuM1-K394, LDHM1-L381, PDS19-K364, P6S21-Y153, Rib30sM1-E125, P4G22-K274, PE22-160, PF12-293, and HapE523-L1036) (10 nM) to a panel of fixed pulmonary epithelial cell lines immobilized on 96-well plates: A549 (A), Detroit 562 (B), and NCI-H292 (C) as measured by cell enzyme-linked immunosorbent assay. HP09011 was included as negative control. Recombinant EF-Tu, LDH, PD, and P6 bound to the cell lines to the similar degree as the well-established NTHi Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), whereas the negative control protein HP09011 did not. Preincubation of cell lines with antilaminin antibodies (equal mixture of antilaminin-111 polyclonal antibodies (Sigma) and antilaminin-332 monoclonal antibodies [AbCam]) as previously described [9] reduced NTHi Lbps binding compared to without preincubation with antilaminin antibodies. Of note, the presence of cellular laminin in A549, Detroit 562, and NCI-H292 was confirmed (data not shown) as previously described before performing protein-cell binding assays [9]. (D) Adhesion assay with NTHi 3655 wild-type (wt), isogenic mutants and NTHi 3655+anti-EF-Tu to the alveolar epithelial cell line A549 upon supplementation of soluble laminin as analyzed by a colony counting method. Data are presented as percentage of bacterial adhesion to cells relatively to the NTHi 3655 wt without preincubation with lamininmur that was set as 100%. Bacterial preincubation with laminin promoted NTHi 3655 wt and NTHi 3655Δhpd adhesion, but not to other Lbp-deletion mutants and NTHi 3655+anti-EF-Tu. All data in A–D represent mean values of three independent experiments, and error bars indicate standard deviations. Statistical differences between preincubation with and without antilaminin antibodies in A–C, or between preincubation with or without laminin in D, were calculated by a two-way ANOVA. *, P ≤ .05; **, P ≤ .01; and ***, P ≤ .001.