CEACAM1 binding properties of <i>M. catarrhalis</i> strain 035E and its derivatives.

Abstract

<p>A) Western blot of Mx strains from right to left MX2 (used for comparison), 035E and 035E D2 and D1 overlaid with CEACAM1-Fc (CC1) or SIGLEC10-Fc (SIGLEC) as described in the methods. As expected, MX2 UspA1 bound to CEACAM1-Fc. No CEACAM binding protein was observed for 035E while its transformants D1 and D2 both bound to CEACAM1. Of these proteins, D2 migrated at a much higher molecular weight than expected for UspA1. B) SDS-PAGE gels stained with Coomassie (Gel) and corresponding Western blots (CC1 and CD33) overlaid with CEACAM1-Fc and CD33-Fc respectively. Bacterial lysates of 035E D1 and D2 were preincubated without (−FA) or with 70% formic acid (+FA) and then heated at 100°C for 5 min. In the case of D1, a high molecular weight band (*) is no longer seen in the gel after formic acid treatment and one prominent CEACAM-binding band was observed in the Western blot with or without prior formic acid treatment. Thus heat alone (−FA) appears to be sufficient to induce a level of dissociation of the protein and so affect the migration of the protein, whereas formic acid treatment results in its complete dissociation (+FA). In the case of D2, formic acid treatment was required for the dissociation of the D2 high molecular weight band in the gel and correlated with the appearance of a lower molecular weight CEACAM binding band (>). Note the laddering effect on the D2 CEACAM-binding blot in the absence of formic acid is characteristic of some oligomeric coiled coil adhesins. Whilst regions of interest are presented here, full gel and blot images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045452#pone.0045452.s001" target="_blank">Fig. S1</a>.</p