Moraxella catarrhalis binding to host cellular receptors is mediated by sequence-specific determinants not conserved among all UspA1 protein variants

Abstract

The Moraxella catarrhalis ubiquitous surface proteins (UspAs) are autotransporter molecules reported to interact with a variety of different host proteins and to affect processes ranging from serum resistance to cellular adhesion. The role of UspA1 as an adhesin has been confirmed with a number of different human cell types and is mediated by binding to eukaryotic proteins including carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), fibronectin, and laminin. A distinct difference in the ability of prototypical M. catarrhalis strains to adhere to CEACAM-expressing cell lines prompted us to perform strain-specific structure-function analyses of UspA1 proteins. In this study, we characterized CEACAM binding by a diverse set of UspA1 proteins and showed that 3 out of 10 UspA1 proteins were incapable of binding CEACAM. This difference resulted from the absence of a distinct CEACAM binding motif in nonadhering strains. Our sequence analysis also revealed a single M. catarrhalis isolate that lacked the fibronectin-binding motif and was defective in adherence to Chang conjunctival epithelial cells. These results clearly demonstrate that UspA1-associated adhesive functions are not universally conserved. Instead, UspA1 proteins must be considered as variants with the potential to confer both different cell tropisms and host cell responses.

FIG. 1.

Cellular tropism of the M. catarrhalis O35E UspA1 protein. (A) Attachment of wild-type strain O35E.118 and mutant strain O35EΔUspA1 to A549 and Chang cells in vitro using a 30-min incubation period for attachment. (B) Expression of CEACAMs by Chang and A549 cells determined by Western blot analysis. Whole-cell lysates of these two human cells lines were probed with antibody to CEACAM (panel 1) or glyceraldehyde-3-phosphate dehydrogenase (panel 2), followed by HRP-conjugated secondary antibody. (C) Attachment of wild-type M. catarrhalis O35E.118 and recombinant E. coli DH5α strains containing either pELU1-10G, which expresses the O35E UspA1 or the empty pACYC184 vector to A549 and Chang cells using a 30-min incubation period for attachment. (D) Effect of rabbit antibody to CEACAM (αCEACAM) and normal rabbit serum (NRS) on the binding of the two recombinant E. coli strains to Chang cells. Representative experiments are shown (A, C, and D). Statistical analysis of attachment data involved the use of one-way or two-way ANOVA with a Bonferroni posttest.

FIG. 2.

CEACAM binding by recombinant E. coli expressing UspA1 variants. (A) E. coli whole-cell lysates were Western blotted and then probed to detect UspA1 protein (top) with MAb 24B5, and soluble CEACAM5 binding (bottom) was detected with anti-CEACAM (Dako). CEACAM5 binding was associated with UspA1 in 7 of 10 strains, while no binding was evident in the other three strains. (B) E. coli cells expressing the indicated UspA1 variants were spotted onto nitrocellulose and then incubated with soluble CEACAM5, followed by antibodies to detect bound CEACAM5. Ig, immunoglobulin. (C) E. coli cells expressing the indicated UspA1 variants were adsorbed to ELISA plates and then incubated with soluble CEACAM5, which was then detected using specific antibodies.

FIG. 3.

M. catarrhalis wild-type (wt) or UspA1-deficient (Δ) strain binding to CEACAMs. M. catarrhalis whole-cell lysates were separated by electrophoresis and were immunoblotted with MAb 24B5 to detect UspA1 (A and B, top) or binding to soluble CEACAM5 (A, bottom) or CEACAM1 (B, bottom). UspA proteins were detected using Cy5-conjugated secondary antibodies, while bound CEACAM was using Bodipy-conjugated antisera. M. catarrhalis ferric binding (Fbp) was probed using mouse anti-Fbp antiserum as a loading control (bottom). (C) M. catarrhalis strains were adsorbed into ELISA plates and then incubated with soluble CEACAM1 or CEACAM5. The bound CEACAMs were then detected using CEACAM cross-specific antisera (Dako CEA), followed by HRP-conjugated goat anti-rabbit immunoglobulin. Abs, absorbance.

FIG. 4.

UspA1 protein sequence alignments. (A) Alignment of variant sequences spanning the CEACAM-binding region of UspA1, defined by Hill et al. (12), which is delineated with a horizontal black line above the sequences. CEACAM binding by M. catarrhalis and/or recombinant E. coli strains, as determined in the aforementioned studies, is indicated. +, CEACAM binding; −, does not bind CEACAM. (B) Alignment of UspA1 variant sequences spanning the region reported to contribute to fibronectin and Chang cell binding (42). Relevant peptides from the UspA1 and UspA2 proteins of M. catarrhalis strain Bc5, which was originally used to define the fibronectin-binding sequences, are included for comparison with the sequences considered in this study. (C) Attachment of recombinant E. coli cells expressing UspA1_O35E or UspA1_{ATCC 43617} to Chang conjunctival epithelial cells in the presence or absence of FCS. The negative control is E. coli containing the pCC1-Kan construct (Kan).