Carcinoembryonic antigen family receptor recognition by gonococcal Opa proteins requires distinct combinations of hypervariable Opa protein domains

Abstract

Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.

FIG. 1.

Binding of soluble CEA to gonococci expressing chimeric Opa proteins. Bacteria were incubated with CEA in HEPES buffer, spun, and washed. Cell pellets were boiled, separated by SDS-PAGE, blotted onto nitrocellulose, and probed with rabbit polyclonal anti-CEA antibodies, followed by protein A-HRP. Detection was with the ECL kit from Amersham. Numbers on the left are molecular masses in kilodaltons.

FIG. 2.

Predicted two-dimensional structure of OpaB protein (23). Thick lines in each loop indicate which portion of the loop was deleted or replaced. Amino acids that are different in the modular OpaB protein compared to native OpaB are in squares, with the native residues shown in circles on the left. Sequences of HV loops that were introduced in the modular OpaB protein are given in Table 3. CL, conserved-loop domain.

FIG. 3.

Expression of deletion and chimeric Opa proteins in gonococcal strain MS11. (A) Expression and surface exposure of chimeric Opa proteins determined by limited trypsinolysis. Whole bacteria were exposed to trypsin as described in Materials and Methods. Cell lysates were analyzed by SDS-PAGE and stained with Coomassie brilliant blue. Molecular mass standards are indicated on the left in kilodaltons. Arrow, porin protein; *, Opa proteins; − and +, absence or presence of trypsin (tryp), respectively. (B) Detection of Opa proteins by immunoblotting with anti-Opa antibody 4B12. Samples are identical to those shown in the gel in panel A. (C) Expression of deletion Opa variants. Whole-cell lysates were separated by SDS-PAGE, and Opa proteins were detected by immunoblotting with anti-Opa antibody 4B12. _, deletions of HV regions (i.e., m_B = mBB with its HV-1 loop deleted; mB_ = mBB with its HV-2 loop deleted).

FIG. 4.

Crystal violet-stained HeLa-Neo cells infected with E. coli expressing the mIB protein in the absence (A) or presence (B) of heparin.

FIG. 5.

Binding of CEA N domain to gonococci expressing chimeric Opa proteins. Bacteria were incubated in HEPES buffer containing His₆-tagged CEA N domain (8) for 20 min at 37°C, spun, and washed. Cell pellets were boiled, separated by SDS-PAGE, blotted onto nitrocellulose, and probed with a mouse monoclonal anti-His₆ antibody, followed by protein A-HRP. Detection was with the ECL kit from Amersham. Numbers on the left are molecular masses in kilodaltons.