CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains

Abstract

Neisseria gonorrhoeae strain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed in Escherichia coli reflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants, suggesting that the differential recognition of CD66 receptors by Opa variants is dictated by the presence of specific binding regions on the N-domain of the receptor.

Figure 1

Amino acid alignment of CD66 N-domains used in this study. Protein sequences were deduced from DNA sequencing of the clones. Dots indicate residues identical to those in CD66e. Arrows indicate the regions corresponding to the primer binding sites for PCR amplification of N-domains from HeLa-CD66 DNA. The location of the Cac8I site present in the DNA used to construct CD66b/e chimeric N-domains is indicated at the corresponding residue 59.

Figure 2

Interaction of MS11 Opa variants with CD66e glycoforms. (A) Immunoblot showing binding of CD66e glycoforms by gonococcal Opa variants. CD66e glycoforms were isolated from CD66e-transfected CHO cell lines by using PI-PLC (lanes 1–3). Binding of CD66e glycoforms to Opa⁻ (-) or OpaI (I) expressing gonococci is shown in lanes 4–9. Bound CD66e was detected with anti-CEA rabbit serum. M_r markers (× 10⁻³) are indicated on the left. The cell types from which CD66e was derived are indicated above the blot. (B) Adherence and entry of OpaI variants to Pro5, Lec2, and Lec8 cells and their CD66e-expressing counterparts. GC, gonococci. Data are means ± SE of three independent experiments.

Figure 3

Effect of deglycosylation of CEA on recognition by MS11 Opa variants. (A) Immunoblot showing native CEA (CEA) and CEA treated with TFMSA (dg-CEA). Each lane contains 50 ng of material, which was detected with anti-CEA rabbit serum. M_r markers (× 10⁻³) are indicated on the left. (B) Binding of MS11 Opa variants to native and deglycosylated CEA. Radiolabeled bacteria were added to the wells of ELISA plates containing native (CEA) or deglycosylated (dg-CEA) human tumor CEA for 1 hr at 37°C. Nonadherent bacteria were removed by washing and the number of bound bacteria was determined by liquid scintillation counting of detached bacteria. GC, gonococci. Data are means ± SE of three independent experiments.

Figure 4

Binding of E. coli-expressed N-domains of CD66e and CD66b by MS11 Opa variants. Opa variants were incubated with cleared lysates of E. coli containing the appropriate N-domains and processed for immunoblotting. The N-domains were detected with anti-His antibody. Lanes labeled lys show the cleared lysate, in an amount representing 100% binding; lanes - and I show the amount of N-domain bound by Opa⁻ and OpaI variants, respectively. The figure is representative of three independent experiments.

Figure 5

Binding of E. coli expressed N-domains of different CD66 receptors by recombinant MS11 Opa variants. Gonococci expressing different Opa proteins were incubated with cleared lysates of E. coli containing the N-domains of CD66e, -d, or -a and processed for immunoblotting. Bound N-domain was detected with anti-His antibody. The + and - designations refer to the results of our previous study, where interaction of Opa variants with CD66e, -d, and -a expressed on HeLa cells was studied: + indicates recognition; - indicates no recognition (12). Opa protein expression of the variants was evaluated by immunoblotting with 4B12 antibody and is shown in Bottom. Below the designation of the recombinant Opa variants used the nomenclature of wild-type Opa homologs is indicated in parentheses. The figure is representative of three independent experiments.

Figure 6

Interaction of Opa₅₄/F and Opa₅₉/H variants with CD66e and recombinant CD66e-N-domain. (A) Binding of CD66e, derived from Pro5-CD66e cells through PI-PLC treatment, by recombinant Opa variants and their wild-type counterparts. Bound CD66e was detected with anti-CEA rabbit serum. Below the designation of the recombinant Opa variants the nomenclature of the wild-type Opa homologs is indicated in parentheses. (B) Binding of recombinant CD66e-N-domain by wild-type Opa variants. Opa⁻, F, H, and I variants were incubated with cleared lysates of E. coli containing the CD66e N-domain and processed for immunoblotting. Bound N-domain was detected with anti-His antibody. Results with Opa₆₀/I are shown as a positive control. The figure is representative of three independent experiments.

Figure 7

Binding of E. coli-expressed CD66e, CD66e/b, and CD66b/e N-domain chimeras by recombinant MS11 Opa variants. Opa variants were incubated with cleared lysates of E. coli expressing the indicated N-domains and processed for immunoblotting. Bound N-domain was detected with anti-His antibody. The lane designated lys contains only the E. coli lysate. Below the designation of the recombinant Opa variants used the nomenclature for wild-type Opa homologs is indicated in parentheses. The figure is representative of three independent experiments.