Characterization of complement factor H binding to Yersinia enterocolitica serotype O:3

Abstract

A number of bacteria bind factor H (FH), the negative regulator of the alternative complement pathway, to avoid complement-mediated killing. Here we show that a gram-negative enteric pathogen, Yersinia enterocolitica serotype O:3, uses two virulence-related outer membrane (OM) proteins to bind FH. With Y. enterocolitica O:3 mutant strains displaying different combinations of surface factors relevant to complement resistance, we demonstrated that the major receptor for FH is the OM protein YadA. Another OM protein, Ail, also contributes to FH binding provided that it is not blocked by distal parts of the lipopolysaccharide (i.e., the O antigen and the outer core hexasaccharide). Importantly, we demonstrated that surface-bound FH was functional; both YadA- and Ail-bound FH displayed cofactor activity for factor I-mediated cleavage of C3b. With truncated recombinant FH constructs, we located the binding site of Ail specifically to short consensus repeats 6 and 7 of FH, while YadA showed a novel type of FH-binding pattern and appears to bind FH throughout the entire FH molecule. We thus conclude that Y. enterocolitica, via YadA and Ail, recruits functionally active FH to its surface. FH binding appears to be an important mechanism of the complement resistance of this pathogen.

FIG. 1.

FH binding to Y. enterocolitica O:3. (A and B) FH binding to YadA-positive and -negative strains, respectively, from 50% HIS was quantitated by ELISA. The wild-type strain FH-binding level was set to 100% (black bars), and those of the mutant strains are expressed relative to it. The data were obtained from three independent experiments. Error bars represent standard deviations. The factors expressed by the strains tested are marked as follows: YadA, Y; Ail, A; OAg, O; OC, C; the strains of the same phenotype obtained by different genetic methods are indicated by the subscript numbers 1, 2, and 3 (see Table 1 for details). The average OD₄₉₂ value for wild-type strain YeO3 was 0.74 ± 0.26 (range, 0.35 to 1.36). (C) Comparison of the ELISA and immunoblotting analyses of FH binding to selected Y. enterocolitica O:3 strains. (D) Presence of cross-reacting anti-YadA antibodies in goat antiserum against human FH (Quidel). Detection of FH bound to wild-type YeO3 bacteria by immunoblotting with the antiserum before and after adsorption with YadA-expressing bacteria is shown.

FIG. 2.

The cofactor activity of FH bound to the bacterial surface. In the left and middle panels, bacteria were preincubated with FH (final concentration, 30 μg/ml), washed extensively, and exposed to FI and C3b. Bacterial surface-bound FH is indicated by the asterisk, while in the control panel at the right, reactions were carried out without bacteria. C3b and its cleavage products were detected in the supernatants with a mixture of polyclonal antibodies against C3c and C3d. Inactivation of C3b is demonstrated by the reduced intensity of the C3b α′ chain and the appearance of the α′ chain cleavage fragments of 67, 43, and 41 kDa. FH bound to bacteria was detected in pellets by immunoblotting with polyclonal antiserum against human FH. A nonspecific bacterial protein band recognized by the anti-FH antiserum is shown as a loading control (LC). The factors expressed by the tested strains are marked as in Fig. 1.

FIG. 3.

Binding of FH to YadA. (A) Binding of purified FH to YadA shown by affinity blotting. Triton X-114 membrane protein extracts from E. coli expressing YadA (Tx-YadA) or from E. coli carrying the empty vector (Tx-Ctrl) were subjected to 8% SDS-PAGE and transferred onto nitrocellulose membrane. The part of the membrane containing the proteins with molecular masses greater than 160 kDa was incubated with purified FH. Binding of FH was detected by immunoblotting with preadsorbed goat anti-human FH and HRP-conjugated rabbit anti-goat antibodies. Binding of FH to the YadA band is indicated by the arrow. (B) Divergent effects of gelatin and collagen on FH binding to YadA. Binding of purified FH to YadA-expressing wild-type Y. enterocolitica bacteria was tested in VBS (gelatin free), GVB (contains 0.1% gelatin), or VBS supplemented with type I collagen (0.01%). After extensive washings, bound FH was identified by immunoblotting with nonadsorbed polyclonal antiserum against human FH. The use of nonadsorbed serum allowed the simultaneous detection of the YadA band (indicated by the arrow), which served as a loading control.

FIG. 4.

(A) Mapping of the binding region on FH for YadA and Ail of Y. enterocolitica O:3. Strain YeO3 (SP YAOC), YeO3-Ail (SP Y-OC), YeO3-c-OCR (SP -A--₂), and YeO3-c-Ail-OCR (SP ---) bacteria were incubated with truncated recombinant FH constructs representing SCRs 1-5, 1-6, 1-7, 8-11, 11-15, and 8-20. Following incubation, the bacteria were washed and whole-cell lysates were run in 12% SDS-PAGE and analyzed by immunoblotting with goat anti-FH and HRP-conjugated rabbit anti-goat antibodies. The factors expressed by the strains tested are marked as in Fig. 1. (B) Detection of recombinant FH constructs with goat anti-FH antiserum and HRP-conjugated rabbit anti-goat antibodies.

FIG. 5.

Characterization of FH interaction with Y. enterocolitica. The effects of NaCl (A) and heparin (B) on the binding of purified FH to Y. enterocolitica O:3 strains YeO3 (SP YAOC), YeO3-Ail (SP Y-OC), YeO3-c-OCR (SP -A--₂), and YeO3-c-Ail-OCR (SP ---) are shown. Bacteria were incubated with FH (final concentration, 30 μg/ml) in 1/3 PBS with various amounts of NaCl (100 to 650 mM) or heparin (0 to 1,000 μg/ml). Following incubation, the bacteria were washed and whole-cell lysates were subjected to 8% SDS-PAGE and immunoblotting with the nonadsorbed goat antiserum against human FH. Quantitative analysis of FH binding was performed with the NIH ImageJ software. The intensities of the FH protein bands were quantitated, and the influence of loading differences was eliminated by relating the values to the intensity of a nonspecifically reacting bacterial protein band in the same lane (loading control [LC]). The FH-binding level of the bacteria incubated in 1/3 PBS alone was set to 100, and the FH-binding level of the bacteria in 1/3 PBS supplemented with NaCl (100 to 650 mM) and heparin (1 to 1,000 μg/ml) was expressed relative to the FH-binding level of the bacteria in 1/3 PBS alone. The factors expressed by the strains tested are marked as in Fig. 1.