Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein

Abstract

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

Figure 1. Binding of ¹²⁵I-C4bp to Y. enterocolitica serotypes O:3, O:8, O:9 and biotype 1A.

Indicated strains were incubated with ¹²⁵I-C4bp for 30 min at 37°C. Subsequently, they were centrifuged through 20% sucrose to separate bacteria-bound ¹²⁵I-C4bp from the unbound protein. Radioactivity was measured with a γ-counter. The binding of ¹²⁵I-C4bp is expressed as percentage of the total radioactivity. Mean±SD values from two experiments performed in duplicate are shown.

Figure 2. Cofactor activity of Y. enterocolitica-bound C4bp for C4b cleavage.

Y. enterocolitica serotype O:3 wild-type bacteria (YeO3), YadA-negative less virulent biotype 1A strain 27675 (BT 1A) and Ail-expressing strain YeO3-028-OCR were preincubated with C4bp and after extensive washings exposed to factor I and C4b. C4b and its cleavage products from the supernatants were detected using polyclonal antibodies against C4c. As positive and negative controls, assays containing C4b and FI with or without C4bp, respectively, were included. Inactivation of C4b is demonstrated by the appearance of C4b cleavage fragments (indicated with arrows).

Figure 3. Binding of serum C4bp to the wild type and YadA-negative Y. enterocolitica serotypes O:3 and O:8.

Wild type bacteria (Ye03 and 8081) and their YadA-negative derivatives (YeO3-028 and YeO8-116, respectively) (3×10⁸) were incubated in 5% heat-inactivated serum (HIS) for 30 min. Bacteria washed in 1/3 PBS were subjected to elution with PBS while those washed with PBS were subjected to elution with 0.1 M glycine-HCl (pH 2.7). The 1/3 PBS or PBS wash fractions (w1 and w2, respectively) and PBS or 0.1 M glycine-HCl (pH 2.7) elute fractions (e1 and e2, respectively) were separated by 8% non-reducing SDS-PAGE and analyzed by immunoblotting using a sheep anti human-C4bp antiserum.

Figure 4. Binding of ¹²⁵I-C4bp to Y. enterocolitica serotype O:3 strains.

The bacteria were incubated with ¹²⁵I-C4bp as described in Fig. 1. The factors expressed by Ye O:3 strains are marked as follows: YadA, Y; Ail, A, O-ag,•; OC, C. Strains carrying the pYV plasmid are marked with +.

Figure 5. Binding of ¹²⁵I-C4bp to Ail.

The strain YeO3-c-Ail-OCR was complemented in trans with pTM100-ail expressing the cloned ail gene (YeO3-c-Ail-OCR/pTM100-ail). YeO3-c-Ail-OCR/pTM100 was used as the vector control. The bacteria were incubated with ¹²⁵I-C4bp or ¹²⁵I-BSA as described in Fig. 1.

Figure 6. Affinity blotting analysis of C4bp-binding to Triton X-114-extracted YadA.

Membrane proteins were extracted from E. coli JM103/pYMS4450 expressing YadA of Y. enterocolitica serotype O:3 (Tx-YadA) and E. coli JM103/pL2.1 carrying the empty vector (Tx-Ctrl). Extracts were subjected to SDS-PAGE and proteins were transferred onto nitrocellulose membrane. The membrane was blocked in 5% skimmed milk in PBS and incubated with 5% normal human serum. After washing C4bp-bound to extracted proteins was detected using rabbit anti-human C4bp antibody (anti-C4bp). The YadA-bound C4bp is indicated with an arrow. In parallel, YadA protein on the membrane was detected by the monoclonal antibody 3G12 (anti-YadA).

Figure 7. The effect of NaCl (A), heparin (B), unlabeled C4bp (C) or BSA (D) on binding of ¹²⁵I-C4bp to Y. enterocolitica.

Wild type (YadA+ Ail+, YeO3) and YadA-negative (YadA- Ail+, YeO3-028-OCR and YadA- Ail+ (complemented), YeO3-c-Ail-OCR/pTM100-ail) bacteria were incubated with ¹²⁵I-C4bp as described in Fig. 1. The binding is presented as percentage of the ¹²⁵I-C4bp binding occurring in the presence of 50 mM NaCl without any additives. In the wild type strain Ail protein is masked by O-ag and OC and thus the binding represents properties of the YadA-C4bp interaction.

Figure 8. Schematic model of C4bp binding to Y. enterocolitica surface structures.

Y. enterocolitica expresses outer membrane proteins, YadA and Ail, that are able to capture C4bp. Ail, however, is masked by LPS O-ag chains and OC branches. Hexasaccharide OC is abundant on the Y. enterocolitica surface and forms a branched structure with O-ag. Removal of the O-ag and OC can unveil Ail and promotes Ail-mediated C4bp binding. C4bp ,, YadA , Ail, outer membrane and O-antigen (1 nm per sugar residue) are drawn to scale.