Functional mapping of YadA- and Ail-mediated binding of human factor H to Yersinia enterocolitica serotype O:3

Abstract

Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37 degrees C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding.

FIG. 1.

Characteristics of YadA deletion mutants. A schematic representation of the in-frame deletions generated within the YadA neck and stalk is shown at the top. Whole-cell lysates of the strains were subjected to SDS-PAGE, and the gels were either stained with Coomassie blue (top row) or processed for immunoblotting or affinity blotting (four middle rows). Parts of the gels or immunoblots with the trimeric YadA bands and, in the case of nonadsorbed polyclonal anti-FH antiserum (that cross-reacts with YadA) (see Materials and Methods), also the monomeric band are shown. An aliquot of the lysates was analyzed for MAb 2A9 reactivity by dot blotting (bottom row). Affinity blotting was used to test the ability of YadA deletion mutants to bind type I collagen. The bound collagen was detected using type I collagen-specific MAb. wt, wild type.

FIG. 2.

Effect of deletions on the structural model of the YadA stalk. (A) Ribbon presentation of the full-length wild-type YadA structure. (Modified from reference with permission of the publisher.) (B and C) Comparison of the trimeric coiled-coil stalks of the wild type and four deletion mutants. Two of the mutants shown were predicted to alter the periodicity of the coiled-coil (B), and the other two were predicted to retain the wild-type periodicity of the stalk (C). The deleted regions in each stalk pair are indicated for the wild-type stalks.

FIG. 3.

Deletion mapping of YadA for CR determinants. The resistance of the mutants to CP/AP- and AP-mediated killing is presented for the 2-h time point. For the same strains, the FH binding from HIS was determined by ELISA (for each strain, the transparent columns with standard deviation bars represent the average FH binding for three independent duplicate experiments) and by immunoblotting (superimposed gray columns) (quantitation of the FH band intensities was performed with the Typhoon 9400 Image Quant analyzer). Survival and FH binding values of the strain expressing wild-type YadA (YeO3-c/pYMS4505) were set to 100, and the survival percentage and FH binding of the mutants are expressed relative to those of the wild type. For the calculation of statistical significances between the deletion mutants and wild-type YadA, the actual survival percentages for each experiment were used. The average YeO3-c/pYMS4505 survival value for AP killing in serum pool 1 was 76.1 ± 22.1, and that in pool 2 was 103.7 ± 27.7; the value for the CP/AP killing in serum pool 1 was 49.4 ± 17.4, and that in pool 2 was 73.3 ± 7.2. In ELISA, the average optical density at 492 nm value for strain YeO3-c/pYMS4505 was 0.47 ± 0.19 (range, 0.27 to 0.92). The strain with a frameshift mutation in the yadA gene (YeO3-c/pYMS4505:C), unable to express YadA, was included as a negative control.

FIG. 4.

Comparison of FH binding to bacteria expressing wild-type YadA to that of bacteria expressing mutated YadA during a 5-min incubation at 37°C in NHS, EGTA-Mg-treated serum, or HIS.

FIG. 5.

Cofactor activity of FH bound to bacteria expressing wild-type or mutated YadA. Bacteria were preincubated with FH (30-μg/ml final concentration) (left) and washed. Subsequently, the bacteria were exposed to factor I and C3b. The bacterial surface-bound FH is indicated by the asterisk, while in the control panel (right), reactions were carried out without bacteria. C3b and its cleavage products from the supernatants were detected using a mixture of rabbit anti-C3c and anti-C3d antisera. Inactivation of C3b can be seen by the reduced intensity of the C3b α′-chain and the appearance of α′-chain cleavage fragments of 67, 43, and 41 kDa. FH bound to the bacteria was detected from pellets by immunoblotting using preadsorbed goat anti-FH antiserum.

FIG. 6.

Binding of recombinant FH fragments to bacteria expressing wild-type or mutated YadA. Bacteria were incubated with truncated recombinant FH constructs representing SCRs 1 to 5, 1 to 6, 1 to 7, 8 to 11, 11 to 15, and 8 to 20. Following incubation, bacteria were washed, and whole-cell lysates were run on 12% SDS-PAGE gels and analyzed by immunoblotting using goat anti-FH and HRP-conjugated rabbit anti-goat antibodies for detection. The intensities of the bands for SCRs 1 to 6, 1 to 7, and 8 to 11 were measured. The intensity of the wild-type band was adjusted to 100, and those of the mutants are relative to that of the wild type. The relative intensity values are indicated above the bands.

FIG. 7.

Effect of loop 2 and loop 3 substitutions of Ail on CR. Wild-type (wt) and mutated Ail proteins were expressed in strain YeO3-c-Ail-OCR. The survival percentages of the Ail mutants to CP/AP- and AP-mediated killing are presented for the 0.5-h and 2-h time points. The columns with standard deviation bars show the averages of data for three independent duplicate experiments. pTM100-ail1, His65Ala substitution in loop 2; pTM100-ail4, Ser100Asp substitution in loop 3.