YadA and Ail mediate the binding of FH to Y. enterocolitica
). In this study, we further characterized regions on YadA and Ail involved in the serum resistance and interaction with FH.
YadA is the main FH receptor on the surface of Y. enterocolitica
, and YadA-bound FH retains its biological function as an FI cofactor for C3b cleavage (3
). To identify YadA regions involved in serum resistance and FH binding, short deletions were introduced into the neck and stalk, taking into consideration the proposed coiled-coil structure of the trimeric stalk (22
). The YadA head domain was previously shown not to confer resistance against complement-mediated killing and was not mutagenized for this reason (43
). None of the in-frame deletions within the stalk interfered with the surface exposure of the mutated YadA, except for a deletion of 22 residues overlapping the junction between the right-handed and left-handed parts of the stalk. YadA was not detected in this mutant, consistent with the work by Roggenkamp et al. demonstrating that this part of the stalk is indispensable for YadA translocation across the outer membrane (43
). Additionally, all the stalk mutants bound collagen type I, indicating that the deletions did not affect the head and neck domains involved in collagen binding.
Functional analysis employing the YadA deletion mutants revealed that deletions in the N-terminal part of the stalk resulted in increased FH binding and CR. The reason for this is not clear. It is possible that in these deletions, FH gained better access to its binding site; however, since our stalk deletions were designed such that they left the collagen-binding head domain of YadA intact, the removal of the head was not the reason. A more likely possibility remains that the N-terminal part of the stalk contains the target site(s) for other serum proteins that could slow down or compete with FH binding. The deletion of these sites would then result in the facilitated access of FH to its binding site(s) on the YadA stalk. On the other hand, some deletions within the middle and C-terminal 15- and 7-mer regions of the stalk reduced the CR. In general, all serum-resistant YadA deletion mutants bound FH. The binding of FH to YadA seemed to depend on the recognition of several complementing and discontinuous structural YadA motifs rather than on one segment of the stalk, and the interaction seemed to be sensitive to both the conformation and the spacing of these motifs. This reasoning is based on the following observations.
Firstly, although most AP-sensitive mutants had not lost the FH binding activity, decreased FH binding was associated with decreased CR for some mutants. Since Δ261-277 disturbs both the pitch and the angle of the supercoil in the stalk, one could suggest that the reduction the FH binding of this mutant was due to a change in the protein conformation. Deletions Δ263-277 and Δ301-330, however, being in a pentadecad register, do not distort the supercoil. We can only speculate that the reduced ability to bind FH by these mutants is due to a change of a distance between the putative FH binding subsites on YadA. In addition, these three deletions were partially included in some other mutants (Fig. ) whose ability to bind FH was not affected. Thus, the reduced FH binding to these three mutants was most probably not due to a deletion of the actual FH binding subsites of YadA.
Secondly, deletions Δ308-323, Δ323-363, and Δ371-377, which reduced CR but not FH binding, affected the MAb 2A9 epitope (see above), showing that the deletions introduced close to the C terminus could affect the N-terminal conformation of the YadA stalk. Even though FH bound strongly to these mutants, it was not protective. The cofactor assay, however, showed that FH bound to these mutants retained its cofactor activity (Fig. ). Moreover, these mutants, similarly to wild-type YadA, bound FH fragments representing the SCRs of the entire polypeptide chain of FH (Fig. ). We are forced to speculate that although FH bound to these mutants displays the cofactor activity, its ability to interfere with the formation of the alternative pathway convertases and/or to accelerate their decay must be affected. In addition, these mutants could be more susceptible to complement-mediated killing due to the defect(s) in the binding of other serum factors crucial for CR, e.g., C4bp. C4bp, the classical and lectin pathway regulator, has recently been shown to bind to YadA (21
). The reduced binding of this regulator to bacteria could have an impact on CR, especially since C4bp is also able to stimulate the FI-mediated cleavage of C3b (6
). Alternatively, the excessive binding of FH by the mutants could lead to the gradual depletion of FH from serum and, as a result, provoke an uncontrolled activation of the complement system.
Thirdly, although the YadA stalk has been shown to mediate serum resistance (43
) and deletions generated in this study covered the whole stalk, none of these deletions, even those with broken periodicity (Fig. ), totally abolished FH binding. This suggests that there is no single FH-binding site on the YadA stalk but instead that the binding is mediated by several conformational and discontinuous sites.
The notion of multiple FH binding sites on YadA may not be surprising given the very large size and extended nature of the FH protein. FH contains 20 short consensus repeats of different activities, and we have shown that SCRs throughout the length of FH are involved in the FH-Y. enterocolitica
). Thus, the abolishment of FH binding to one of the YadA sites involved in the interaction does not necessarily affect the other FH regions to find their complementary structural motifs elsewhere on YadA. We also note that the right-handed part of the YadA stalk is formed of 15-residue repeats and has a clear internal sequence symmetry so that a binding site could occur in similar forms several times along the stalk.
YadA deletion mutants proved to be useful for the epitope mapping of three MAbs specific for YadA (3G12-15, 2A9, and 2G12). Previous attempts to map the epitopes of these MAbs using overlapping 16-mer YadA peptides were not successful (10
). The deletion mutants generated in this study allowed us to localize the epitopes within the N terminus of the stalk. This suggested that the epitopes are conformational, as several overlapping deletions destroyed the epitopes, and the MAb 2A9 epitope seemed to be affected by deletions several nanometers apart. This long-distance effect was shown by a reduced signal in the dot blotting of the C-terminal deletions Δ308-323, Δ323-363, and Δ371-377 (Fig. ). In the case of Δ371-377, this could be due to problems of protein folding and/or trimerization, also reflected as a lower level of collagen binding (Fig. ). Our modeling analyses suggested that deletions Δ308-323 and Δ323-363, however, would affect the periodicity of the stalk and thus the supercoil structure. Interestingly, MAb 2A9 binding was much less influenced by two other deletions, Δ247-262 and Δ261-277, which also perturb the supercoil and are located even closer to the actual MAb A9 epitope than Δ308-323 and Δ323-363. The reason for this observation is unclear to us at present.
The conformational and discontinuous nature of FH binding was reported previously for OspE and BBA68 of Borrelia burgdorferi
and FhbA of Borrelia hermsii
). All three proteins were predicted to form coiled coils, and these structural motifs were shown to be involved in forming the binding site for FH. The distortion of one of the three widely spaced coiled coils of OspE resulted in the attenuation or complete abolishment of FH binding (29
), while the destabilization of the BBA68 coiled coils resulted in a reduced FH binding capacity (28
). Interestingly, FhbA shares some similarity with YadA, as a deletion of the N-terminal coiled-coil segment of FhbA did not affect FH binding. The FhbA region responsible for the interaction with FH was mapped to the C-terminal coiled coils and the loop that they flank (19
). Although the loop might be a contact point for FH, the proper presentation of this region by the flanking coiled coils seems to be critical for the interaction to occur (19
). The binding of FH to streptococcal M protein was also shown to involve coiled-coil conformation (13
); hence, several FH binding proteins appear to present the conformational and discontinuous epitope for FH.
Ail acts as a receptor for FH on Y. enterocolitica
provided that it is well surface exposed. Moreover, FH bound to Ail showed cofactor activity for FI (3
). An attempt to locate Ail regions involved in FH binding was undertaken in this work. Amino acids shown to be crucial for serotype O:8 Ail-mediated serum resistance (32
) were substituted in loop 2 or loop 3 of serotype O:3 Ail, and the mutants showed decreased CR in both NHS and EGTA-Mg serum. Despite this, their FH binding was not affected. This suggests (i) that these Ail mutants would bind FH in a way that prevents the biological activity of FH, resulting in reduced serum resistance, or, more likely, (ii) that these Ail mutants fail to interact with another serum factor(s) important for CR of Y. enterocolitica
. In support of the latter hypothesis, we have evidence that, similar to YadA, Ail is also able to bind C4bp (21
). However, a more comprehensive set of Ail mutants is needed to elucidate the structural requirements of Ail-mediated FH and C4bp binding and the relative importance of these binding activities to Ail-mediated CR. On the other hand, the biological significance of FH binding by Ail, a factor promoting Yersinia
entry into tissue culture cells in vitro (34
), could also be related to bacterial adherence to the host cells as FH binds to glycosaminoglycans and sialic acids.
In conclusion, we have demonstrated that FH binding seems to involve multiple higher-order structural motifs on the YadA stalk. As a rule, YadA deletion mutants that bound FH survived well in serum. The FH binding-deficient deletion mutants identified in this study can be used to study the biological significance of this interaction in vivo. On the other hand, our results demonstrated that the two serum-sensitive Ail mutants were not affected in their abilities to bind FH. Thus, it appears that FH binding might not be the main mechanism of Ail-mediated CR.