Identification of FH receptor(s) on Y. enterocolitica O:3.
To find a bacterial receptor responsible for FH binding, a set of 23 Y. enterocolitica
O:3 strains, expressing YadA, Ail, OAg, and OC in all possible combinations was used (7
). In a previous work, we demonstrated that the level of expression of these factors by the mutant strains was not affected by the type or number of mutations introduced (7
). For clarity, we have given the strains a letter code for the expressed surface phenotypes (SPs) of the strains (Table ) as follows: YadA, Y; Ail, A; OAg, O; OC, C. Thus, SP YAOC means that all four factors are expressed and SP Y--C means that YadA and OC are expressed and Ail and OAg are not. Identical SP codes of different strains are differentiated by subscript arabic numerals (Table ). This set of strains was tested for the ability to bind FH from 50% HIS both by ELISA (Fig. ) and by immunoblotting (Fig. and data not shown) with goat antiserum against human FH for detection. In immunoblotting, we noticed that the antiserum contained YadA-specific antibodies; however, these were efficiently removed by adsorption with YadA-expressing bacteria (Fig. ). Unless otherwise indicated, the preadsorbed antiserum was used for FH detection in all of the experiments.
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 (more ...) FH ELISA.
The results of our FH-binding analysis by ELISA are shown in Fig. . In general, all strains expressing YadA were able to bind serum FH (Fig. , 53 to 326% of the wild-type level) while much less FH binding to most other strains occurred (Fig. , to 47% of the wild-type level). The difference in FH binding between these two groups was statistically significant (P < 0.0001).
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 (more ...)
As demonstrated in Fig. , expression of YadA in the absence of OAg resulted in increased binding (SP YA-C, SP Y--C, SP Y--1
, SP Y--2
, and SP Y---), perhaps due to better exposure of YadA on the bacterial surface. Interestingly, most of the YadA-expressing strains bound as much FH as their Ail-lacking counterparts (Fig. , compare SP YAOC versus SP Y-OC, SP YA-C versus SP Y--C, and SP YAO-1
versus SP Y-O-), suggesting that YadA is the major FH-binding OM protein. The contribution of Ail to FH binding when YadA was expressed could only be observed when both OAg and OC were not produced. Thus, SP YA-- strains YeO3-OCR and YeO3-trs11-R, missing both OAg and OC, bound significantly more FH (P
< 0.029 and P
< 0.001, respectively) than the SP Y--- strain, the Ail-negative derivative YeO3-Ail-OCR (Fig. ). It is worth noting that the latter strain, expressing only YadA, was still able to bind more FH than the wild type (P
< 0.001). Thus, in the absence of OAg, YadA is likely to be better exposed and this could favor FH binding. On the contrary, lack of OC in YadA- and OAg-expressing SP YAO-1
and SP Y-O- strains (YeO3-OC and YeO3-Ail-OC) did not greatly affect the bacterial capacity to bind FH. Interestingly, SP YAO-2
strain YeO3-trs11, which, as shown earlier (7
), expresses less OAg than its phenotypic counterpart SP YAO-1
strain YeO3-OC, bound significantly more FH (P
< 0.0046). This further supports the hypothesis that in the absence of the OAg blocking effect, FH binding to better-exposed YadA would be facilitated.
LPS blocking effect.
To support the conclusion of the OAg blocking effect on YadA, we examined whether OAg could block the access of YadA-specific MAbs 2A9, 3G12, and 2G12 to their epitopes. These MAbs recognize epitopes within the neck and the very N-terminal region of the YadA stalk (M. Biedzka-Sarek et al., submitted for publication). The concentrations of the MAbs used were adjusted so that most of the MAb was adsorbed by YadA-expressing bacteria under the adsorption conditions. The MAbs were incubated with the YadA-positive strains expressing or lacking OAg (SP YAOC and SP YA-C), and their YadA-negative counterparts (SP -AOC and SP -A-C) were used as negative controls. Following incubation, bacteria were centrifuged and the remaining amount of MAbs in the supernatants was semiquantitated by dot blotting from 1:2 dilution series. Although differences in the adsorption of MAbs between the YadA-expressing and YadA-negative strains were clear, no difference between the YadA-positive strains expressing or lacking OAg could be observed (data not shown). These results indicated that OAg did not block the MAb epitopes located in the N-terminal end of the stalk; however, the possibility remains that it could block the YadA regions that are involved in FH binding and located closer to the C terminus and thus the OM (Biedzka-Sarek et al., submitted).
Both YadA and Ail bind FH.
The YadA-negative mutants also provided evidence for the involvement of YadA in FH binding (Fig. ). Almost all YadA-negative strains bound less FH than the wild-type strain. The exceptions were SP -A--1,2
strains YeO3-c-trs8-R and YeO3-c-OCR, which express Ail in the absence of both OAg and OC (Fig. ). These strains bound amounts of FH comparable to that bound by the wild-type strain. These observations suggest that to bind FH, Ail needs to be very well exposed on the OM since neither the removal of OAg in the SP -A-C1,2
strains (YeO3-O28-R and YeO3-R1) nor that of OC in the SP -AO-1-3
strains (YeO3-O28-OC, YeO3-c-trs8, and YeO3-c-OC) was enough to facilitate Ail-mediated FH binding (Fig. ). To demonstrate the masking potential of LPS, we compared the sensitivities of different Y. enterocolitica
O:3 strains, grown at RT, to bacteriophages
R8-01 (Table ). The results obtained supported the LPS masking hypothesis.
R1-37 could reach its OC receptor only in the absence of blocking OAg, and
R8-01 infection took place exclusively when both OAg and OC were not expressed (Table ). This suggests that the still unknown receptor of
R8-01 does not protrude far from the OM and is efficiently masked by both OC and OAg. While
R1-37 infectivity was not affected by the growth temperature of the host bacteria, bacteriophage
R8-01 produced almost no plaques on bacteria grown at 37°C (data not shown). Thus, the
R8-01 receptor is either strongly downregulated at 37°C or masked by surface structures expressed under these conditions (other that OAg or OC).
Characterization of the LPS masking effect by analysis of the phage sensitivities of bacteria grown at 22°C
In general, the immunoblotting and ELISA results correlated well (Fig. and data not shown). Only YeO3-c-Ail-OCR, YeO3-trs11, and YeO3-Ail-R showed relatively higher FH binding in ELISA than in immunoblotting. We can only speculate that this may be due to some nonspecific reactivity in ELISA.
Collectively, these results demonstrated that Y. enterocolitica could acquire FH from human serum and that YadA was the main FH receptor on the bacterial surface. In addition to YadA, Ail was able to bind FH but solely when not masked by LPS OAg and OC.
FH bound to Y. enterocolitica is functional.
FH functions in the negative regulation circuit of AP activation. Specifically, it acts as a cofactor in FI-mediated progressive cleavage of the C3b α′ chain into several fragments (67, 43, 41, and 30 kDa). With the cofactor assay, we aimed to examine whether YadA- and Ail-bound FH remains functionally active. Y. enterocolitica bacteria were incubated with purified FH, and after intensive washes, bacteria were incubated with purified FI and C3b. Following incubation, the samples were centrifuged and supernatants were analyzed for C3b cleavage with the mixture of anti-C3c and anti-C3d antibodies that recognizes both the intact C3b α′ chain and its cleavage products while the lysed pellets were examined for FH deposition on bacteria with goat anti-FH antiserum (Fig. ).
Similar to FH binding from HIS, purified FH could be detected on YadA-expressing bacteria while Ail bound FH only in the absence of LPS (Fig. and ). Bacteria that expressed Ail in the presence of LPS or were YadA and Ail negative (SP --OC and SP ---) bound negligible amounts of FH. This further indicated that neither OAg nor OC directly binds FH. On the other hand, we could also see slightly more binding of purified FH to YeO3-c-Ail-OCR (SP ---) than to YeO3-c-Ail or YeO3-028 (SP --OC and SP -AOC2, respectively; Fig. ). This could also mean that the loss of the LPS surface structures might have generated or exposed novel FH-binding specificities such as the LPS inner core or small OM proteins.
The ability of Y. enterocolitica strains to cleave C3b in the presence of FI was reflected in their ability to bind FH. FH bound to the wild-type (SP YAOC), SP Y-OC, and SP -A--2 bacteria retained its cofactor activity, as shown by the cleavage of the C3b α′ chain into 67-, 43-, and 41-kDa fragments (Fig. , left panel; note the reduction of the intact α′ band intensity and the simultaneous appearance of the α′ cleavage fragments). No or very modest cofactor activity was observed with SP -AOC1, SP -AOC2, SP --OC, and SP --- bacteria displaying no or scarce FH on their surface.
These results showed that YadA- and Ail-bound FH functions as an FI cofactor for C3b cleavage.
Purified FH binds directly to YadA.
As the analyses described above suggested that Y. enterocolitica binds purified FH via both YadA and Ail, we wanted to further show that the interaction between YadA, the main FH receptor on Y. enterocolitica, and FH is direct. To this end, Tx-114 membrane protein extracts from E. coli expressing YadA (Tx-YadA) and E. coli carrying the empty vector (Tx-Ctrl) were prepared. The extracts were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. The fragment of the membrane containing the proteins with molecular masses greater than 160 kDa was cut out and incubated with purified FH. As shown in Fig. , binding to Tx-YadA, but not to Tx-Ctrl, was detected.
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 (more ...)
Since demonstration of direct binding of purified FH to Y. enterocolitica
I-labeled FH was not successful earlier (46
), we also repeated 125
I-labeled FH-binding experiments by following a protocol successfully applied to streptococci and Borrelia
). These experiments, however, failed to demonstrate any FH binding (data not shown). To verify whether 125
I labeling affected FH tyrosines involved in the interaction with Y. enterocolitica
, the binding experiment was repeated with FH labeled with nonradioactive I. I-labeled FH, however, was acquired by Y. enterocolitica
O:3 as efficiently as the nonlabeled regulator (data not shown). We then examined the effect of the buffers commonly used in the 125
I-labeled FH-binding assay, such as VBS and GVB. In the latter, gelatin is used to nonspecifically prevent bacterial aggregation. With YadA-expressing Y. enterocolitica
O:3, however, the effect was quite the opposite and strong aggregation of bacteria was observed. This resulted in a significant reduction of the binding of nonlabeled FH (Fig. ). Gelatin is a denatured form of collagen and has been shown to inhibit 50% of YadA-mediated collagen binding (13
). Thus, the lack of 125
I-labeled FH binding to Y. enterocolitica
O:3 in GVB could result from the blocking of FH-specific sites on YadA by gelatin and/or by the strong gelatin-induced aggregation. Interestingly, when the gelatin in the buffer was replaced with collagen, a significant increase in the amount of Y. enterocolitica
O:3-bound FH was observed. Our preliminary studies show that collagen per se is able to bind FH (Fig. ). Thus, by binding to collagen, Y. enterocolitica
could also indirectly acquire FH, thereby increasing its chances to avoid complement attack. More studies elucidating the role of collagen as a shield protecting Y. enterocolitica
against complement attack are warranted.
Locations of YadA- and Ail-binding sites on FH.
To identify the FH region(s) involved in binding to Y. enterocolitica O:3, we examined the binding of truncated recombinant FH fragments representing SCRs 1-5, 1-6, 1-7, 8-11, 11-15, and 8-20 to wild-type (SP YAOC), SP Y-OC, SP -A--2, and SP --- bacteria.
Bacteria were incubated with truncated fragments of FH and washed, and whole-cell lysates were subjected to SDS-PAGE and immunoblotting with a goat anti-FH antiserum that recognizes all of the FH fragments tested (Fig. ). As shown in Fig. , all of the FH fragments bound to wild-type bacteria and the YadA-expressing strain of SP Y-OC. The Ail-expressing strain, SP -A--2, on the other hand, specifically bound only SCRs 1-6 and 1-7. Ail binding to SCRs 1-5, 8-11, and 8-20 was at the level of the negative control SP --- strain and thus was not considered specific (Fig. ).
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 (more ...)
On the basis of these results, we conclude that the binding site for Ail on FH is located within SCRs 6 and 7 while YadA appears to bind throughout the entire FH.
Effects of salt and heparin on FH binding to Y. enterocolitica.
The nature of FH binding to YadA and Ail was examined by using the wild-type strain and strains expressing either Ail (the SP -A--2 strain) or YadA (the SP Y-OC strain). Bacteria were incubated with purified FH in 1/3 PBS or 1/3 PBS supplemented with NaCl to create a salt concentration gradient ranging from 50 to 650 mM. After incubation with FH, the bacteria were washed, whole-cell lysates were run in SDS-PAGE, and the bound FH was detected with goat antiserum against human FH. The results showed that FH binding to Ail by the SP -A--2 strain was maximal at the lowest salt concentration and decreased dramatically with increasing salt concentrations (Fig. ). To the contrary, YadA-mediated FH binding was more salt resistant and only a twofold decrease in FH binding to the SP Y-OC strain was observed at an NaCl concentration of 250 mM (Fig. ). FH binding to the wild-type bacteria under these conditions was not affected and dropped slightly only at an NaCl concentration of 650 mM (Fig. ).
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 (more ...)
Heparin interaction sites for FH have been mapped to SCRs 7, 13, and 20 (8
). We tested whether heparin inhibits the binding of FH to YadA- or Ail-expressing bacteria (Fig. ). Bacteria were incubated with purified FH in the presence of heparin (0 to 1,000 μg/ml) in 1/3 PBS, and the bound FH was detected by immunoblotting as described above. Heparin efficiently inhibited FH binding to the SP -A--2
strain, such that even at the lowest heparin concentration used, 1 μg/ml, FH binding was almost completely abolished (Fig. ). This indicates that the binding sites for heparin and Ail on FH are likely to be identical or at least overlap. This also corroborates our above observation that the Ail-binding site on FH resides on SCRs 6 and 7. The YadA-mediated FH binding determined with the SP Y-OC strain was only affected at a heparin concentration of 100 μg/ml and did not drop further at 1,000 μg/ml, the highest concentration of heparin used. This finding is supported by the ability of YadA to bind all of the truncated FH fragments (Fig. ). As wild-type bacteria express both YadA and Ail but the latter is likely to be blocked by the LPS, binding of FH occurs mainly via YadA. Thus, similar to the SP Y-OC strain, FH binding to the wild-type bacteria was slightly reduced only at the highest heparin concentrations used, 100 to 1,000 μg/ml (Fig. ).
FH binding to all of the three strains tested was affected only by the highest BSA concentration used (1,000 μg/ml, 25% of control binding; data not shown), indicating that YadA- and Ail-mediated binding to FH is specific.
In conclusion, the FH-Ail interaction appears to depend on ionic interactions between the proteins and Ail shares the binding site on FH with heparin. On the contrary, the YadA-FH interaction is more salt and heparin resistant.