The complement system is an essential part of host defense against many microorganisms. A number of pathogens, however, have evolved mechanisms to subvert complement activation at different steps of the cascade. To survive and establish infection in the gut and surrounding tissues, Y. enterocolitica
must resist complement-mediated opsonization and lysis. It is thus equipped with surface factors that confer resistance to serum, such as the outer membrane proteins, YadA and Ail 
. Although it has been shown that Ail promotes resistance to complement killing, the mechanism of Ail-mediated serum resistance has remained unknown. YadA, in turn, has been shown to be the major serum resistance determinant of Y. enterocolitica 
. Thus, not surprisingly, mechanisms underlying YadA-mediated resistance have for long been of interest. It has been speculated that the formation of YadA-composed velvet-like coat on the bacterial surface could by itself act as a shield protecting against complement 
. There is evidence, however, for YadA-mediated binding of the alternative pathway regulator FH and inhibition of the complement cascade at both C3 and C9 levels 
. This manifests as a reduced binding of C3b and as a failure of the membrane attack complex to incorporate into the outer membrane of Y. enterocolitica 
. Lipopolysaccharide O-ag and OC are involved in complement-resistance indirectly 
. They block outer membrane proteins, such as small-sized Ail, thereby having a negative influence on bacterial resistance to serum 
This study demonstrated a novel immune evasion mechanism of Y. enterocolitica, C4bp binding by YadA and Ail proteins. All serotypes tested, O:3, O:8 and O:9, were shown to bind the host complement regulator C4bp to avoid opsonophagocytosis and bactericidal action of serum (). The bacteria also acquired this CP-inhibitor from serum, as demonstrated by serum adsorption assays (). In addition, binding of purified radiolabeled C4bp to Y. enterocolitica could be observed ( and ). This shows that the binding is direct and does not involve other serum proteins. Importantly, FI cofactor assay showed that C4bp bound to Y. enterocolitica surface was functionally active (). By binding C4bp Y. enterocolitica can thus inhibit antibody-mediated CP, and the LP.
C4bp receptors on Y. enterocolitica
surface were identified using a set of serotype O:3 mutants expressing YadA, Ail, O-ag and OC in different combinations (). Analyses of 125
I-labeled C4bp binding to Y. enterocolitica
O:3 strains showed that YadA was crucial for capturing this CP regulator. Therefore, YadA- or pYV-negative mutants bound only marginal amounts of C4bp, exceptions being the strains expressing Ail in the absence of O-ag and OC (). Thus, Ail could bind C4bp solely when accessible on the outer membrane. This was additionally confirmed by trans
in a strain missing all four factors (YeO3-c-Ail-OCR). Since this strain lacks O-ag and OC, C4bp binding to Ail was strongly favored (). The demonstration of C4bp receptors as YadA and Ail correlated with the previously published serum resistance results of these Ye O:3 strains 
. These results revealed that the major serum resistance determinant of Y. enterocolitica
was YadA and that the removal of LPS O-ag and OC potentiated Ail-mediated complement resistance of YadA-negative strains 
. It is possible that during infection the production of LPS, O-ag, and OC is suppressed. Similar phenomenon has been observed for Salmonella 
. In addition, LPS was shown not to contribute to serum resistance directly. Accordingly, in the present work we observed binding of C4bp neither to O-ag nor to OC ().
YadA is a member of a large family of surface proteins of Gram-negative bacteria. These trimeric autotransporter proteins exert many functions and are required for full virulence of pathogenic species. Some of these proteins, such as Actinobacillus actinomyctemcomitans
Omp100, E. coli
EibD, Haemophilus ducreyi
DsrA and Moraxella catarrhalis
UspA1 and UspA2, confer resistance to serum 
. Interestingly, DsrA, UspA1 and UspA2 have been shown to capture C4bp 
. Apparently, C4bp-binding is a mechanism shared by multiple members of this family of autotransporters. The interaction between YadA and C4bp appeared to be ionic strength-dependent (). Interestingly, salt inhibited YadA-C4bp interaction similarly to that between C4bp and C4b 
. The fact that the positively charged cluster of amino acids between CCP1 and CCP2 is involved in C4b binding 
would suggest that these CCPs are needed also for the YadA-C4bp interaction. Heparin inhibition assay, however, showed discordant results (). The electronegative polysaccharide, heparin, alike C4b, binds to CCP1-2, and thus partially competes with C4b for C4bp binding 
. Heparin inhibition data showed an initial increase in C4bp binding to YadA at low heparin concentrations (). This could be theoretically explained by the binding of C4bp oligomers, formed in the presence of heparin, to YadA. Higher doses of heparin inhibited rather weakly C4bp binding to YadA, and 50% inhibition of the binding could only be observed at the highest heparin concentration of 1 mg/ml (). Thus, the C4bp binding sites for C4b and YadA do not seem to be identical, but most likely are overlapping. Electrostatic forces thus mediate the YadA-C4bp complex formation.
Ail belongs to a family of β-barrel outer membrane proteins that include Salmonella enterica
serovar Typhimurium PagC and Rck, and Enterobacter cloacae
. These proteins, though highly similar in structure, do not appear to share many of their functions. The only protein sharing serum resistance phenotype with Ail is Rck 
. Here we provided evidence for Ail-mediated C4bp binding. The mechanism of C4bp binding to Ail appeared to be different from that of C4b and YadA, as heparin efficiently and dose-dependently inhibited the binding of C4bp to Ail (). This observation suggested that Ail-binding involved the CCP1-3 domains of C4bp α-chain. The Ail-C4bp interaction was also less sensitive to salt when compared to that of YadA-C4bp () or C4b-C4bp interactions 
. Thus, other than electrostatic forces, e.g. hydrophobicity, could also be involved in this interaction. Ail binding sites on C4bp, however, are most likely not fully equivalent, though possibly overlapping, with those involved in C4b-C4bp or YadA-C4bp interactions.
, the causative agent of plague, has been shown to be resistant against complement-mediated killing and to bind C4bp 
. In contrast to Y. enterocolitica
, the surface protein Ail (also called OmpX) seems to be solely responsible for the serum resistance property in Y. pestis 
. Y. pestis
Ail protein shares about 70% sequence identity with Y. enterocolitica
Ail. As a result of several gene mutations, however, Y. pestis
does not express YadA or O-ag 
. Here we have shown that in YadA-negative Y. enterocolitica
strain, removing the Ail-masking O-ag and OC greatly enhances the binding of C4bp (). Based on these previous findings and our current results about Ail binding C4bp, it is probable that constitutively expressed Ail binds C4bp also on the surface of Y. pestis
and is, at least partly, responsible for the high complement resistance.
In summary, this study provides the first evidence that Y. enterocolitica acquires the CP regulator C4bp in a functionally active form able to promote degradation of C4b. The binding depends on the two outer membrane proteins YadA and Ail, the latter binding C4bp only when well surface-exposed, i.e., not blocked by O-ag or OC (). Y. enterocolitica is thus able to take advantage of the captured C4bp and is likely to be able to prevent both C4b-mediated opsonization and formation of the CP C3-convertase (C4bC2a). Consequently, the bacteria can avoid complement-mediated lysis and increase their chances to survive in the human host. It is also remarkable that Y. enterocolitica uses both YadA and Ail to recruit both the AP and CP regulators FH and C4bp, respectively. This way the pathogen can ascertain that it will be protected from complement activation during different phases of infection.
Schematic model of C4bp binding to Y. enterocolitica surface structures.