In the previous work, we identified a highly conserved glycine residue (G389) within the second beta-sheet of the YadA membrane anchor domain and demonstrated that it is involved in YadA autotransport and trimer stability and in YadA-mediated serum resistance when expressed in E. coli
). In the present study, we wanted to clarify two issues, as follows. (i) What is the impact of G389 on the YadA-associated in vitro
virulence functions of Y. enterocolitica
? To address this, we introduced the G389 substitution mutations G389A, G389S, G389T, G389N, and G389H into Y. enterocolitica
and tested the bacteria for YadA expression, trimer stability, trypsin accessibility, outer membrane localization, autoagglutination, adherence, host cell cytokine induction, serum resistance, and binding of the serum complement regulatory components factor H and C4BP. (ii) Are the in vitro
virulence assays performed with the YadA point mutants relevant to the virulence of Y. enterocolitica
in the mouse model? To elucidate this, we infected mice with Y. enterocolitica
wild-type bacteria and mutants expressing the YadA point mutants and compared bacterial burdens in several organs, serum cytokine levels, and morphological changes in the spleen.
The most salient finding of this study is that all YadA mutants displayed a reduction of trimer stability; however, their abilities to mediate autoagglutination, adherence, serum resistance, and virulence differed significantly in the various yadA
mutant strains (a summary of the phenotypes of all strains is given in Table ). Moreover, the data obtained by both the intravenous and orogastric mouse infection models suggest that only a few of the in vitro
virulence traits actually are closely associated with the virulence of Y. enterocolitica
in mice, thus challenging their meaningfulness. In fact, apparently only trimer stability and serum resistance, not the other in vitro
virulence traits, are closely associated with the virulence functions in vivo
. Finally, we can conclude that minor changes in the membrane anchor of YadA may directly or indirectly affect the complement resistance of Y. enterocolitica
, the key virulence function mediated by YadA. However, at present, we can only speculate about the molecular basis for the reduced virulence and serum resistance. The current YadA membrane anchor model is based on the Hia structure (19
) and suggests that at position G389, larger side chain residues could be accommodated. The reasons why and how the larger side chains disturb the protein's stability and autotransport function need further investigation. Thus, the molecular structure of the YadA membrane anchor needs to be elucidated either by X-ray crystallography or by nuclear magnetic resonance (NMR) experiments. From the high level of conservation of the Gly residue (19
), it can be assumed that the absence of a large side chain might be very important either in the folding process, in the autotransport process, or for final thermodynamic stability (or a combination of these factors). How mutations of this conserved residue finally may affect the recruitment of complement regulatory factors remains to be elucidated.
Summary of phenotypes of various mutantsa
In analyzing the expression of YadA, we were not able to detect the G389N, G389T, and G389H mutants by Western blotting of whole-cell lysates of Yersinia
. When the SDS gels were overloaded, occasionally very faint bands of YadA trimer or monomer could be observed in nonheated, but not heated, samples of the G389N, G389T, and G389H mutants (data not shown), suggesting that the G389N, G389T, and G389H mutants have reduced trimer stability and are readily degraded in Y. enterocolitica
. Nevertheless, we could demonstrate expression of the G389N, G389T, and G389H mutants by immunofluorescence microscopy and flow cytometry, indicating that these methods are more sensitive than Western blotting. In E. coli
, we were able to rescue expression of yadA
G389 mutants by knocking out the periplasmic protease DegP (19
). DegP is part of the periplasmic stress response and is responsible for degradation of misfolded proteins, including autotransporter proteins accumulating in the periplasm (22
). Analogous to our experiments with E. coli
, future experiments with a Y. enterocolitica
mutant strain will have to demonstrate whether the closely related periplasmic protease HtrA (28
) is involved in degradation of YadA G389 mutants.
YadA is the major adhesin of Y. enterocolitica
and has been demonstrated to mediate autoagglutination (43
). The exact mechanism of this phenomenon is not understood but is most likely due to the hydrophobic nature of the bacterial surface and due to homologous interaction of highly concentrated outer membrane proteins, as YadA-expressing bacteria readily settle out of a suspension. However, the role of autoagglutination per se
for virulence of Y. enterocolitica
is not known. Bacteria bearing the pYV virulence plasmid and expressing YadA in 3D collagen gels grow as densely packed microcolonies, while bacteria carrying the pYV plasmid but lacking YadA grow as loosely packed microcolonies (15
). There appears to be a correlation between the autoagglutination phenotype and growth behavior in collagen gels (15
). Formation of densely packed microcolonies, however, did not depend on the ability of YadA to bind collagen (15
). It is conceivable that densely packed microcolonies might protect bacteria from complement attack and from phagocytosis. The results of the present study clearly demonstrate that autoagglutination may also occur in the absence of YadA and that the autoagglutination of Y. enterocolitica
wild-type or mutant strains is not associated with the ability to exert a systemic infection in an experimental mouse infection model or to induce tissue abscesses including microcolonies in vivo
. In fact, Y. enterocolitica
WAP, WAP Inv−
, and YadAwt and the G389A and G389S mutants displayed autoagglutination, suggesting that YadA, but not invasin, contributes to this effect. The G389T, G389N, and G389H mutants showed no significant sedimentation, most probably due to the small quantity of YadA available for hydrophobic interactions. Y. enterocolitica
YadA0, however, displayed rapid autoagglutination in our hands. To rule out the possibility that this rather unexpected result emanated from our experimental setting, we repeated the experiment under serum-free conditions with minimal medium and LB but gained the same outcome. Therefore, we can exclude the possibility that autoagglutination of Y. enterocolitica
YadA0 is mediated by serum-originating components. We rather think that there are other factors on the surface of Y. enterocolitica
which are also capable of mediating autoagglutination and that these factors are masked by YadA. If there are only some residual molecules of YadA present, as in the G389T, G389N, and G389H mutants, then efficient interaction of these unknown autoagglutination factors is blocked. The identification of these factors is the subject of future experiments.
, YadA induces cytokine production (e.g., IL-8 production) in host cells (39
). This host cell response can be suppressed by injection of Yops via the type III secretion system into the host cells. To our surprise, a YadA-deficient strain also efficiently suppressed secretion of IL-8 by HeLa cells. Accordingly, none of our mutant strains, differing only in the amount of YadA presented on the surface, resulted in significant IL-8 secretion, suggesting that few adhering bacteria translocated enough effector proteins to exert inhibition of IL-8 secretion. Thus, in vitro
YadA does not play an essential role in Yop translocation if sufficient adherence is ensured. This result is in accordance with the observation that small amounts of YadA can mediate adhesion to host cells. Assuming that YadA is a major determinant for virulence and that a YadA-deficient Y. enterocolitica
strain might also be able to translocate Yop effectors efficiently in vivo
but is nevertheless severely attenuated, other YadA-mediated effects must be decisive for YadA-dependent survival of Y. enterocolitica
in the mouse model. Surprisingly, all of our mutant strains displayed comparable invasion behaviors. This might be explained by the fact that invasion reflects the net balance of adhesion (triggering uptake) and Yop injection (preventing uptake by disrupting the host cell actin cytoskeleton). Although we observed significantly reduced adhesion to HeLa cells with some of our mutants, these strains seemed to have injected enough Yops to efficiently suppress internalization.
The action of complement is one of the first host immune barriers to invading Y. enterocolitica
. Complement resistance is thus a crucial feature of invasive, extracellularly located pathogens such as Y. enterocolitica
. Consequently, numerous pathogenic microorganisms have developed mechanisms to evade the complement system (26
). An important function of YadA is the prevention of membrane attack complex (MAC) formation on the bacterial surface. Y. enterocolitica
evades the complement system by the recruitment of two major complement regulatory proteins, namely, factor H and C4BP. Factor H prevents C3 deposition on the bacterial surface in vitro
by inhibiting the binding of factor B to C3b, by supporting the dissociation of the C3bBb complex (decay accelerating activity), and by its function as a cofactor for the cleavage of C3b by factor I (1
). C4BP is a fluid-phase complement regulator that downregulates classical and lectin pathway complement activity by preventing the assembly and accelerating the decay of the C3 convertase.
It has been shown in vitro
that both the stalk and the membrane anchor are involved in serum resistance (34
). By means of the Y. enterocolitica
YadA G389 mutants, we were able to test whether the quantity of YadA is decisive for complement resistance. Serum resistance levels of Y. enterocolitica
WAP and Y. enterocolitica
YadAwt were comparable. All of the other mutant strains had significantly reduced serum resistance, irrespective of the amount of YadA displayed on the outer membrane. Therefore, it is assumed that the exchange of G389 abrogates efficient interaction of YadA with complement regulators and thus leads to complement killing of Y. enterocolitica
. Obviously, this complement regulatory factor (CRF) is neither factor H nor C4BP, because both are bound to almost wild-type levels by the G389A mutant and at least factor H exhibits full cofactor activity when bound to this strain. Nevertheless, interaction with other CRFs could be disturbed by modulation of binding to YadA. It has been shown for Ail that single point mutations within loop 2, which is exposed on the surface, can abrogate serum resistance (31
). Studies by Ackermann et al. (1
) support a scenario where specific interaction between the C terminus of YadA and complement inhibitory factors is inhibited. Analyses of serum resistance of Y. enterocolitica
strains expressing no YadA, wild-type YadA, or chimeras revealed that the exchange of the YadA C terminus with that of UspA1 (Moraxella catarrhalis
), EibA (Escherichia coli
), or Hia (Haemophilus influenzae
), respectively, resulted in a significant loss of survival in normal human serum. These results demonstrate that the C-terminal membrane anchor domain is involved in serum resistance either directly or indirectly, a finding also supported by this study.
Systemic infection of mice with Y. enterocolitica YadAwt and the G389 mutant strains revealed that only YadAwt was highly pathogenic in vivo and was associated with a high splenic bacterial burden, abscess formation, and high CXCL1 serum levels, while Y. enterocolitica YadA0 and the YadA G389T, G389N, and G389H mutants did not induce significant changes. Both the YadA G389A and G389S mutants were attenuated but retained the ability to induce abscesses and had increased CXCL1 serum levels. Thus, Y. enterocolitica YadA0 and the YadA G389T, G389N, and G389H mutants are rapidly killed by the first line of host defense (most likely complement), are not able to establish abscess formation, and do not lead to major inflammatory events, including cytokine production. The YadA G389A and G389S mutants cannot be killed as efficiently, probably due to their still-present, though reduced, serum resistance. In an orogastric mouse infection model, Y. enterocolitica YadAwt and the G389A and G389S mutants were able to colonize the Peyer's patches even though the YadA G389A and G389S mutants were found in slightly reduced numbers. Interestingly, only Y. enterocolitica YadAwt, not the G389A and G389S mutants, was found in mesenteric lymph nodes and could disseminate into the spleen at the time points investigated. Although this may be the result of (i) less efficient uptake by M cells, (ii) more efficient killing by the host, or (iii) delayed bacterial growth within the host tissue, scenario ii might be the most tempting one and will be pursued by exploring the virulence of YadA mutants in mice deficient in complement system components.
In summary, we demonstrated that exchange of the highly conserved single amino acid glycine within the YadA membrane anchor abrogates serum resistance. Moreover, virulence tests in the mouse model have shown that YadA-mediated serum resistance is decisive for Y. enterocolitica virulence, rather than other YadA-mediated functions, such as adhesion, which were demonstrated in vitro. These data suggest that in vivo assays provide the most sensitive assays to address minor changes in virulence factors.