The major goal of this work was to examine the hypothesis that the interaction of LcrV and TLR2 contributes significantly to Y. pestis
virulence, whether via the induction of IL-10 or by any other means. In the most direct interpretation, this model predicts that TLR2−/−
mice will show (i) enhanced resistance to Y. pestis, (ii) enhanced inflammation at foci of infection, and (iii) decreased IL-10 and enhanced proinflammatory cytokine production during Y. pestis
infection. The results of our experiments with TLR2−/−
mice did not confirm these predictions. We observed no significant differences in survival, mean time to death, or bacterial load; no evidence of enhanced inflammation at foci of infection; and an increase, rather than a reduction, in IL-10 titers. We did observe a slight increase in TNF titer, but this occurred in the presence of enhanced rather than reduced IL-10 levels. The other proinflammatory cytokine measured, IL-6, showed no change. In comparison with survival experiments, small numbers of animals were utilized for bacterial titer and cytokine measurements, and we may have failed to observe modest differences. For example, for bacterial titers and IL-6 titers, the two measurements for which no significant differences were observed, power analysis using a sample size of five and the measured variances of our observations yields an 80% probability of detecting differences of about twofold relative to the mean values, with a correspondingly lower probability of detecting smaller differences. Another limitation of these experiments is that measurements were made at a single time point, 2 days postinfection, and we cannot be certain that our results reflect conditions pertaining at earlier stages of infection. However, both absolute survival and survival kinetics were remarkably consistent at both doses tested, indicating that any influence of TLR2 on the course of infection must be minor. It should be noted that the virulent Y. pestis
-mouse infection model is a very sensitive one, in that specific genetic modifications of bacteria or mice often have large effects on virulence. For example, otherwise virulent mutants lacking the Pla protease (27
) and mutants with defects in iron acquisition (31
) show an increase in 50% lethal dose of several orders of magnitude, as do strains modified to produce highly stimulatory LPS (15
). TLR4-deficient mice are highly susceptible to a strain producing stimulatory LPS, while wild-type mice are highly resistant (15
). In both of these instances, reduced virulence (or enhanced resistance) was also associated with marked enhancement of inflammation at foci of infection. Thus, it is clear that experimental manipulations in this system do indeed have very large effects when they are related to functional differences in the interaction between the bacteria and host defenses. Our failure to observe any indication of enhanced resistance or improved inflammatory response in TLR2 deficient mice must therefore be regarded as strong evidence that this receptor does not play a significant role in interactions contributing to virulence of Y. pestis
during infection. Because we did not carefully examine the progress of infection at multiple time points, it remains possible that there are subtle differences in the development of infection between the genotypes that do not affect survival or time to death.
The literature regarding the induction of IL-10 by the LcrV of the yersiniae presents a somewhat confusing picture. Sing et al. reported that specific residues are required for induction of IL-10 by LcrV of Yersinia enterocolitica
and that peptides containing these residues are effective inducers (23
). However, they also found that a peptide from the cognate region of Y. pestis
and Y. pseudotuberculosis
LcrV has little activity (23
). On the other hand, Overheim et al. reported that regions of LcrV entirely distinct from that defined by Sing et al. are required for IL-10 induction by Y. pestis
). While Sing et al. provided evidence from a well-established in vivo model supporting a role for the TLR2-LcrV interaction during Y. enterocolitica
), these results are unfortunately dependent on the mouse strain employed (26
). No similar evidence has been published previously regarding Y. pestis
. For this species, currently available in vivo data are indirect in that all the relevant experiments involve injection of mice with various forms of rLcrV, followed by measurement of cytokine levels and/or challenge with LPS, attenuated Y. pestis
, or other unrelated pathogens (18
). While the differences in IL-10 levels that we observe are not consistent with the LcrV-TLR2 model (levels were not reduced in TLR2-deficient animals), IL-10 is clearly elevated in mice with well-developed Y. pestis
infection. Thus, we cannot rule out the possibility that immunosuppression due to elevated IL-10 levels induced by a TLR2-independent mechanism plays an important role in plague. Indeed, it is possible that the unusual combination of proinflammatory stimuli presented by Y. pestis
, which as we have shown previously activates TLR4 very poorly (15
), results in an aberrant cytokine profile that may compromise innate defenses.
A variety of fusion proteins have been used to demonstrate the immunosuppressive properties of LcrV. For example, Overheim et al. utilized an N-terminal decahistidyl tag (22
). Motin et al. fused a 34-kDa fragment of protein A to the N terminus of a truncated LcrV lacking the first 67 residues (16
). The three-dimensional structure of LcrV was determined from a fusion protein containing five residues fused to an LcrV N terminus beginning at residue 28 and a C-terminal addition of four residues plus a hexahistidyl tag (6
). This structure shows that both the N and C termini are very flexible and are located near each other, external to one of the LcrV globular domains. The flexibility and location of these termini are consistent with tolerance for additions and deletions. Thus, it is unlikely that the three-residue N-terminal and the eight residue C-terminal additions (the latter including a six-residue His tag) present in our LcrV construct are less reflective of the properties of native LcrV than those employed by others. Moreover, we have shown directly that the rLcrV protein of Overheim et al. (22
) behaves similarly to our own. It should be noted that the cytokine-inducing properties of native LcrV purified from Y. pestis
or any other Yersinia
species have not been studied.
Our results with His-tagged Y. pestis rLcrV are consistent with those of others in that we do observe stimulation of TLR2 in vitro. However, we also show that the ability of this material to stimulate TLR2 is unexpectedly complex at the biochemical level. The major forms of rLcrV in our preparations, dimer and tetramer, have no TLR2-stimulating activity. Such activity is detected only in high-molecular-weight multimers or aggregates. Although the data from experimental infections discussed above argue strongly against a role for this activity in Y. pestis virulence, we consider three alternative hypotheses regarding TLR2 stimulation by LcrV, one of which implies physiological significance.
First, the stimulatory activity may result from the presence of a potent TLR2-activating contaminant (e.g., a lipoprotein, lipopeptide, peptidoglycan, etc.) constituting a small proportion of the aggregate and not from rLcrV per se. Such contamination is both common in material purified from whole-cell extracts and notoriously difficult to exclude. TLR2-stimulating activity initially attributed to what were thought to be highly purified materials has later been shown to result from such contamination (for example, see references 10
, and 33
). There is no general method to ensure freedom from such contaminants in protein preparations. Note that vulnerability to such contaminants is greatly increased when cells expressing a variety of TLRs, such as mouse macrophages, are the targets of stimulation.
A second possibility is that the stimulatory activity is indeed due to rLcrV in the aggregates/multimers but that this material is nonphysiological and is an artifact of overexpression and purification techniques. The tendency of recombinant proteins expressed at high levels in E. coli to form high-molecular-weight aggregates is well established. Exposure of mature rLcrV to cell extracts, as occurs during purification, could also be critical to formation of the stimulatory material. In Y. pestis the level of LcrV expression is much lower, and exposure of mature LcrV to concentrated cell extracts does not occur. In this view, the stimulatory aggregates/multimers are purely an in vitro artifact and have no physiologic relevance.
A third possibility is that the stimulatory fractions contain structures that are, or resemble, physiological multimeric species that are normally detected by TLR2 as an indicator of pathogens with TTSS machinery. Micrographs of LcrV arranged at the tips of secretion needles (17
) suggest to us an octamer composed of four dimers. Perhaps such structures are occasionally released from bacteria in vivo and elicit proinflammatory responses via TLR2. However, there is no evidence to support this idea from infection experiments with yersiniae. Also, PcrV, a related protein from Pseudomonas aeruginosa
, does not stimulate TLR2 (25
The association of TLR2 stimulation with aggregates/multimers of rLcrV suggests that individual rLcrV molecules may interact weakly with TLR2 and that activation results from clustering of the receptor. This would explain the failure of low-molecular-weight forms to cause activation. Other groups have not reported the molecular sizes of the active fractions in their rLcrV preparations under nondenaturing conditions. Consequently, the presence of a similar stimulatory high-molecular-weight aggregate in their experiments cannot be excluded. The specific activity of our preparations is similar to that reported by others; if the activity that they observe is not due to aggregates, which we find to constitute a small fraction of total protein, then the activity of rLcrV in their preparations on a per-molecule basis must be correspondingly low. The clustering hypothesis suggests that rather than disrupting a specific TLR2-LcrV interaction, mutations which give rise to inactive rLcrV preparations may interfere with formation of aggregates capable of activating TLR2.
Overheim et al. (22
) have presented data suggesting that LcrV deletion mutants failing to stimulate IL-10 production are more effective immunogens, presumably due to the elimination of immunosuppressive activity. Our results show that highly purified LcrV preparations containing only dimers and/or tetramers lack TLR2-stimulating activity and hence may also have improved performance in vaccine applications. However, we have previously shown that with DNA vaccines, production of LcrV multimers in vivo was critical to providing an effective protective response and also biased the response toward TH1 compared with the response produce by constructs producing only rLcrV monomers (30
). While we do not know the extent of multimerization occurring in vivo, the association of multimerization with TLR2 stimulation suggests the possibility that large multimers form in vivo and provide adjuvant activity in the context of the live vaccine through stimulation of TLR2, rather than immunosuppression. This adjuvant effect is more consistent with current understanding of TLR2 function than is an immunosuppressive effect.
In summary, our investigation provides no support for the hypothesis that activation of TLR2 by LcrV contributes to the virulence of Y. pestis
via immunomodulation. In a sensitive infection model using virulent Y. pestis
, elimination of TLR2 has no effect on the course of disease and little on cytokine levels observed in vivo. The bulk of rLcrV protein has no TLR2-stimulating activity in vitro, and such activity is restricted to high-molecular-weight aggregates/multimers which contain LcrV but are of undetermined composition. Given the well-established sensitivity of the Y. pestis
mouse infection model, its lack of response to TLR2 deficiency must be regarded as strong evidence that TLR2-induced immunomodulation does not have a significant role in plague. The early observations suggesting a direct immunosuppressive role for LcrV were based on direct injection of rLcrV preparations into mice, resulting in immunosuppression and elevated levels of IL-10 (19
). A TLR2-independent mechanism of IL-10 induction would be consistent with these early observations.
Detailed infection experiments have also been conducted with Yersinia enterocolitica
and Yersinia pseudotuberculosis
by Victoria Auerbuch and Ralph Isberg (2a
). They also observed no differences in the course or pattern of disease, or in cytokine levels, between TLR2-sufficient and -deficient mice. The conflicting results of Sing et al. imply that LcrV-TLR2-mediated immunosuppression may operate under certain limited circumstances (i.e., with specific combinations of Y. enterocolitica
strains and mouse strains), but, given the present weight of evidence, it is unlikely to be a phenomenon of general importance to virulence in the yersiniae.