Unlike other toxins, CyaA has been considered for a long time as independent of any receptor binding. This was based on the observations that CyaA can intoxicate a wide variety of cell lines from various origins 14
and binds to Jurkat cells and sheep erythrocytes in a nonsaturable fashion 44
. These observations established that nonspecific adsorption of CyaA to lipid membranes leads to some translocation of the catalytic domain into the cytosol. However, they did not rule out the existence of a specific receptor. Indeed, in vivo studies showed that during murine respiratory infection with Bordetella
species, CyaA specifically destroyed leukocytes (especially macrophages) without dramatically damaging epithelial cells 631
. In this study, we hypothesized that this specificity might be linked to the recognition of a receptor specifically expressed by certain leukocytes. We showed, using myeloid cell lines, that the binding and the toxic properties of CyaA are dependent on its interaction with the integrin CD11b/CD18. Efficient and saturable binding of CyaA correlates with the expression of CD11b and is fully and specifically blocked by anti-CD11b mAbs. Moreover, expression of CD11b/CD18 in CHO cells dramatically enhances the binding of CyaA and its cytotoxicity. Our results are the first evidence supporting the interaction of CyaA with a cell-surface molecule specifically expressed on leukocytes. The nearly complete inhibition of CyaA binding by anti-CD11b mAbs and anti-CD18 for human cells suggests that CD11b/CD18 is the main receptor for CyaA in the cell lines tested. The lack of efficient binding to CD11c/CD18 transfectants, or CD11a/CD18 expressing cells such as EL4 or LB27.4, also suggests that CD11b/CD18 is the only integrin of the β2 family involved in the binding of CyaA to the target cells. However, the weak binding of CyaA to LB27.4 cells may suggest an ancillary role for CD11d 45
or another surface ligand.
This study clearly shows that the interaction of CyaA with CD11b+
cells is strictly dependent on Ca2+
, as could be expected since CyaA is a calcium-binding protein 18
ions bind to the RTX repeat motifs with a weak affinity and trigger a conformational change that allows the toxin to bind to target cell membranes and to translocate its catalytic domain into the cytosol 131822
. More surprisingly, our study also established that the binding of CyaA to CD11b+
cells is independent of Mg2+
. To our knowledge, most known ligands (fibrinogen, iC3b, intercellular adhesion molecule 1) of CD11b/CD18 bind to the I domain of CD11b in a Mg2+
) dependent fashion 43
. Structural and functional analyses have shown that the integrin I domain contains a Mg2+
coordination site at its surface (MIDAS) that is critical for ligand binding 464748
. Therefore, CyaA might be the first example of a novel class of Mg2+
independent CD11b ligand. These results also suggest that CyaA binding to CD11b/CD18 might occur independently of the CD11b I domain. Indeed, regions outside the I domain have been shown to contribute partially to the binding of some proteinaceous ligands like factor X 49
. Moreover, CD11b/CD18 displays other binding sites like the well-characterized lectin-like binding site involved in the binding of β-glucans 50
and possibly in the lateral association of CD11b/CD18 with FcγRIII 51
Experiments are currently underway to map the regions of interaction between CyaA and CD11b/CD18. At present, we can only exclude the possibility of CyaA binding via an arginine-glycine-aspartate motif since CyaA does not contain such a sequence and an argine-glycine-aspartate peptide does not inhibit CyaA binding to CD11b+ cells (data not shown).
In line with previous studies (for a review, see reference 14
), we observed a detectable binding of CyaA to all cell lines tested. Furthermore, at high concentrations CyaA triggered a small but detectable cAMP increase in mock-transfected CHO cells that is not associated with cell lysis. Thus, although at high concentration CyaA can bind and enter into a wide variety of cell lines, at low concentrations only CD11b-expressing cells will be intoxicated by CyaA.
Previous studies, performed mainly on erythrocytes (devoid of CD11b) led to a two-step model for translocation of CyaA across membranes 22
. First CyaA binds to the membrane via its COOH-terminal moiety without any receptor involvement, then the NH2
-terminal catalytic domain translocates across the plasma membrane to the cytosol 22
, coincidentally with a conformational change triggered by Ca2+
binding to the RTX repeats located in the COOH-terminal domain of CyaA 18
. The interaction between CyaA and CD11b/CD18 could favor the membrane insertion of CyaA, or, alternatively, CyaA may bind first to lipid membrane, and then CD11b may participate in stabilizing its interaction with membranes, allowing a more efficient intoxication.
The ability to translocate directly across the plasma membrane is a peculiarity of CyaA, which is considered to be the only toxin known to reach the cytoplasm without going through vesicular trafficking. Indeed, after binding to a specific receptor at the cell surface, other toxins acting on cytosolic targets are taken up together with their receptor by endocytic pathways. Some toxins like the Bacillus anthracis
lethal factor or the diphtheria toxin translocate into the cytosol from acidic vesicles of the endocytic pathway. Others like the Shiga
toxin, the cholera toxin, or the Pseudomonas
exotoxin A are routed via a retrograde endocytic pathway to the endoplasmic reticulum from which they reach the cytosol 52
. Unlike CD11a/CD18, which does not undergo endocytosis efficiently and may retain RTX toxins at the membrane of the cell, CD11b/CD18 can be internalized by endocytosis 5354
. This calls into question the physiological relevance of the current model of direct translocation of CyaA from the plasma membrane to the cytosol. Current experiments are in progress to determine whether CyaA is taken up by endocytosis together with CD11b and to characterize the precise location of the membrane translocation events.
Binding of CyaA to a member of the β2 integrin family is reminiscent of the behavior of other RTX toxins which were recently found to interact with these molecules 323334
. The E. coli
HlyA, which shares a strong homology with CyaA, forms cationic pores at the plasma membrane. HlyA exhibits a specificity for leukocytes but only at a low concentration 55
. This relative specificity was shown to be mediated by its interaction with the integrin CD11a/CD18 32
. Besides that, Actinobacillus actinomycetemcomitans
and Pasteurella haemolytica
leukotoxins (LtxA and LktA, respectively) are RTX toxins specific for human and bovine leukocytes, respectively, but also interact with CD11a/CD18 323334
. Despite its strong homology with HlyA, CyaA recognizes another β2
integrin (CD11b/CD18) whose cellular distribution is more restricted. Indeed, CD11b is expressed mostly on macrophages, neutrophils, and dendritic cells, but not on the majority of T and B cells, whereas CD11a is expressed on all leukocytes including T and B lymphocytes.
The restricted specificity is in agreement with the effect of CyaA in the early phases of infection on bactericidal phagocytes such as macrophages 629
and neutrophils 31
. Such specific targeting of CyaA toxicity to CD11b-expressing cells may represent an escape mechanism against the early steps of the innate immune response. Indeed, this could allow the specific elimination of these cells 62930
and/or eventually the inhibition of their oxidative microbicidal functions in the case of sublethal intoxication 132526
. The killing of lung dendritic cells, which are known to express CD11b, may also have indirect effects on the development of protective adaptative responses. Finally, the specificity of CyaA for CD11b+
cells reinforces the hypothesis that the effect of CyaA on immune effector cells participates in the inhibition of antibacterial responses to bystander pathogens during B. pertussis
infection as originally proposed by Confer and Eaton 13
The filamentous hemagglutinin (FHA) of B. pertussis
has also been shown previously to interact with CD11b/CD18 56
leading to the internalization of a subset of bacteria during the course of infection 57
. Moreover, FHA also interacts with a complex containing the leukocyte response integrin and the integrin-associated protein CD47 that is known to upregulate the expression of CD11b/CD18 58
. It is striking that such different factors, both in terms of structure and function, target the same molecule on leukocytes. One could imagine that they may cooperate to efficiently deplete the CD11b/CD18-expressing leukocytes. FHA may, for example, act first to bind to cells and facilitate the interaction of CyaA with this receptor. Such a hypothesis was suggested previously in a study showing that both CyaA and FHA are necessary to inhibit monocyte function 59
. Therefore, the interplay between FHA and CyaA remains to be determined.
This selective binding of CyaA to CD11b-expressing cells might be exploited to specifically target this particular subset of cells. Detoxified mutants of CyaA might be used to deliver pharmacologically active molecules to CD11b+
cells, without noticeably affecting other cell types. In fact, we recently developed such an approach by genetic insertion into detoxified CyaA of viral 60
or tumoral peptides 61
. Once delivered into the cytosol of APCs and after proteasome-dependent intracellular processing, the inserted epitopes are presented by MHC class I molecules to CD8+
T cells 62
and in vivo, stimulate protective CTL responses against viral challenge 60
or tumor graft 61
. The present results highlight the strong potential of delivery vectors based on such CyaA detoxified mutants.