Commensal microbes drive intestinal inflammation by IL-17–producing CD4+ T cells through ICOSL and OX40L costimulation in the absence of B7-1 and B7-2

The costimulatory B7-1 (CD80)/B7-2 (CD86) molecules, along with T-cell receptor stimulation, together facilitate T-cell activation. This explains why in vivo B7 costimulation neutralization efficiently silences a variety of human autoimmune disorders. Paradoxically, however, B7 blockade also potently moderates accumulation of immune-suppressive regulatory T cells (Tregs) essential for protection against multiorgan systemic autoimmunity. Here we show that B7 deprivation in mice overrides the necessity for Tregs in averting systemic autoimmunity and inflammation in extraintestinal tissues, whereas peripherally induced Tregs retained in the absence of B7 selectively mitigate intestinal inflammation caused by Th17 effector CD4⁺ T cells. The need for additional immune suppression in the intestine reflects commensal microbe-driven T-cell activation through the accessory costimulation molecules ICOSL and OX40L. Eradication of commensal enteric bacteria mitigates intestinal inflammation and IL-17 production triggered by Treg depletion in B7-deficient mice, whereas re-establishing intestinal colonization with Candida albicans primes expansion of Th17 cells with commensal specificity. Thus, neutralizing B7 costimulation uncovers an essential role for Tregs in selectively averting intestinal inflammation by Th17 CD4⁺ T cells with commensal microbe specificity.Immune activation is stringently controlled to balance mobilization of protective components while simultaneously silencing detrimental responses that cause harm to host tissues. One means of regulation is the additional necessity for B7-1 (CD80)/B7-2 (CD86) costimulatory signals, along with T-cell receptor stimulation, in T-cell activation (1). Reciprocally, soluble recombinant formulations of the natural high-affinity B7 ligand—cytotoxic T-lymphocyte antigen 4 fused with human Ig (CTLA4-Ig), which blocks B7 costimulation—are efficacious in neutralizing aberrant T-cell activation in autoimmune disorders such as rheumatoid arthritis and juvenile idiopathic arthritis (2). Ongoing studies suggest that these therapeutic benefits also extend to many other types of autoimmunity including psoriasis, systemic lupus erthematosus, multiple sclerosis, and type 1 diabetes (3–6). Interestingly, however, the protective benefits of B7 blockade are not universal as CTLA4-Ig is distinctively nonefficacious for inflammatory bowel disease (7) and can induce intestinal inflammation among individuals with unrelated autoimmune disorders (8).Given that B7 costimulation required for T-cell activation also sustains accumulation of immune-suppressive regulatory T cells (Tregs) essential for averting fatal systemic autoimmunity (9, 10), this discordance in therapeutic efficacy with B7 blockade may reflect tissue-specific differences in necessity for Treg suppression in the absence of B7 costimulation. Here, unique features of the intestine that include high-density commensal bacteria colonization or expression of the accessory costimulatory molecules ICOS ligand (ICOSL) or OX40 ligand (OX40L) may foster susceptibility to inflammation despite B7 deprivation (11–15). To investigate these possibilities, the interplay between Tregs and B7, ICOSL, and OX40L costimulation in autoimmunity was evaluated after targeted ablation of each individually or concurrently. Our results show that B7 deprivation overrides the necessity for Tregs in averting systemic autoimmunity and inflammation in extraintestinal tissues, whereas enteric commensal microbes drive inflammation restricted to the intestine through ICOSL/OX40L stimulation when Tregs and B7 are simultaneously eliminated. These results illustrating persistent intestinal inflammation despite B7 deprivation may explain why inflammatory bowel disease, compared with other forms of autoimmunity, is distinctively resistant to B7 blockade.     

Seminal CD38 is a pivotal regulator for fetomaternal tolerance

A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4⁺ forkhead box P3⁺ (Foxp3⁺) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38^−/− males was high compared with that in females mated with Cd38^+/+ males, and this was associated with a reduced proportion of Tregs within the CD4⁺ T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.Seventy-five percent of pregnancies that are lost represent failure of implantation and are therefore not clinically recognized as pregnancies (1). Recurrent miscarriage (the spontaneous loss of three or more consecutive pregnancies) is a significant health issue for 1–2% of women, with no identifiable biological cause and no effective treatment. During early stages of pregnancy, complex processes help to create a uterine environment that is conducive to a successful pregnancy. These include immunological adaptation to the semiallogeneic fetus. Tolerance to paternal alloantigens is critical for successful reproduction in placental mammals (2, 3). Many studies have proposed that regulatory T cells (Tregs) play an essential role in the development of fetomaternal tolerance in mice and humans (4–7). Seminal plasma contains potent immunoregulatory molecules that contribute to the induction of tolerogenic DCs (tDCs) and ultimately Treg expansion, which is necessary to establish maternal tolerance against paternal antigens (8–10). However, the specific molecules in semen that are responsible for expansion of Tregs and establishment of maternal tolerance remain undefined.CD38, a mammalian prototype of ADP ribosyl cyclases (ADPRCs), is a type II transmembrane (TM) glycoprotein expressed in many cell types and seminal fluid (11–16). CD38 produces calcium-mobilizing second messengers, cyclic ADP ribose, and nicotinic acid adenine dinucleotide phosphate (11, 12). We previously showed that intact CD38 in prostasomes assists progesterone-induced sperm Ca²⁺ signaling (13). In addition to its enzymatic role for Ca²⁺ signaling, CD38 may also have a nonenzymatic role through its interaction with CD31 (17, 18). CD31, a type I TM homophilic or heterophilic receptor, is expressed in endothelial cells and a variety of immune cells (19) and is involved in attenuating the inflammatory response in a variety of inflammatory diseases (20–23). For example, recombinant CD38 inhibits LPS-induced inflammatory signals in mouse macrophages and human DCs through an interaction with CD31 (24, 25).In this study, we found that CD38 is truncated and released into the seminal plasma from seminal vesicles (SVs) as a soluble form (sCD38) in humans and mice. This finding prompted us to examine whether CD38 plays a role in maternal immune tolerance during pregnancy. We found that sCD38 present in seminal plasma was crucial for the induction of uterine tDC and Tregs, which are responsible for the development of the fetomaternal tolerance.     

Inflammatory monocytes are potent antitumor effectors controlled by regulatory CD4+ T cells

The present study evaluates the impact of immune cell populations on metastatic development in a model of spontaneous melanoma [mice expressing the human RET oncogene under the control of the metallothionein promoter (MT/ret mice)]. In this model, cancer cells disseminate early but remain dormant for several weeks. Then, MT/ret mice develop cutaneous metastases and, finally, distant metastases. A total of 35% of MT/ret mice develop a vitiligo, a skin depigmentation attributable to the lysis of normal melanocytes, associated with a delay in tumor progression. Here, we find that regulatory CD4⁺ T cells accumulate in the skin, the spleen, and tumor-draining lymph nodes of MT/ret mice not developing vitiligo. Regulatory T-cell depletion and IL-10 neutralization led to increased occurrence of vitiligo that correlated with a decreased incidence of melanoma metastases. In contrast, inflammatory monocytes/dendritic cells accumulate in the skin of MT/ret mice with active vitiligo. Moreover, they inhibit tumor cell proliferation in vitro through a reactive oxygen species-dependent mechanism, and both their depletion and reactive oxygen species neutralization in vivo increased tumor cell dissemination. Altogether, our data suggest that regulatory CD4⁺ T cells favor tumor progression, in part, by inhibiting recruitment and/or differentiation of inflammatory monocytes in the skin.The concept of suppressive T cells, which was first described in the early 1970s, remained poorly investigated until Sakaguchi et al. demonstrated the suppressive functions of a subset of CD4⁺ T cells now called regulatory T cells (Tregs) (1). Tregs express Foxp3 (2) and high surface levels of the α-chain of the IL-2 receptor (CD25) and are the main mediators of peripheral tolerance under physiological settings (1). Their role in the suppression of antitumor immunity was originally described in the 1980s (3) but remained, at first, largely underestimated. The demonstration that systemic depletion of Tregs favors tumor rejection in mouse models highlighted their contribution in tumor progression (4). Whereas it is established that Tregs potently interfere with tumor-specific T cells (5–8), their impact on innate immune cells, in particular, on myeloid cells, has been clearly less investigated in the context of tumor development (9, 10).To decipher the role of Tregs in the relationship between cancer immunity and autoimmunity, we used mice expressing the human RET oncogene under the control of the metallothionein promoter (MT/ret mice) (11). In this model, the primary uveal tumor disseminates early but remains dormant for several weeks (12, 13). MT/ret mice then develop cutaneous metastases and finally distant (i.e., pulmonary, visceral, and mediastinal adenopathy) metastases (14). A significant proportion of MT/ret mice spontaneously develops vitiligo-like depigmentations. The tumor progression is significantly delayed in mice harboring vitiligo compared with mice without depigmented skin (14). The development of vitiligo in melanoma patients has also been associated with a better prognosis (15–18). It occurs most often in immunotherapeutic trials, although vitiligo may also develop in patients treated with surgery, radiotherapy, or chemotherapy.Consistent with previous data obtained in melanoma patients (19, 20), we found that Tregs accumulated in tumor-draining lymph nodes (TdLNs) of MT/ret mice. Treg depletion led to a decreased occurrence of cutaneous metastases and an increased percentage of mice developing vitiligo. Interestingly, inflammatory (Ly-6C^high) monocytes and dendritic cells (DCs), initially described as important cells during inflammatory and infectious processes, appeared to be critical effectors during the early phase of the antitumor response, in particular by killing disseminated malignant melanocytes. Altogether, our results suggest that Tregs prevent the recruitment and/or differentiation of Ly-6C^high monocytes in the skin, subsequently favoring tumor progression.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Induction of autoimmune disease by deletion of CTLA-4 in mice in adulthood

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is essential for immunological (self-) tolerance, but due to the early fatality of CTLA-4 KO mice, its specific function in central and peripheral tolerance and in different systemic diseases remains to be determined. Here, we further examined the role of CTLA-4 by abrogating CTLA-4 expression in adult mice and compared the resulting autoimmunity that follows with that produced by congenital CTLA-4 deficiency. We found that conditional deletion of CTLA-4 in adult mice resulted in spontaneous lymphoproliferation, hypergammaglobulinemia, and histologically evident pneumonitis, gastritis, insulitis, and sialadenitis, accompanied by organ-specific autoantibodies. However, in contrast to congenital deficiency, this was not fatal. CTLA-4 deletion induced preferential expansion of CD4⁺Foxp3⁺ Treg cells. However, T cells from CTLA-4–deficient inducible KO mice were able to adoptively transfer the diseases into T cell-deficient mice. Notably, cell transfer of thymocytes de novo produced myocarditis, otherwise not observed in donor mice depleted in adulthood. Moreover, CTLA-4 deletion in adult mice had opposing impacts on induced autoimmune models. Thus, although CTLA-4–deficient mice had more severe collagen-induced arthritis (CIA), they were protected against peptide-induced experimental autoimmune encephalomyelitis (EAE); however, onset of protein-induced EAE was only delayed. Collectively, this indicates that CTLA-4 deficiency affects both central and peripheral tolerance and Treg cell-mediated suppression.Cytotoxic T lymphocyte antigen-4 (CTLA-4), a member of the CD28 family, is a vital coinhibitory molecule, up-regulated by activated conventional T cells (Tconv cells) and constitutively expressed by Foxp3⁺ Treg cells (1–3). Germ-line haploinsufficiency of CTLA-4 causes lymphoproliferation and autoimmunity in humans (4, 5), and CTLA-4 polymorphisms are associated with several human autoimmune-disorders, including Graves disease (6), systemic lupus erythematosus (7), type I diabetes (8), and rheumatoid arthritis (6, 9). Moreover, anti–CTLA-4 antibody (Ab) treatment enhances tumor immunity in metastatic melanoma patients (10) but also elicits autoimmunity (11, 12). In mice, Ctla4 germ-line deficiency produces a lymphoproliferative disorder hallmarked by multiorgan lymphocyte infiltrations, especially in the heart and pancreas, that is lethal 3–4 wk after birth (13, 14). Moreover, CTLA-4 deficiency specifically in Foxp3⁺ Treg cells is sufficient to cause lymphoproliferation and autoimmune diseases including myocarditis, which is fatal at around 8 wk of age. It also leads to enhanced tumor immunity in vivo and abrogated suppressive function induced by allo-antigen in vitro. These findings collectively indicate CTLA-4 as a key molecule for Treg cell-mediated suppression (15). However, recent reports have shown that ctla-4–deficient Treg cells exert effective suppression in mouse models of colitis and experimental autoimmune encephalomyelitis (EAE), whereas they failed to be suppressive in an adoptive transfer model of type I diabetes (16–19). These apparently conflicting results suggest that the role of CTLA-4 might vary depending on disease condition.CTLA-4 has been shown to control immune activation via both cell autonomous and nonautonomous pathways (20). Autonomous mechanisms include among others internal signaling that leads to cell cycle arrest (21, 22), as well as competition with CD28 for interaction with CD80 and CD86 on antigen presenting cells (APCs) (23, 24). In contrast, several studies show that CTLA-4–sufficient T cells, primarily Treg cells, can keep other T cells inert (1, 25–28). This nonautonomous CTLA-4–mediated regulation is at least partly dependent on control of the antigen presenting capacity of the APCs by CD80 and CD86 modulation (29, 30) or by induction of the immune suppressive agent kynurenine (31). Apart from peripheral tolerance, it is controversial whether CTLA-4 has a role in central tolerance and selection of thymic T cells and Foxp3⁺ Treg cells. Some studies have not detected any differences (19, 32), whereas others have reported an impact on negative selection (27, 33–35). CTLA-4 has also been suggested to affect thymic quantitative output of Foxp3⁺ Treg cells and to broaden the T-cell receptor (TCR) repertoire of Tconv cells in TCR transgenic mice specific for myelin basic protein (17, 36). Taken together, the function of CTLA-4 is complex and likely operates by different mechanisms on different levels of tolerance via different cell populations. Hence better-defined ways to study CTLA-4 in vivo are needed to elucidate its multiple roles.To this end, we bred mice in which ctla-4 can be conditionally deleted by tamoxifen treatment in adulthood, thus circumventing the critical period shortly after birth when murine T cells migrate and populate the periphery. In contrast to a recent report by Paterson et al. (18), who used a similar way of CTLA-4 deletion but saw no overt disease, we found that CTLA-4 deletion in adult mice rapidly induced aberrant immune activation, multiorgan lymphocyte infiltration, and auto-antibody production, but only mice born with CTLA-4 deficiency developed myocarditis and succumbed to fatal pancreatitis. Furthermore comparison of protein-induced EAE or collagen-induced arthritis (CIA) with peptide-induced EAE revealed opposing effects of CTLA-4 deletion in adulthood. Collectively, our results show that abrogation of CTLA-4 expression in adult mice induces autoimmune diseases in otherwise unmanipulated mice, that CTLA-4 has a role in regulating both central and peripheral tolerance, and that its function in autoimmune diseases differs depending on underlying disease-specific mechanisms.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.