Mechanisms of V.cholerae Cytolysin (VCC)

Mechanisms of V.cholerae Cytolysin (VCC)V.cholerae cytolysin (VCC) is a focalize- conditioning toxin secreted by many a(prenominal) pathogenic strains of the Gram-negative bacteria V.cholerae the causative pathogen of diarrheal disease cholera. VCC display potent cytotoxic use against the erythrocytes and mammalian cellphones. It is also account to possess enterotoxin activity in enclosures of inducing bloody legato accumulation in the rabbit ileal loops. Based on these observations, VCC has been believed as a potential virulence doer of V.cholerae. VCC, in starticular, secreted by the pathogenic strain lacking cholera toxin, the primary virulence factor of V.cholerae that credi cardinalrthy for inducing the massive dehydrating diarrhea disease during V.cholerae infection.VCC is encoded by the hlyA gene stand for in V.cholerae chromosome two. VCC toxin is synthesized as a 81 kDa protein, called Pre-Pro-VCC. During the secretion of toxin, the N- entrepot signal peptide co mposed of 25-residue is removed to show a non operable precursor skeletal frame of the VCC blood cell, named as Pro-VCC. Following, 15 kDa N-terminal ecological successions from Pro-VCC is proteolytically deleted that resulting the formation of the functional mature form of the toxin. Proteolytic activation of the toxin is mediated by the HA/protease, which display the major extracellular proteolytic activity of V.cholerae. Conversion from Pro-VCC into the mature pass on of the VCC drive out also be obtained in vitro by other proteases like trypsin, chymotrypsin, and subtilisin. It has been account that the activation of the Pro-VCC can be resulted by the proteolytic activity of the proteases present on the surface of the arse host cell tissue layer as well.Functional mature form of the toxin has been displaying to induce lysis of the erythrocytes and other eukaryotic cells by generating the heptameric oligomeric digest organise of 1-2 nm diameters. The functional tiss ue layer permeabilization ability of the toxin could also be resembled in the membrane lipoid bilayer of the man-made liposomes. Along with its membrane permeabilization activity, VCC toxin also displayed a prominent lectin-like activity by binding to the complex glycoproteins and glyco lipides with the terminal 1-galactosyl component. VCC is characterized as a member of -PFTs and reported that the toxin follow the overall scheme of the generalized -PFT mode of action. morphological Features of VCCVCC is secreted as a water- dissolvable monomeric form of the toxin, which after the removal of the N-terminal Pro- field of study gets converted into the mature functional form of the molecule. VCC induces lysis of its target cells by generating heptameric oligomeric contracts on the membrane. The high-resolution organize of the water-soluble, monomeric precursor maintain Pro-VCC toxin has been incurd. Heptameric transmembrane structure of the VCC has also been determine late(a)ly. Many foregoing studies confirmed that the VCC is a -PFTs family member, and the toxin employed pore-forming activity by generating the transmembrane heptameric -barrel pores on the target cell membrane. Consistent with the -PFTs tramsmembrane pore structure, pore complex of VCC represent a mushroom-shaped organization, which can be divided into two parts (a) transmembrane b-barrel morphologic, and (b) membrane interacting rim orbit. morphological analysis of the VCC molecule highlights many unique features which are non reported in the prototypic member of -PFTs family. Consistent with the morphological of archetypical -PFTs, VCC harbors a central cytolysin athletic field that constitutes the heart and soul structure of the mushroom-shaped oligomeric transmembrane pore structure. The cytolysin humans of a function contains the pore-forming ancestor-loop of the toxin. apart from cytolysin subject area, VCC structure also contains three additional morphologic domain wh ich are not commonly documented in any other member of b-PFTs family an N-terminal Pro-domain in the in lively Pro-VCC precursor state of the toxin, and two lectin-like domain name -Trefoil domain and -Prism lectin-like domain at the C-terminal cheek of the cytolysin domain.Cytolysin domainThe VCC molecular structure contains 325 amino acid long cytolysin domain that structurally similar with the cytolysin domains present in the member of -PFTs like S. aureus -hemolysin. Cytolysin domain of the VCC during the membrane pore-formation lick inserts its pre- base region into the lipid bilayer and generates -barrel structure on the membrane and provides the central scaffold of the pore structure. VCC generate mushroom-shaped oligomeric transmembrane pore structures that can be classify into two major parts (a) transmembrane region that make the -barrel pore structure, and, (b) membrane interacting rim-domain that interact with the membrane surface. The membrane inserted -barrel struct ure of the VCC pore structure is composed solely of the central cytolysin domain of the toxin. Notably, the majority of the rim-domain is also generated by the cytolysin domain.Cytolysin domain of the toxin harbors the 42-residue long pore-forming loop loop that involve in the formation of the transmembrane -barrel pore structure. In the water soluble monomeric form of the toxin, this region remains completely folded against the cytolysin domain, in the form of a so named pre- block theme. During the process of the functional pore-formation, the pre-stem loop from each of the participating protomers undergoes enormous structure recognition to obtain a so-called stem configuration, and inserted into the lipid bilayer of the membrane. Stem region from each of the protomers contributes two -strands towards the formation of the stem region of the heptameric -barrel pore structure. Heptameric oligomer highlights that the stem regions make the extensive interaction between the neighbor ing protomers and hence contribute towards the robust stability of the transmembrane oligomeric assembly. Apart from the pore-forming stem-loop segment, other part of the cytolysin domain contains the membrane-proximal rim-domain of the transmembrane pore structure. morphologic analysis of the -PFTs pore, suggests that the membrane-proximal rim-domain work as the structure motif for transmembrane pores. Rim-domain acts as structural scaffolds that mediate interaction of the protein with the lipid head-group of the target membrane lipid bilayer. Cytolysin domain of the VCC contributes towards the interaction of the toxin with the lipid head-group of the membrane.Pro-domainAs mentioned previously, VCC toxin is secreted by yet bacteria as the water-soluble inactive precursor state called Pro-VCC. The high resolution three-dimensional structure of Pro-VCC molecule shown the presence of 15 kDa Pro-domain, which make contact to the N-terminal of the consequence cytolysin domain by din t of a 29-residue long flexible linker.The linker region harbors amino acid long structural motif that act as the cleavage site(s) for a group of proteases. Proteolytic removal of the Pro-domain at this linker sequence resulted in the generation of a mature form of the toxin. The presence of the Pro-domain in the precursor form of the toxin has been reported to be critical for the efficient secretion and the appropriate folding of the VCC molecule. One earlier study has been reported that the recombinant V.cholerae cells, containing the deleted variant of hlyA gene lacking the sequence for the Pro-domain, unable to secrete the protein outside the bacterial cells. In vitro denaturation/renaturation, checkout adjudge demonstrated that without the Pro-domain VCC fails to refold back to its active conformation, whereas Pro-VCC can obtain proper refolding. Recent study on Pro-domain, suggested that the presence of Pro-domain increase the unfolding property of the Pro-VCC molecule in r esponse to many denaturing conditions, whereas mature active form of the toxin display considerable resistant towards the unfolding of the toxin. Overall, these studies suggested, the Pro-domain show an intramolecular chaperone-like activity in term of providing large level of structural plasticity in the VCC structure, which probably essential for the efficient secretion of the toxin in its precursor from across the bacterial membrane. However, its not clear so far how the presence of the Pro-domain bind the protein in its precursor form.-Trefoil lectin-like domainVCC harbors a -Trefoil lectin-like domain ( 15 kDa) at the C-terminal edge of the center cytolysin domain. This -Trefoil lectin-like domain is also present in related cytolysin from Vibrionaceae bacteria, but not present in the archetypical -PFTs protein for congresswoman S. aureus -hemolysin. The -Trefoil lectin-like domain is associated with the cytolysin domain through a short linker sequence constitute of Gly-Gly-A rg-Pro. The -Trefoil lectin-like domain of VCC display structural similar to the carbohydrate-interacting domain of the plant toxin ricin, and featured the presence of the QXW conserved carbohydrate-interacting motif (s) observed in the archetypical -Trefoil lectin domains of carbohydrate binding lectins. However, the carbohydrate binding propensity of the -Trefoil domain of VCC has not been elucidated. Also, the implications of the -Trefoil domain in the structure-function weapon of the VCC need to be explored in future.-Prism lectin-like domainThe VCC harbors an additional 15 kDa domain that is linked to the C-terminal of the -Trefoil domain through the long linker sequence.This domain is not present in any other member of the -PFTs family, including the cytolysin secreted by V.vulnificus and Aeromonas hydrophilia.The C-terminal domain of the VCC display structural similarity to several -Prism lectins including jacalin and Maclura pomifera agglutinin (MAP). VCC -Prism lectin-lik e domain possess a binding scoopful similar to the carbohydrate-binding site of the jacalin and MPA lectins. Recently, we have conclusively established the role of -Prism domain in the lectin activity of the toxin. In the absence seizure of the -Prism domain, VCC toxin did not show lectin activity towards -1 galactosyls terminated glycoconjugates. We have identified the critical site at bottom the -Prism domain which responsible for the lectin activity of the toxin. We reported that the amino acid tried (composed of Asp617, Tyr654, and Tyr679) located within the putative(prenominal) carbohydrate-interacting pocket generate the crucial element for the VCC lectin activity. Overall, it has been established that the -Prism domain of the VCC act as structural scaffold playing a critical role in the lectin-like activity of the toxin. During the process of functional pore-formation in the lipid bilayer of the target host membrane, VCC molecule undergoes enormous structural reorganisatio n. The -Prism domain of the VCC obtained two different positions with respect to the core cytolysin domain, in the monomeric precursor form (Pro-VCC) and the transmembrane pore structure. In monomeric water-soluble inactive precursor Pro-VCC, the -Prism domain positioned on the opposite side of the Pro-domain on top of the pre-stem region, whereas in the transmembrane pore structure it is relocated in the endow of the Pro-domain. This structural rearrangement of the -Prism domain is mandatory for the membrane insertion, and the functional oligomeric pore-formation procedure. In the absence of such structural reorganization of the -Prism domain, it would be located in such a behavior that would generate steric hindrance between the bring protomers and subsequently block the oligomerization of the toxin. Also, without such reorganization of the -Prism domain, the pre-stem loop would not be able to unfold for the membrane insertion of the toxin. Overall it appears that the -Prism domain-mediated lectin activity of the toxin might act as a triggering mechanism to allow such structural reorganization of the -Prism domain with respect to core cytolysin domain. Our study suggested that the presence of the -Prism domain in VCC molecule is critical for the efficient membrane pore-formation of the toxin. The -Prism domain truncated variant of the toxin display abortived membrane pore-formation. However, in the absence of -Prims domain, VCC molecule could generate membrane-associate oligomers but does not show any functional membrane pore-forming activity.Structural reorganizations during oligomeric pore-formationStructural analysis of the water-soluble monomeric form and the transmembrane oligomeric structure of VCC reveal that the VCC molecule undergoes structural reorganization within the toxin monomer during the process of the oligomeric transmembrane pore-forming procedure. The most critical structural change is the unfolding the pre-stem region from the cytoly sin domain, and its insertion into the lipid bilayer to generate stem configuration. In the water soluble monomeric structure of Pro-VCC, the pre-stem region remains packed between the b-Prism domain and the cytolysin domain of the toxin. Hence, the movement of -Prism domain is essential for the conversion of pre-stem to the stem region of the toxin. During the formation of the functional pore-formation of the toxin on the membrane, the -Prism domain of the toxin reorients with respect to the central cytolysin domain by almost 180o angle, and attends the location where the Pro-domain was located in the Pro-VCC molecule structure. This reorganization of the -Prism domain of the VCC represents the second most critical structural change involved in the membrane pore-formation of the VCC toxin. The structural change in the position of the -Prism allows the pre-stem to undergo the reorganization for the following membrane insertion and the functional heptameric pore-formation process.Str uctural Features of the VCC -Barrel Pore primarily study based on the Transmission electron microscopy (TEM) characterized the transmembrane oligomer of VCC as typical ring-like structures with the interior(a) diameter of almost 1-2 nm. Inhibitions in the cell cytotoxic ability by the osmoprotectants of defined molecular sizes have also advised similar pre diameter for VCC oligomer pore. Single channel conductance measurement by using the VCC oligomeric pore generated in the synthetic lipid bilayer suggested that VCC produce anion-selective diffusion channels. This analysis also indicated that the VCC pore is having asymmetric pore geometry larger opening in the cis-side than in the trans-side with a narrow region at the central part of the human. The high-resolution structure of the VCC oligomer suggests cup-shaped lumen geometry of the pore. Analysis of the oligomeric pore structure also suggests that the narrow constriction salutary the central of the pore lumen is generated by the aromatic ring of a tryptophan residue contributed by each of the participating protomers during heptameric pore-formation.Mechanism of Membrane Pore-formationThe functional pore-formation of the -PFTs involve on the membrane lipid bilayer of the target cells involves three distinct steps (i) interaction of the water-soluble monomeric form of the toxin towards the target cell membrane (ii) self-assembly of the membrane-associated monomeric toxin to generate the intermediate pre-pore oligomeric assembly on the membrane surface (iii) conversion from the transient pre-pore oligomeric assembly to the functional transmembrane pore structure. During the process of the pore-formation, the pore-forming stem-loop of the toxin inserted into the membrane lipid bilayer and generates the transmembrane -barrel structure. Many structural studies reported that the member of -PFTs follow the similar way of pore-formation on the membrane. However, each member of the -PFTs family differs from each other in the definite step towards the pore-formation process. Membrane interaction step displays enormous range of variation in term of receptor specificity, the role of different lipid component and presence of specific carbohydrate receptor on the membrane. Notably, the molecular mechanism that involve the separate steps for the functional pore-formation are not properly elucidate for most of the -PFTs members. The pore-formation on the membrane by VCC can resemble in the synthetic lipid bilayer liposomes indicating that the membrane association step does not critically required any particular non-lipid components. However, the membrane pore-formation is reported more efficient in the biomembrane as compared to that in the synthetic lipid bilayer of the liposomes, indicating the role of extra molecule present on the cell membrane plays significant role in the pore-formation process. For example, erythrocytes are more susceptible compared to the liposome. Notably, VCC toxin disp lays a different level of hemolytic activity towards the erythrocytes of the different species. Rabbit erythrocytes are form to exhibit more sensitive as compared to the human erythrocytes. Earlier studies have suggested the role of many cell surface receptor proteins (e.g., glycophorin B on the human erythrocytes) as a potential receptor molecule for the VCC toxin. VCC displayed potent lectin-like activity towards the interacting with the cell membrane. However, the specific receptor for the VCC has not been identified. The formation of the transmembrane oligomeric pore structure can be induced in the presence of the synthetic lipid bilayer liposomes. A previous study suggested that the binding of the VCC molecule with the liposomes driven mostly by global amphiphilicity of the monomeric state of the toxin. However, the self-assembly of the toxin and membrane pore-formation has been observed more specific events required the specific components of the membrane. More importantly, t he presence of the cholesterol in the lipid bilayer of the membrane has been reported to play critical role in the membrane pore-formation of the toxin. In our recent study, we identified the specific lipid-binding structure motif present within the cytolysin domain of the toxin. However, our study suggested that the specific motif is responsible for the lipid association in general not specific towards the cholesterol presence in the lipid bilayer of the membrane. In the pore-formation process of the VCC, the pore-forming loop of the toxin unfolds and insert into the membrane toward the generation of the functional pore-formation. It is reported that the trapping of the pore-forming stem-loop in its pres-stem configuration through engineered disulfide linkage could arrest the toxin in its pre-pore oligomeric assembly. Also, a VCC variant without the pre-stem loop is found to remain arrest in the pre-pore oligomer on the membrane surface. Overall these studies suggested that the VCC follows the archetypical -PFTs mechanism of pore-formation. A previous study indicated that the membrane interaction of the VCC precedes membrane oligomerization. Many environmental factors also affect the binding and oligomerization events of the toxin. For example, membrane association can occur even at a low temperature of 4 oC while the membrane oligomerization and functional pore-formation blocked under the similar condition. This observation clearly indicated that the association of the toxin with the target cell membrane is distinct step from its subsequent oligomerization and pore-formation steps.