Binding efficiencies of carbohydrate ligands with different genotypes of ...

J Mol Model (2012) 18:1–10 DOI 10.1007/s00894-010-0947-6

ORIGINAL PAPER

Binding efficiencies of carbohydrate ligands with different genotypes of cholera toxin B: molecular modeling, dynamics and docking simulation studies Mobashar Hussain Urf Turabe Fazil & Sunil Kumar & Rohit Farmer & Haushila Prasad Pandey & Durg Vijai Singh

Received: 29 September 2010 / Accepted: 27 December 2010 / Published online: 16 March 2011 # Springer-Verlag 2011

Abstract Vibrio cholerae produces cholera toxin (CT) that consists of two subunits, A and B, and is encoded by a filamentous phage CTXΦ. The A subunit carries enzymatic activity that ribosylates ADP, whereas the B subunit binds to monosialoganglioside (GM1) receptor in epithelial cells. Molecular analysis of toxigenic V. cholerae strains indicated the presence of multiple ctxB genotypes. In this study, we employed a comparative modeling approach to define the structural features of all known variants of ctxB found in O139 serogroup V. cholerae. Modeling, molecular dynamics and docking simulations studies suggested subtle variations in the binding ability of ctxB variants to carbohydrate ligands of GM1 (galactose, sialic acid and N-acetyl galactosamine). These findings throw light on the molecular efficiencies of pathogenic isolates of V. cholerae harboring natural variants of ctxB in causing the disease, thus M. H. U. T. Fazil : D. V. Singh (*) Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India e-mail: [email protected] S. Kumar Bioinformatics Centre, Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India R. Farmer Department of Computational Biology and Bioinformatics, Jacob School of Biotechnology and Bioengineering, Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad 211007, India H. P. Pandey Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

suggesting the need to consider ctxB variations when designing vaccines against cholera. Keywords Cholera toxin B . GM1 receptor . Galactose . Molecular dynamics . Protein modeling . Docking simulations

Introduction Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. Of the 209 serogroups, only the O1 (which is further classified into the two biotypes El Tor and Classical) and O139 serogroups of V. cholerae have the potential to cause epidemic and pandemic cholera [1]. The colonization, subsequent production and secretion of cholera toxin (CT) by V. cholerae in the intestinal cells lead to diarrhea, which can be lethal if left untreated. The CT-encoding genes, ctxAB, are found within a filamentous bacteriophage CTXΦ [2]. Based on the variations in the repressor protein, rstR, CTXΦ has been classified into three major types: CTXClassΦ (classical type), CTXETΦ (El Tor type), and CTXCalcΦ (Calcutta type) [1]. In V. cholerae O1 El Tor and O139, multiple CTXΦ prophages are sitespecifically inserted near the terminus of the large chromosome, whereas classical prophages insert at the termini of both chromosomes [3, 4]. The characterization of ctxAB mutants presented evidence that the toxin is advantageous to the growth of the bacterium in intestinal milieu [5]. It was demonstrated that the ctx mutants colonized less efficiently in rabbit intestines than the parental strain. CT enhances intestinal colonization either by compensating for nutritional deficiency or by suppressing the bactericidal activity produced by epithelial cells [6].

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Cholera toxin is a hexameric A-B5 type toxin. The toxicity is attributed to the enzymatic activity of the A subunit, which catalyzes ADP ribosylation of the α subunit of GTP binding protein, GS, leading to activation of adenylate cyclase and a concomitant elevation in cAMP levels, in turn causing the hypersecretion of Cl− ions and water, and ultimately profuse diarrhea. Cholera toxin A subunit (CTA) consists of two polypeptide chains, CTA1 and CTA2. CTA1 confers CT-mediated toxigenicity, whereas CTA2 acts as a linker between CTA1 and cholera toxin B subunit (CTB). The five B subunits of the toxin bind principally to monosialoganglioside (GM1) receptors that are present on the surfaces of mammalian cells. The B subunit is considered a molecular recognition unit and delivery vehicle for the A subunit [7, 8]. Although the action of CT is conserved among classical and El Tor strains, the CTB sequence differs among the two biotypes, which serves as the basis for ctxB genotyping. Based on nonrandom base variations, three types of ctxB genes have been described, due to changes in the deduced amino acid sequence positions at 39, 46 and 68 [9]. Genotype 1 is found in classical biotype strains worldwide and in US Gulf Coast strains, genotype 2 in El Tor biotype strains from Australia, and genotype 3 in El Tor biotype strains from the seventh pandemic and the Latin American epidemic. New ctxB variants showing additional polymorphism at amino acid positions 28 and 34 have recently been described in V. cholerae O139 strains [10]. Cholera toxin B subunit is used to develop the oral cholera vaccine. When administered orally, CTB induces immunogenicity at mucosal surfaces [11, 12]. This event is believed to be the result of CTB binding to eukaryotic cell surfaces via GM1, which elicits a mucosal immune response against the pathogen and an enhanced immune response when coupled chemically with other antigens. Previous studies have indicated that amino acid sequence diversity at positions not involved in receptor binding can lead to epitope variations of CTB, and therefore must be considered during the development of synthetic and natural vaccines against cholera [13]. Mutation analysis of CTB revealed variations in immunoreactivity, hemolysis and GM1 binding ability for the toxin subunit [14, 15]. However, the role of natural variations in CTB and their advantages, if any, to this organism in terms of survival or pathogenesis have barely been explored. In this study, we carried out molecular modeling, dynamics simulations and docking studies of all natural CTB variants. Monosaccharide libraries have previously been used to explore binding sites in order to develop simple and easily synthesizable molecules against E. coli heat labile toxin, a close homolog of CT [16]. In this regard, monosachharide components of GM1 receptor, namely galactose, sialic acid, N-acetyl galactosamine and glucose, were employed individually

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for docking simulations. The information generated should prove useful for establishing the enterotoxic potentials of the CTB variants, if any, and for determining the relationships between variants of CTB and disease severity, aside from defining the requisite binding sites of drug-like molecules.

Materials and methods Bacterial strains Seven representative strains, of which five were V. cholerae O139 strains isolated from diarrheal patients from Kerala, southern India, were included in the study (Table 1). Reference strains of V. cholerae O395 (O1 classical Ogawa), N16961 (O1 Biotype El Tor) provided by Dr. R. K. Bhadra of the National Institute of Cholera and Enteric Diseases (NICED), Kolkata, were used as control. All strains were maintained in tryptic soy broth (Difco) supplemented with 30% glycerol at –80°C or in Luria Bertani (LB) agar stab culture at room temperature. DNA isolation and sequencing Chromosomal DNA from V. cholerae strains grown overnight was isolated following the method of Ausubel et al. [17]. To generate a sequencing template from strains carrying either the El Tor or the Calcutta CTXΦ, the DNA sequence between the rstR gene and the 3′ end repeat of attRS was amplified. PCR products obtained from these reactions were then used as templates to get a ctxB gene product via the method described earlier [18]. PCR products were purified using QIAquick gel extraction kit (Qiagen) and sequences for both the DNA strands were determined using a CEQ 8000 automated DNA sequencer (Beckman Coulter, Brea, CA, USA). Modeling of CTB variants The experimentally generated crystal or NMR structures for all known variants in V. cholerae are not yet available. Therefore, we built molecular models for all the reported variants of the ctxB gene (Table 1) by means of comparative modeling. The crystal structure of the cholera toxin B pentamer complexed with GM1 pentasaccharide (PDB code: 2CHB) was selected as template for all modeling procedures. Sequence alignment was achieved using the Clustal W [19] (http://www.ebi.ac.uk/clustalw) software. Since the first 21 amino acids belonging to the Nterminal region of the target protein did not have an equivalent region in the crystal structure, modeling was carried out using the academic version of MODELER9v6

J Mol Model (2012) 18:1–10 Table 1 Amino acid variations among the ctxB genotypes present in different strains of V. cholerae O1 and O139

3 Strains

Phage type

ctxB genotype

Amino acid position variation

References

Type 1 Type 2

28 D D

[10]

34 H H

39 H H

46 F L

68 T T

El Tor, N16961 Type 3 D H V. cholerae O139, (2005) Type 6 D P ctxB selectively amplified from CTXΦ prophages of V. cholerae O139 V. cholerae O139 AL49 El Tor Type 4 D H V. cholerae O139 AL40 El Tor Type 5 A H V. cholerae O139, AL72 Calcutta Type 1 D H

Y Y

F F

I T

Y H H

F F F

T T T

V. cholerae O139, TV169 V. cholerae O139, TV258

Y H

F F

T T

Classical, 569B El Tor, Australia

Calcutta Calcutta

(http://salilab.org/modeler) [20] from amino acid positions 22 to 124 of the CTB protein. A total of 20 models were generated by MODELLER, among which the one with the best PROCHECK [21] G-score and VERIFY3D [22] profile was subjected to MD simulations and energy minimization. A nonbinding cut off of 14 Å, the CHARMM force field [23] and CHARM-all-atom charges were employed, and a steepest descent algorithm was used to remove close van der Waals contacts. This was followed by conjugate gradient minimization until the energy exhibited stability during sequential repetition. All hydrogen atoms were included during the calculation. The energy minimization started with the side chain first, and then all of the atoms of the protein were relaxed during optimization. All calculations were performed by using the ACCELRYS DS modeling 2.0 software suite (Accelrys Inc., San Diego, CA, USA). VERIFY3D (a structure evaluation server) was used to check the residue profiles of the three-dimensional models obtained. In order to assess the stereochemical qualities of the three-dimensional models, PROCHECK analysis was performed. Quality evaluation of the model for the environment profile was also predicted using the ERRAT (structure evaluation server) software [24]. Molecular dynamics simulations All atom MD simulations of CTB protein in explicit water were carried out using the GROMACS 4.0.6 software and the GROMOS96 [25] force field for a time scale of 1 ns. Threedimensional periodic boundary conditions were imposed, enclosing the molecule in a truncated octahedron (0.5 nm thick) solvated with the SPC water model [26] provided in the GROMACS package. The system was neutralized with a single Cl− counterion. The 3D molecule was locally minimized in order to provide a first minimization of the rough geometry derived from homology modeling using 1000 steps of steepest descent energy minimization. The electrostatic terms were described using the particle mesh Ewald algorithm. The LINCS [27] algorithm was

Type 4 Type 5

D A

H H

This study

used to constrain all bond lengths and cut-off distances for the calculation of the coulombic and van der Waals interactions at 1.0 nm. The system was equilibrated by 100 ps of MD runs with position restraints on the protein to allow the relaxation of the solvent molecules at 300 K and normal pressure. The system was coupled to the external bath by the Berendsen thermostat [28] using a coupling time of 0.1 ps. The pressure was maintained by coupling to a reference pressure of 1 bar and V-rescaling [29] the temperature with a coupling constant of 0.1 ps. The final MD calculations were performed for 1 ns under the same conditions, except the position restraints were removed. The results were analyzed using the standard software provided by the GROMACS package. In silico site-directed mutagenesis In silico site-directed mutagenesis has been widely used to identify the critical residues responsible for binding to a ligand. The in silico mutations performed using the mutate _model command of MODELLER are listed in Table 1. Amino acid variations affecting the stability, structure and function of the protein were calculated by the SDM server (http://mordred. bioc.cam.ac.uk/∼sdm/sdm.php). SDM uses a statistical potential energy function to predict the effect of amino acid polymorphisms on the stability of proteins. The percentage variations in solvent accessibility at positions of mutation along with the differential free energies of folding for mutants were readily generated. Protein ligand docking studies The chemical structures of monosaccharides were extracted from pubchem (http://pubchem.ncbi.nlm.nih.gov). The structures of all four carbohydrate ligands (glucose, galactose, N-acetyl neuraminic acid, N-acetyl galactosamine) included in binding to CTB were retrieved in the two-dimensional MDL/SDF format. Three-dimensional coordinates for these molecules were generated using the

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CORINA [30] software. The molecules were then read into Discovery Studio 2.0 for further treatment of energy minimization for 100 steps with the CHARMM force field. Genetic Optimization for Ligand Docking (GOLD) version 4.1.1 (Cambridge Crystallographic Data Centre, Cambridge, UK) was used for docking 50 times in the standard default settings. The standard default settings (population size 100, selection pressure 1.1, niche size 2, migrate 10, crossover 95, number of operations 100,000, number of dockings 10) were adopted for GOLD docking. For ligand–protein binding, 10 docking conformations (poses) were tested and the best GOLD score selected for studies. GOLD uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein [31]. The docking procedure consisted of three interrelated components: (a) identification of the binding site; (b) the use of a search algorithm to effectively sample the search space (the set of possible ligand positions and conformations on the protein surface); (c) the application of a scoring function. To estimate the protein–ligand complexes, the scoring function for the GOLD score was employed on the basis of four components: (a) protein–ligand hydrogen bond energy (external H-bond); (b) protein–ligand van der Waals energy (external vdw); (c) ligand internal van der Waals energy (internal vdw); (d) ligand intramolecular hydrogen bond energy (internal H-bond). The binding affinity between the protein and ligand was estimated using the consensus scoring function X-Score V2.1 [32]. The ligand that presented the greatest interaction with the protein was plotted using the program LIGPLOT [33]. Hydrogenbonding interactions were double-checked with the software GETNEARES, which is available with the program DOCK [34].

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was selected, and the resultant trajectory of simulations was analyzed to study the physical properties of the protein. The time evolution of the root mean square deviation (RMSD) was computed for the simulated structure by taking the modeled protein as the initial structure (Fig. 1). Based on intrinsic dynamics, structural stability and the improved relaxation of the modeled protein, the energy of the structure and the radius of gyration were calculated (Fig. 2a, b). The energy and RMSD calculations for CTB demonstrated that the protein is not very flexible over the timescale of the MD simulations. The model obtained after rigorous refinement by means of MD and EM was subjected to mutational studies. We generated the variants of CTB corresponding to the reported classification of genotypes of V. cholerae [10] harboring the ctxB genes using the final model. The mutant structures were subjected to another round of EM and then used for docking. Protein structure validation To validate the homology-modeled CTB, a Ramachandran map was drawn and the structure was analyzed by PROCHECK, a well-known protein structure checking program. It was found that the phi/psi angles of 93.7% of the residues fell in the most favored regions, 6.3% of the residues fell in the additional allowed regions, 0% fell in the generously allowed regions, and none of the residues fell in the disallowed regions (Fig. 3). The overall PROCHECK G factor for the homology-modeled structure was −0.10. A decrease in the overall G factor was observed after MD simulation. This indicates that an increase in the number of bad dihedral angles of the modeled structure had occurred, possibly due to the MD simulation causing an unfavorable dihedral angle, which allowed the protein to overcome high energy barriers. The final structure was

Results and discussion ctxB gene sequencing The sequencing of representative V. cholerae O139 strains carrying either El Tor or Calcutta type CTXΦ prophages revealed variations in ctxB genes corresponding to amino acid positions 28, 34 and 68, and belonging to new genotypes (Table 1). Taking into account variations in ctxB, including the ones already reported, the translated amino acid sequences were used in protein structure generation and in silico mutational studies. Molecular dynamics simulations One of the 20 modeled structures of the CTB protein obtained by MODELLER9v6 that retained binding properties identical to the crystal structure along with the G factor

Fig. 1 Calculated root mean square deviation versus time graph for the modeled CTB protein

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Fig. 2 a Calculated energy versus time plot for MD simulations of CTB using GROMACS software. b Radius of gyration over the timescale of MD simulations in CTB

validated by an ERRAT graph. The quality factor of 95.74 indicated good quality, as scores of >50 are acceptable for a reasonable model. These observations thus indicated that the obtained structure was of good quality. Mutations and docking simulations The translated protein sequences of the ctxB genotypes were utilized to perform protein model development, and the model used in silico mutagenesis. The structure of the CTB subunit predominantly consisted of β sheets sur-

rounded by α helices. The conserved Val 52–Ile 58 region encompassing a flexible loop plays an important role in the action of cholera toxin. We generated binding regions for the carbohydrates of GM1 pentasaccharide using all-atom superimposition of the template, which included His13, Asn14, Glu51, Gln56, His57, Gln61, Trp88, Asn90, and Lys91 [35]. The amino acids His13 and Asn14 fall in the loop region following a helix at the N-terminal region. Gln56 and His57 belong to the conserved flexible loop central to the action of the toxin, whereas Glu51 and Gln61 flank the loop region. Trp88, Asn90 and Lys91 were included in a β hairpin bend, and are known to be important in ligand binding. Though the mutations described here are not part of the flexible loop or the β hairpin bend (Fig. 4), we found differences in ligand binding among the variants of CTB, as presented below. Ligplots are provided in order to show variable hydrogen-bonding patterns among different CTB genotypes, which were considerably different from the template structure according to site-directed mutagenesis software. Protein–ligand interactions at energy minima representing the best docking poses were generated in accordance with the GOLD software. These complexes were used for ligplots that indicated probable hydrogen bonding patterns (indicated by dotted lines) in the respective variants (Fig. 5, 6, 7, 8). Mutations that had deleterious effects without being among the catalytic or substrate-binding residues were known to cause variations by triggering changes in structural features, such as alterations in surface charge allocation and the disruption of packing in the protein core region. Genotype 1

Fig. 3 Ramachandran map of the CTB protein. Calculations for the three-dimensional (3D) model of the CTB protein were done with the PROCHECK program

ctxB genotype 1 has been reported in V. cholerae of the classical biotype carrying classical CTXΦ prophages. The

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used as the standard against which the other types are compared. Genotype 2 Genotype 2 showed the replacement of phenyl alanine with leucine at position 46, which occurs at the center of a β sheet. Docking simulations with galactose, sialic acid and N-acetyl galactosamine showed that in each case three hydrogen bonds formed between the ligand and the CTB protein, while 5–9 amino acids were involved in hydrophobic interactions. There were small increases in the percent solvent-accessible surface (+0.7) and the free energy of unfolding (+0.786 kcal mol−1) compared to classical CTB for amino acid variation at this position. This variant does not show much of a difference in hydrogen bonding ability from classical CTB. Fig. 4 CTB model labeled with mutations corresponding to different variants

amino acids at positions 39 (histidine), and 68 (threonine) are employed to classify the genotype of classical ctxB. Galactose, which is a major component of GM1 pentasaccharide, formed five hydrogen bonds in total, and five amino acids were involved in hydrophobic interactions within a radius of 10 Å from the binding pocket (Fig. 5a). Sialic acid and N-acetyl galactosamine each formed three hydrogen bonds with other amino acids coordinating hydrophobic interactions (Fig. 5b,c). Although glucose is a carbohydrate component of the GM1 receptor, no direct hydrogen bonding was seen among amino acids of the CTB. For all practical purposes, genotype1, corresponding to classical CT with the published crystal structure, will be

Genotype 3 Amino acid variations at positions 39 (histidine to tyrosine) and 68 (threonine to isoleucine) are the characteristic feature of genotype 3. Interestingly, docking simulations with galactose and N-acetyl galactosamine show only one hydrogen bond with each carbohydrate ligand (Fig. 6a,c), but four hydrogen bonds formed with sialic acid (Fig. 6b). The changes in solvent accessibility for amino acid substitutions at positions 39 and 68 for tyrosine and isoleucine were −7.9% and +28.9% compared to classical CTB. However, the free energy difference between the two proteins for unfolding was 3.040 kcal mol−1. Therefore, this protein can be assumed to be physically more distinct than the other described genotypes.

Fig. 5 Molecular interaction plots between carbohydrate ligands and genotype 1: a galactose, b sialic acid, c N-acetyl galactosamine

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Genotype 4 Genotype 4 is characterized by an amino acid variation at position 39 from histidine to tyrosine. This variant formed three hydrogen bonds with each of the ligands employed, although the binding conformation was very different from that of genotype 1 at minimal energy. A decrease in the percent solvent accessible area (−4.7) along with a change in the differential free energy of unfolding of 1.82 kcal mol−1 was observed. However, this mutant was classified as neutral compared to classical CTB. Genotype 5 This genotype was recently identified, and is associated with an amino acid change from aspartate to alanine at position 28. This CTB genotype also formed three

hydrogen bonds with all ligands except for glucose, which makes it similar to classical CTB in hydrogen-bonding ability (Fig. 7a–c). However, a decrease in the percent solvent accessible area (−9.1) was observed for this model compared to classical CTB. Also, the differential free energy of folding rose to 2.055 kcal mol−1, thus distinguishing this variant of CTB. Genotype 6 Genotype 6 was classified on the basis of variation at amino acid positions 34 (histidine to proline) and 39 (histidine to tyrosine) with respect to genotype 1. With galactose, this variant formed five hydrogen bonds, in a similar manner to classical CTB (Fig. 8a). The interactions between sialic acid and type 6 CTB were stronger with respect to hydrogen bonding (Fig. 8b). Aside from Asp90, Glu56 and Gln61 (as


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in genotype 1), Asn14 and Trp88 were also involved in direct hydrogen bonding between the protein and ligand. Although equivalent numbers of hydrogen bonds were observed for genotypes 1 and 6, their ligand binding characteristics with N-acetyl galactosamine differ, since about eight amino acids were involved in stabilizing the hydrophobic interactions, in contrast to classical CTB (Fig. 8c). Changes in the percent solvent accessibility at positions 34 and 39 were +21.8% and – 4.7% for this variant with respect to classical CTB. The free energy difference between the two proteins was 3.429 kcal mol−1 for unfolding. These findings thus suggest that this CTB variant has a superior binding ability compared to other known CTB genotypes. The classical type of CTB, designated genotype 1, was produced by the V. cholerae harboring the classical type of CTXΦ prophages. The El Tor type of CTXΦ prophages produces the El Tor type of cholera toxin, which includes two variants of CTB, designated genotypes 2 and 3. The O139 V. cholerae possesses both the El Tor and Calcutta types of CTXΦ prophages, and includes genotypic variants 4, 5 and 6. Sequencing data indicate that the majority of the V. cholerae O139 El Tor phages belong to genotype 4, Table 2 Variable docking scores of carbohydrate ligands with cholera toxin B subunit variants

Cholera toxin genotype

CT CT CT CT CT CT

B1 B2 B3 B4 B5 B6

whereas the Calcutta-type phages are mostly of genotype 5. However, both variants of CTB produce the classical type of cholera toxin B (Fazil et al., unpublished data). Docking simulations studies suggested that the ability of the type 1 CTB to interact with galactose and N-acetyl galactosamine is much greater than that of type 3 found in V. cholerae biotype El Tor (Table 2). Among the CTB variants, V. cholerae O139 genotype 6—the most recently disovered type—shows the most impressive bonding characteristics with galactose, sialic acid and N-acetyl galactosamine of all known CTB genotypes. V. cholerae O1 biotype El Tor, the causative organism of the ongoing seventh pandemic, produces CT of genotype 3, while the classical biotype produces CT of genotype 1. The CT genotype of the El Tor strains currently associated with cholera in the Indian subcontinent has shifted from genotype 3 to genotype 1. Thus, the currently circulating El Tor strains that cause cholera have characteristics of the El Tor biotype but possess classical CTB [36, 37]. In terms of the chronology of evolution in ctxB genotypes, type 1 CTB was the first genotype of classical V. cholerae to be reported. The phage that encoded this CTB genotype was found to be integrated into host Docking score with respective carbohydrate ligand Galactose

Sialic acid

Galactosamine

32.51 30.49 24.53 29.85 30.35 32.34

37.56 36.6 37.15 38.23 38.15 38.74

28.52 28.52 25.32 28.89 28.83 29.92

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chromosomes and unable to replicate independent of the host, while the El Tor type phages replicated in the host to produce functional phages [38]. V. cholerae of the classical biotype are known to cause more severe cholera [39], while El Tor biotype strains survive better in aquatic environments [40]. The results of this study indicate decreased binding efficiency of El Tor CTB compared to the classical cholera toxin towards various carbohydrate ligands. The data generated suggest that new ctxB genotypes of V. cholerae O139, similar to classical CTB, possessed enhanced potential to bind carbohydrate ligands. Recent variations in CTB employed by the microbe indicate that V. cholerae produces a potent toxin that is more proficient at hydrogen bonding and thus forming stable complexes than the seventh pandemic clone of biotype El Tor. This could be a reason to worry, because current strains of V. cholerae O1 and O139 host to CTX prophages that have acquired the capacity for independent replication and infection, and could lead to the rapid spread of severe cholera. Apart from the above mentioned ligands, we tested variant genotypes for their abilities to dock with glucose, which is in fact another carbohydrate component of monosialoganglioside. We found no interaction between the ligand and the proteins in any of the variant CTB proteins included in this study. In the case of CTB, it is understood that galactose, which is present at the GM1 terminus, has a potential role to play in interactions with CTB. Therefore, the elaboration of the binding characteristics of this specific carbohydrate would be primarily intended for virulence target initiatives or vaccine design [41]. Several studies have individually studied with galactose derivatives as an aid to the design of inhibitors against this toxin [42]. Hence, it can be concluded that potential derivatives of the carbohydrates used as CT inhibitors may employ the binding characteristics elucidated in this study.

Conclusions The rate of change in the genetic profile of toxigenic V. cholerae has been a cause for concern due to their ability to cause epidemic and pandemic cholera. Among the various markers used to monitor genetic changes, variations in CTB have been studied recently. In this study, we explored the possibility of variable binding efficiencies of CTB variants to constituent carbohydrates of GM1 ganglioside, an important step in causing severe cholera. The results of this study indicate that there are subtle variations in the hydrogen bonding abilities of CTB variants toward the carbohydrate ligands that constitute the GM1 receptor. The data presented indicate the possibility of rapid genetic reassortments among O139 serogroup V. cholerae, thus

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giving rise to multiple variants of CTB. Though there are no phenotypic differences among strains harboring CTB variants, the data provide clues about potential molecular discrepancies in the binding of CTB to its receptor. These differences can be employed in the design of paratopes that can determine the specificity of either a precise biotype or a standard anticholera toxin monoclonal antibody. As CTB is an attractive drug candidate for autoimmune diseases as well as differentiation therapy, the data presented here can be exploited in the design of drugs or vaccines. Acknowledgments This work was supported in part by a grant from the Indian Council of Medical Research, New Delhi (Immuno. 18/11/ 17-ECD-I) to DVS and funds contributed by the Department of Biotechnology, New Delhi, to the Institute of Life Sciences. A senior Research Fellowship awarded by the Indian Council of Medical Research, New Delhi, India, to MHU Turabe Fazil is gratefully acknowledged. The authors thank the reviewers for their suggestions.

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