Structure, Function and Evolution of Clostridium ...

Structure, Function and Evolution of Clostridium botulinum C2 and C3 Toxins: Insight to Poultry and Veterinary Vaccines Chellapandi P* and Prisilla A Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India Tel: +91-431-2407071 Fax:+91-431-2407045 Email: [email protected] *Corresponding author

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Structure, Function and Evolution of Clostridium botulinum C2 and C3 Toxins: Insight to Poultry and Veterinary Vaccines Abstract Clostridium botulinumgroup III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced from this organism arethe most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis tothe development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxinwhereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B.Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships of them are often determined by using evolutionary constraints asvaluable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems to eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection.

Keywords: Veterinary vaccines, Subunit vaccine Clostridium botulinum, Molecular evolution, Immunotoxin, Molecular pathogenesis.

Introduction Clostridium botulinum is a Gram-positive, anaerobic spore forming bacterium playing an important role in clinical applications. C. botulinum spores are found in soil, dust, manure, and slaughterhouse wastes[1, 2,3].The presence of its spores in foodis not of public health significance. However, the spores can germinate, outgrow and multiply into toxin producing vegetative cells, which are ingested into the gastrointestinal tract of avian and mammals[4, 2]. Vegetative cells of this bacterium are capable of producing botulinum neurotoxin (BoNT),

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which acts as a protease to inhibit the pre-synaptic neurotransmitter release, leading causeavian and animal botulism. BoNT is classified into four distinct taxonomic lineages (Groups I-IV) and eight serotypes (A-H) based on its antigenic specificity and DNA relatedness [5, 6,7,8]Groups I, II and IV are generally connected with human botulism while group III is associatedwith avian and animal botulism [9.10,11,12,13]. Group III strains are well-known organism to produce type C or D and C/D or D/C mosaic BoNT[14]. In group III, type C strains are emerging and serious problems in poultry and wild birds whereas type D strains cause botulism,mainly in cattle and sheep [9, 10, 13]Botulism and associated diseases are associated with their prophages or plasmids harboringcytotoxin encoding genes (C2, Cbot3)[15, 16, 17, 18]. These extrachromosomal elements convert the toxigenic strains to non-toxigenic strains and vice versa[13]. A total of 14 genomes have been completely sequencedfor C. botulinum strains from which two genomes are group III strains.Current genomic knowledge would provide an insight of studying theirpathophysiological mechanisms at the systems level[19]. ADP-ribosylation superfamily C. botulinum C2 and C3 toxins are one of the members of ADP-ribosylation superfamily. Both toxins are grouped separately according to their structures and target binding. In the ADPribosylation, an ADP-ribose moiety of NAD+is transferred to specific target proteins in eukaryotic cells [20]. ADP-ribosylation superfamilyis categorized into six families such as NAD: arginine ADP-ribosyltransferase, binary toxin A, diphtheria toxin C, heat-labile enterotoxin α-chain, pertussis toxin S1 and poly (ADP-ribose) polymerase. It is also categorized into four families (type I-IV) based upon the protein target specificities[21,22]. Heat-labile enterotoxin A, cholera toxin A and pertussis toxin S1 are belonged to type I, which target on the heteromeric GTP-binding proteins. Diphtheria toxin A and exotoxin A are members of type II that modifies elongation factor 2. C. botulinum C3 exotoxin belongs to type III that ADP-ribosylates low-molecular-mass GTPases of the Rho subfamily. Binary toxin A belongs to type IV, which acts on actin of eukaryotic cells. ADP-ribosylation reactions are classified into four major groups: mono-ADPribosylation, poly-ADP-ribosylation, ADP-ribose cyclization and formation of O-acetyl-ADPribose. Mono-ADP-ribosylation and formation of O-acetyl-ADP-ribose are conserved reactions occurring across the prokaryotes and eukaryotes [23]Apart from poly (ADP-ribose) polymerase, other ADP-ribosylationfamilies are widely found in 136 bacterial species. ADPribosylationsuperfamily plays a diverse role in molecular pathogenesis and virulence of many pathogenic bacteria. 3

A conservative of amino acid substitution patterns in its catalytic domain that determinesthe family-specific function and disseminatesinto bacterial lineage. Catalytic subunitof this superfamily is conserved within family members, shared a common ancestor and duplicated within closely related organisms [24, 25]. A strong phylogenetic signal is required for domain divergence due to the slow evolutionary process in the conserved functional core found in this superfamily. Simultaneous conformational changes in dynamic subspace, substantial mutations in the functional core and coupled-amino acid mutations maintain the structure-function integrity of this superfamily [26, 27, 25]. Eighty six crystallographic structures are available for the bacterial ADP-ribosyltransferases in which 22 structures accounted for binary toxin A (Table 1). Thus, the structure, function and evolution aspects of this family are extensively reviewed in application to the development of therapeutics. C. botulinum C2 toxin Binary toxin A family comprises of C. botulinum C2 toxin, C. perfringens ι-toxin, C. difficile toxin (Cdt), C. spiroforme toxin, and Bacillus cereus vegetative insecticidal protein (VIP2). This family is typically divided into two functionally distinct subunits such as a catalytic domain and a translocation/cellular binding domain [28] C2 toxin encoding genes are found in the chromosome and plasmids of C. botulinum strains D-1873, BKT028387, (C)-203U28d and BKT015925 [29,30,19,10] C2 toxin induces necrotic-hemorrhagic lesions, vascular permeability and hypertension. It accumulates a lethal fluid in the lungs and intestinal tracts of various animals [31, 32]. It blocks a gelsolin-actin complex for its nucleation activity in intact neutrophils [33,34]. It is also responsible for delayed caspase-dependent apoptotic cell death in human HeLa and Vero cells [35]. Cellular uptake mechanism C2 toxin consists of an enzymatic component C2I with 431 residues (49,306 Da) and a binding/translocation component C2II with 721 residues (80,800 Da). As shown in Fig.1, both of components are assembled to exhibit the cytotoxic activity in the respective hosts [36].Active C2I is delivered into the cytosol from an acidic endosome during endocytosis. This process is physically accelerated by a host cell chaperone (Hsp90) and several protein-folding enzymes such as cyclophilin A, cyclophilin 40 and FK506 binding protein 51 [37,38,39,40]C2II component is cleaved at theLys181-Ala182 residuesin the N-terminal by a gut protease and released as a residual fragment C2IIa [41,42].This residual fragment C2IIabinds with a receptor at the cell surface and mediates the uptake of C2I via clathrin- and RhoA-dependent endocytosis, and dynamin-dependent pathways [43, 44]. C2IIa is trafficked to the lysosome from late endosome via microtubule-based transport [45]. 4

C2IIa oligomer recognizes the asparagine-linked carbohydrates on the surfaces of on the surfaces of target cells, inserts into the membrane, and forms pores under acidic pH in the endosomal compartment [46]. The core structure of asparagine-linked carbohydrate is ((GlcNAc–Man) 2–Man–GlcNAc–GlcNAc–Asn)-). C2IIa binds to the C2I and then translocated into the cytosol, where active C2I performs ADP-ribosylation at residue Arg177 of G-actin [47].G-actin is essential for architecture, morphology and structure of the cells. C2I-mediated ADP-ribosylation reduces the ability of actin to undergo polymerization, leading to disruption of the cytoskeleton architecture in cells becoming rounder and cell death [48, 32] Structure and function of C2I C2I component consists of two domain structures, which are found in the residues 2–221 (Nterminal adapter domain) and 222–431 (C-terminal catalytic domain) (Fig.2a). A segment 29– 86 (α1-helix) in N-terminal adapter domain is required for binding to the pre-pore C2IIa [49,42] C2I structure composes highly conserved central quartets of β-strands with two middle strands It contains four loops at positions 180, 204, 320 and 384 and α1-α2 helices as similar to bacterial ADP-ribosyltransferases[50].C2I requires unfoldingfor translocation that depends more on the contact to the pore than on a low pH [42]. The role of functional residues in the ADP-ribosyltransferase activity of C2I was studied by site directed mutagenesis [51,52] Arg299, Phe358 and Glu389 in the C-terminal domain perform the catalytic function of this enzyme. The side chain of Arg361 adopts a defined conformation of C2 structure [42]. Structure and function of C2II C2II component is composed of five domains (C2II20, D1-D4) as shown in Fig. 2b. The domains D1-D3 are required in the recognition of C2I, pore forming and oligomerization processes [42]. The C-terminal domain D4 has almost exclusively β-strands responsible for receptor binding and internalization [41]. Electron microscopic studies have shown that C2IIaoligomerformsthe annular structurewith an inner diameter of about 2–4 nm and an outer diameter of about 11–13 nm [53].Crystallographic studies demonstrated that there are no large conformational changes occur between pH 4.3 and pH 6.0 as the relative orientation and positions of D1-D4 domains do not alter drastically. Site-mutagenesis studies investigated the channel and pore forming properties of C2II on actin [54,55].A model to the C2IIa pre-pore structure was developed to understand how a pre-pore to a pore conversion is resulted on the membrane composing a large number of asparagine residues at acidic condition. C2IIa performs not only membrane insertion, but also unfolding and/or translocation of C2I due to the conformational changes of C2II-heptamers induced at low pH.C2I translocation channel is

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the abundance of carboxamides from Gln398, Asn378, Asn459, Asn464, Asn466, Asn474 and Asn475 pointing to the lumen of the pre-pore[42]. Catalytic mechanism Site-directed mutagenesis experiments confirmed its substrate specificity and recognition of NAD-binding core and structure-function relationships [29, 52]. Crystallographic studies elucidated its molecular mechanism of C2I and C2IIa interaction and the translocation of C2I into the target cell[42].A catalytic residue Glu387 in EXE motif may deprotonate Arg177 in actin.Glu389 forms a hydrogen bond with the O′2 on N-ribose that stabilizes the oxocarbeniumcation[56, 57, 58, 59, 29, 51] Glu387 and Glu389 are not essential for NADglycohydrolase activity[60].According to a strain-alleviation model, nicotinamide cleavage occurs via a Sn1 reaction induced by a nicotinamide mononucleotide ring-like structure (Fig. 3). The first oxocarbeniumcation intermediate is formed with a strained conformation. The second cationic intermediate is induced by alleviation of strained conformation by NP-NO5 rotation. C2I at the Arg177 of actin nucleophilically attacks the NC1 of the oxocarbenium ion, leading to α-selective ADP-ribosylation[49,60].Thus, an intensive analysis of its structure, function and catalytic mechanism would support to validate this toxin as a subunit-vaccine candidate. Molecular evolution of C2I C2I and C3 retain a similar folding of a core β-strands surrounded by α-helices [61]. The Nterminal adapter domain of C2I and α1-helical structure corresponds to the N-domain and α1 structure of VIP2 and Ia[49, 62, 63]. A serine–threonine–serine sequence (STS) is a common conserved motif in many ADP-ribosyltransferases [20].The catalytic domain of C2I phylogenetically resembles with VIP2, Bcer, Ia and CdtA (Fig. 4a). Evolutionary constraints are

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integrity[25,64]. The phenotypic consequences of mutations may determine its functional specificity and NAD-binding during ADP-ribosylation. Avirulent mutants derived from C2I are structurally stable, followed the common blueprint of folding process and also attained near-native back conformation. Furthermore, compensation mutation and residue-coupling stabilize its local structural environments and retained the avirulent function in the mutant proteins[65]. Molecular evolution of C2II C2II toxin is shown similarity in structure and function of protective antigen (PA) of anthrax toxin and Ib [66, 67] (Fig. 4b). Domain D1 is involved in interaction with C2I, D2 in pore formation, and D3 in oligomerization as analogous to the PA [67]. Domains D1-D3 show 6

sequence similarity with those domains in PA, but D4 has no sequence similarity with D4 of PA, Ib, Cdtb. It indicates that D4 plays an exclusive role in stabilizing a conformation required or receptor binding and cytotoxicity [68, 41]. Glu307 is the only negatively charged amino acid among residues 303-331 of C2II that forms channel likely to the PA of anthrax toxin [69,70,71].The corresponding glutamic acid residue is absent in Ib, but it likewise requires lower pH value to deliver Ia into the cytosol [72,73].The cis-side vestibule of C2II channel contains many negatively charged amino acids as those found in PA, VIP2, and Ib[69, 70, 71].The binding of C2IIa oligomers to C2I is same to that of PA and Ib [45]. It suggests that C2II, Ib and PA are structurally and functionally homologous to each other. C. botulinum C3 exotoxin C3 exotoxin is the prototype of a family of bacterial ADP-ribosyltransferases. C.botulinum (C3bot1; C3bot2), C.limnosum(C3lim), Bacillus cereus (C3cer) and Staphylococcus aureus(C3Stau1; C3Stau2; C3Stau3) [74] are known organisms able to produce this toxin.C.botulinum strains C-Stockholm [19], 08-BKT015925 [10],Eklund-C [75] and South African, 1873, CB16, 4947 [15, 16,17]are studied for the occurrence of C3 exotoxin coding genes [76]Oguma, 1986). Gene coding for this toxin is located in their phages and plasmids [13]. C3 exotoxin exerts a low cytotoxic activity only on avian and mammalian rho subfamily proteins by its active site moiety. Its incidence ofnatural disease is unknown [77]. Ithas been used as a molecular tool forstudying the cellular contribution of rho subfamily proteins in activation of phospholipase D, lymphocyte-mediated cytotoxicity, cell motility and thrombininduced platelet aggregation. It is also used as drug in the treatment of paraplegia [78]. Cellular uptake mechanism C3 toxin exhibits ADP-ribosyltransferaseactivity, but apparently lacks any specific cell binding and transportation unit like a typical binary toxin. However, in addition to the catalytic domain, it contains a unit for cell binding and transportation. C3 toxin enters into the cytosol of monocytes/macrophages by a non-specific pinocytosis through partially acidified early endosome mimicking the endosomal process [79, 80, 81] (Fig. 5). It significantly binds to C3binding sites of proteinaceous structures on the macrophages. The uptake and internalization of it in macrophages occurs via vimentin, a type III intermediate filament protein, and dynamindependent mechanism ([82,83].C3 toxin modifies a low-molecular-mass GTPases of the Rho subfamily at the Asn41 residue. C3-catalyzed ADP-ribosylation of Rho depolymerises the actin cytoskeleton in the cytosol of the macrophages [84, 85, 86, 49, 80]. C3-mediated impairment of Rho-signalling inhibits the migration and phagocytosis function of macrophages. This pinpoints that C3 toxin acts as an immunosuppressive agent [87, 7

88]. Rho proteins are involved in cell and smooth muscle contraction, phagocytosis, polarity, activation of transcription, cell cycle progression and cell transformation [89].All the molecular and cellular functions are terminated by intrinsic hydrolysis of Rho-GTP [88]. C3-mediated ADP- ribosylationblocks the growth factor-stimulated formation of stress fibers and focal adhesions in fibroblasts [90,91]. C-terminal peptide fragment of C3 (covering residues 154182) selectively acts on neurons to enhance the neuronal outgrowth, connectivityand reinnervation [92]. Consequently, C3 toxin/peptide has taken a major importance to enhance neuronal regenerative growth and connectivity [93, 94]. Molecular structure C3 exotoxin is a 25 kDa protein containing the ADP-ribosylation site. Catalytic function and substrate recognition of it are mediated by ADP-ribosylating turn-turn (ARTT) motif (xxQxE) [95, 52, 96, 97, 98]. A conserved β-structure core is assembled by joining β5 to β6,which anchor the ARTT motif (Fig. 6). Two perpendicular anti-parallel β-sheets in its catalytic domain form a cleft at the interface. A catalytic Glu214 is the fourth residue in the second of two turns joining β5 and β6. Four α-helices flank the three-stranded β-sheet and an additional α-helix flanks the five-stranded β-sheet (Han et al., 2002). NAD-binding site in the C3 structure is flanked by a β-sheet core and a α3-helix [99,100]. The substrate recognition specificity of this enzyme can be determined by NAD-binding β-sandwich toxin fold located near to the NAD-binding pocket [57,58,95] In C3 toxin structure, a general flexure is concentrated in two peripheral lobesfor NAD-binding site [96,97]. The side chain of Phe209 in turn 1 interacts with the hydrophobic region adjacent tothe GTP-binding site of the Rho subfamily [99,100,101,]. A phosphate nicotinamide loop (180– 187) conformation recognizes nicotinamidecapping with the side chain of Phe183 [96]. NADinduced ARTT loop conformation formsC3-NAD complex with RhoA in which the α4-helix and β6-strand of RalA are recognized [78, 97,102]. C3 structure contains two segments defining ‘‘in’’ and ‘‘out’’ conformation. The first segment has two conserved anchor hydrophobic residues (I203 and I206). The second segment comprises of two hinge residues, Ser207 and Gln212, which allowschanging the ARTT loop conformations [98]. C3 toxin is a single-domain protein with bifunctional properties encompasses target specificity to the RhoA, RhoA and RhoC out of 160 GTPase of the Ras superfamily. Such target specificity is the outstanding feature of C3 exotoxinfor designing subunit vaccines [102]. Molecular function

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Several mutants of this toxin have been developed by site directed mutagenesis for inactive NAD-glycohydrolase activity [97] and ADP-ribosyltransferase activity [81, 82]. A catalytic residue Glu214 is required for NAD-glycohydrolysis[74,103,104] and Gln212 is essential for ADP-ribose transfer to the target protein [97]. Phe209 is involved in protein target recognition and interaction [85, 86]Amino-acid exchanges in a functional core of this toxin may bring a major reduction in ADP-ribosyltransferase activity and its molecular interaction with Rho GTPases[98, 82]. Most recently, structure-function disparityhas been predicted in C3 toxins obtained from two different C. botulinumtype C phage and type D phage. Avirulent toxins derived from C3 toxin is evaluated for the prioritization and rationalization of subunit vaccines to prevent disease caused by C. botulinumphagesharboring C3 toxins [105]. Catalytic mechanism A catalytic residue Glu173 may deprotonate the target protein substrate, allowing Sn2-type reaction on the nicotinamide-ribose bond [74, 85, 86,].The deprotonated α-carboxyl group of Glu173 serves as an acceptor for a proton released during ADP-ribosylation reaction [103]. The oxocarbenium transition state is stabilized by Glu214, where it forms a hydrogen bond with the O2’ hydroxyl group of the nicotinamide ribose [95]. Arg85 appears as important binding site residue to form π–π interactions with NAD+[86,106].Glu214 makes direct interactions with NAD+ irrespective of ARTT loop conformation [96, 97, 98]. Gln212 sidechain in turn 2 is required not only for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD- ribose, but also to hold the Glu214 catalytic side-chain in the adjacent catalytic pocket [95] (Fig. 7). A solvent-exposed aromatic side-chain of Phe or Tyr is used for substrate recognition by forming hydrophobic interactions. Phe209 is positioned into the hydrophobic pocket consisting of Phe49, Trp58, and Ile124. The main chain carbonyl and amide group of Gly129 form hydrogen bonds to the carboxamide group of NAD+. The catalytic mechanism of C3 toxin was proposed as the basis of its structurefunction integrity [105]. Since, the identification of amino acid residues that interact with NAD+ and the substrate is imperative to validate this toxin as a vaccine candidate. Molecular evolution of C3 A cross evolution between enzymatic and transmembrane protein ancestors is a decisive force to determine its structure-function integrity [18]. C3 toxin shares 63% sequence identity with C3lim and 30% identical to C3cer (Fig. 4). Site directed mutagenesis studies found a common core structure and conserved ARTT motif required for the NAD-hydrolysis step [74,103,104] C3-like toxins have a similar NAD-binding mechanism, but differing mechanisms of target protein binding due to sequence variations within the ARTT motif structural framework 9

[52]Compensation mutations and blueprint of the folding process are very significant to maintain its native local structural environment, enabling to retain the molecular function [105]. Subunit vaccines in research Subunit vaccines are typically but not exclusively protein molecules. Antigenic protein prepared from any causative agent (bacteria or virus) is used to provide immunity against one or several diseases. Antigenic or immunogenic protein antigens stimulate the immune system, instead of dealing with the entire microorganisms.Vaccination combined with antigens produces neutralizing antibodies that will bind and clear the toxins before it blocks neurotransmission.A properly folded conformational epitope is a primary determinant for inducing toxin-neutralizing antibodies [107,108,109].Several vaccines have been successfully developed

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botulism[110,111,112,113,114], but there is no vaccine to prevent the cytotoxin-mediated diseases in poultry and veterinary animals. C2 and C3 toxins as vaccine candidates C2 and C3 toxins are virulence factors ofC. botulinum. Thecellular mode of action and cytotoxic activity of both toxins are a fully understood[61]. Sequence-structure-function of C2 and C3 toxins are evolutionarily conserved for their catalytic function, shared sub-dynamic space for NAD-binding, and expected sequence variability along a lineage with a constant rate of independent substitutions [25, 64, 65,105]. These are the prime targets to screen antigenic determinants from these toxins for vaccine designing and development [115,116]. Off-target toxicity with enhanced potency is added property of a subunit vaccine if a protein does not have a receptor binding capacity [117].C3 toxin has a no separate binding domain, but it has a general mechanism for internalization to the target cells. This is a more additional property for vaccine designing from C3 toxin. A non-catalytic ADP-ribosyltransferase domain is used in the development of cholera toxin gene-mediated adjuvant [95] revealed the structure and function disparity of C3 toxin that provided a guidance for designinga rational-based subunit vaccine from avirulent mutants. Structure-function-evolution link thus provides an insight to engineer avirulent toxin by removing key residues involved in toxic enzymatic activity whilst retaining immunogenicity[65,105] Conclusion Both toxins are playing a very important role in diverse pathophysiological functions and intracellular signalling of the hosts. A unified understandingabout the structure, function and evolution of C2 and C3 toxins is described in this review on the development of subunit vaccine 10

against C. botulinum infections in the birds and veterinary animals. Enzyme-deficient mutants of C2 and C3 toxins are currently used as novel delivery systems (molecular Trojan horses) that allowing selective and specific transport of drug/protein into the cytosol of human macrophages [81,118,119].Engineered C3 toxin is employed as a molecular tool to modulate Rho- and actin-associated cell functions in human monocytes and macrophages [88]. Evolutionary constraints that imposing on the sequence-structure-function link of these toxins is useful biological measures to design subunit (avirulent) toxins by removing key residues involved in toxic enzymatic activity whilst retaining immunogenicity. Computational protein engineering advances the experimental setup to address the challenges associated with utilizing toxic proteins in developing immunotoxins for C. botulinum infections in avian and cattle. Apart from the molecular level, C. botulinumgenomes should be compared together to elucidate their molecular pathogenesis mechanisms by predicting the production capacity of an array of metabolites involved in virulence. A proper reconciliation of metabolic constructions at the genome-scale is crucial for studying their genetic and phenotypic differences within species and to emphasize the metabolic connections to virulence and pathogenicity. The metabolic models of these genomes would help to predict their cellular behaviour before laborious experimental work and also to discover, and design the potential veterinary vaccines and drugs with minimum side effects. Acknowledgements Life Science Research Board-Defense Research and Development Organization (Sanction No. DLS/81/48222/LSRB-249/BTB/2012), New Delhi, India, is duly acknowledged for financial support. Declarations of interest The authors confirm that this article’s content has no conflictsof interest. References [49]Aktories, K.; Barth, H.Clostridium botulinum C2 toxin – New insights into the cellular uptake of the actin-ADP-ribosylating toxin. Int. J. Med. Microbiol.,2004, 293,557–564. [74]Aktories, K.; Jung, M.;Bohmer, J.;Fritz, G.;Vandekerckhove, J.; Just, I. Studies on the active-site structure of C3-like exoenzymes: involvement of glutamic acid in catalysis of ADPribosylation. Biochimie.,1995, 77,326-332. [61] Aktories, K.; Lang, A.E.; Schwan, C.;Mannherz, H.G. (2011). Actin as target for modification by bacterial protein toxins. FEBS. J.,2001, 278, 4526-43. [79] Aktories, K.; Schmidt, G.; Just. I. Rho GTPases as targets of bacterial protein toxins. Biol. Chem.,2000, 381,421–26. 11

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Tables with captions Table 1 Crystallographic structural information of binary toxin A and Clostridiumbotulinum C3 exotoxin to date BCE: Bacillus cereus, C2I: Clostridium botulinumC2toxin enzyme component I, C2II: Clostridium botulinumC2toxin binding and translocation component II, C3I: Clostridium botulinumC3 exoenzymecomponent I, C3II: Clostridium botulinumC3 exoenzymecomponent II, C3lim:Clostridium limosum C3 exoenzyme, C3stau2: Staphylococcus aureusC3 exoenzymecomponent 2 , CBO: Clostridium botulinum, CBOC: Clostridium botulinum type C phage, CBOD: Clostridium botulinum type D phage, CDI: Clostridium difficile, DTa: Clostridium difficile toxin enzyme component A, CDTb: Clostridium difficile toxin binding component B, CLI: Clostridium limnosum, CPF: Clostridium perfringens, Ia: Clostridium perfringens iota-toxin enzymatic component, Ib: Clostridium perfringens iota-toxin binding component , SAU: Staphylococcus aureus

Figures with captions Figure 1Cellular up-take and cytotoxic action of Clostridiumbotulinum C2 toxin Figure 2 Structure and function of Clostridiumbotulinum C2I subunit (a) and C2II subunit (b) Figure 3 Catalytic mechanism of Clostridiumbotulinum C2 toxin. Figure 4 Molecular diversity of Clostridiumbotulinum C2 and C3 toxins. Evolutionhistory of Clostridial binary toxins was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2011). Figure 5Cellular up-take and cytotoxic action of Clostridiumbotulinum C3 toxin Figure 6 Structure and function of Clostridiumbotulinum C3 toxin Figure 7Molecular recognition of Clostridiumbotulinum C3 with NAD-RalA (a), GDP bound conformation of RalA (b) and NAD+ (c).

22

Table 1

Organism

PDB ID

Molecule

Length

PubMed

CBO

2J3V:A

C2I (pH 3.0)

431

[42]

CBO

2J3Z:A

C2I (pH 6.1)

431

[42]

CBO

2J3X:A

C2I (pH 3.0)

431

[42]

CBO

2J42:A

C2II

721

[42]

CBO

1UZI:A

C3 (tetragonal)

211

[96]

CBO

1G24:A

C3

211

[95]

CBO

2BOV:B

C3-RalA

251

[78]

CBOC

1R4B:A

C3 (monoclinic)

204

-

CBOC

1R45:A

C3 (triclinic)

204

-

CBOD

1GZE:A

C3 (L177C)

211

[97]

CBOD

1GZF:A

C3-NAD+

211

[97]

CBOD

2C89:A

C3

211

[98]

CBOD

2C8A:A

C3-Nicotinamide

211

[98]

CBOD

2C8B:X

C3 (Q212A)

211

[98]

CBOD

2C8C:A

C3 (Q212A)-NAD+

211

[98]

CBOD

2C8D:A

C3 (Q212A)

211

[98]

CBOD

2C8E:E

C3 (E214N)

211

[98]

CBOD

2C8F:E

C3 (E214N)-NAD+

211

[98]

CBOD

2C8G:A

C3 (Q182A)

211

-

CBOD

2C8H:A

C3I (Q182A)-NAD+

211

-

CBOD

2A78:B

C-RalA

223

[102]

CBOD

2A9K:B

C3-NAD-RalA

223

[102]

CDI

2WN4:A

CDTa (pH 4.0)

463

[120]

CDI

2WN5:A

CDTa (pH 8.5)

463

[120]

CDI

2WN6:A

CDTa-NADPH

463

[120]

CDI

2WN7:A

CDTa-NAD+

463

[120]

CDI

2WN8:A

CDTa (pH 9.0)

463

[120]

CLI

3BW8:A

C3lim

217

[100]

23

CFE

1GIQ:A

Ia-NADH

413

[100]

CFE

1GIR:A

Ia-NADPH

413

[100]

CFE

3BUZ:A

Ia-Actin

413

[63]

CFE

4GY2:A

Apo-Ia-NAD-Actin

418

[60]

CFE

4H03:A

Ia-NAD-Actin

418

[60]

CFE

4H0T:A

Ia-ADPR-Actin

418

[60]

CFE

4H0V:A

Ia(E378S)-NAD-Actin

418

[60]

CFE

4H0X:A

Ia(E380A)-NAD-Actin

418

[60]

CFE

4H0Y:A

Ia(E380S)-NAD-Actin

418

[60]

SAU

1OJQ:A

C3stau2

212

[96]

SAU

1OJZ:A

C3stau2-NAD+

212

[96]

BCE

1QS1:A

VIP2

462

[50]

BCE

1QS2:A

VIP2-NAD+

401

[50]

Figure 1

24

Figure 2

25

26

Figure 3

Figure 4 (a) 899 519 1000 279 929

996 580 1000 1000 1000

1000 1000

393

1000 39 24 411

C2I C2I Type C C2I C2I C. botulinum C2I Type D C2I C2I VIP2AC BcerA VIP2 Bacillus VIP2 VIP2 Ia Ia Ia Ia CdtA C. difficile Ia Ia Ia Ia 27

Figure 4 (b) 18 22 4 17

100

2 3

33 11 33 50

CdtB CdtB Ib Ib Ib Ib Ib PA C2II C2II C2II C2II C2II

C.difficile

Bacillus anthracis

C.botulinum

(c) 54 90 73

79

100 100 96

96 100 99

C3 C3 C3 C.botulinum C3 C3 C3Stau2 C3Stau1 Staphylococcus aureus C3Stau1 C3cer Bacillus cereus C3cer 2A C3 Paenibacillus C3

28

Figure 5

29

Figure 6

30

Figure 7

31

32