A human-food web-animal interface on the prevalence of food-borne pathogens (Clostridia and Enterococcus) in mixed veterinary farms.

In the present work, we addressed the impact of a human-food web-animal interface on the prevalence of food-borne pathogens in mixed farms of Tamil Nadu, India. We have isolated and identified six strains of Clostridium sp. and five strains of Enterococcus sp. from food and animal sources disposed near to the veterinary and poultry farms. Phylogenetic relationships of these strains were inferred from their homologies in 16S rDNA sequences and rRNA secondary structures. The strain PCP07 was taxonomically equivalent to C. botulinum confirmed by neurotoxin-specific PCR primers, followed by mouse bioassay. Other Clostridial and Enterococcal isolates have shown a phylogenetic similarity to the C. bifermentans and E. durans isolated from veterinary farms, respectively. Results of our study revealed that a human-food web-animal interface has influenced the disease incidence and prevalence of these isolates in the poultry to veterinary farms, where human food acted as a likely transmittance vehicle for their infections.

Cloning and expression of immunogenic Clostridium botulinum C2I mutant proteins designed from their evolutionary imprints.

C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals.

Deciphering structure, function and mechanism of Plasmodium IspD homologs from their evolutionary imprints.

Malaria is a life-threatening mosquito-borne blood disease caused by infection with Plasmodium parasites. Anti-malarial drug resistance is a global threat to control and eliminate malaria and therefore, it is very important to discover and evaluate new drug targets. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) homolog is a second in vivo target for fosmidomycin within isoprenoid biosynthesis in malarial parasites. In the present study, we have deciphered the sequence-structure-function integrity of IspD homologs based on their evolutionary imprints. The function and catalytic mechanism of them were also intensively studied by using sequence-structure homology, molecular modeling, and docking approach. Results of our study indicated that substrate-binding and dimer interface motifs in their structures were extensively conserved and part of them closely related to eubacterial origins. Amino acid substitutions in their coiled-coil regions found to bring a radical change in secondary structural elements, which in turn may change the local structural environment. Arg or Asp was identified as a catalytic site in plasmodium IspD homologs, contributing a direct role in the cytidylyltransferase activity similar to bacterial IspD. Results of molecular docking studies demonstrated how anti-malarial drugs such as fosmidomycin and FR-900098 have competitively interacted with the substrate-binding site of these homologs. As shown by our analysis, species-specific evolutionary imprints in these homologs determine the sequence-structure-function-virulence integrity and binding site alterations in order to confer anti-malarial drug resistance.

Molecular evolution and functional divergence of IspD homologs in malarial parasites.

Malaria is one of the leading parasitic diseases to humans caused by Plasmodium falciparum. It is imperative to discover novel targets for the development of antimalarial drugs. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) in 2-C-methyl-D-erythritol-4-phosphate pathway has been considered as a second in vivo off-target for antimalarial drugs discovery as its essentiality in malarial parasites and devoid in mammals. Our study was intended to reveal the molecular basis of its functional parts by inferring diversity, origin and evolution across important malarial parasites. Phylogenetic analyses revealed its conservation probability and sequence homology among bacterial IspD homologs. It also indicated that Plasmodium IspD homologs were distantly related to each other and their functional counterparts originated from different progenitor genes. Nucleotide-diphospho-sugar transferase fold and conserved domain of them might have evolved from green sulphur bacteria, whereas coiled-coil region and apicoplast targeting signal derived from protozoal origins. These homologs contained prospectively definable motifs subject to neutral or nearly neutral evolution on a scale that were diverged radically and subsequently converged in making spatial structural arrangements. Our genetic diversity analysis has shown a constructive signal for identifying the evolutionary constraints, which has imposed on their functional divergence in malarial parasites. Thus, this study provides a novel insight into our understanding of the molecular basis of the origin and evolution history of IspD homologs across apicomplexa.

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 and C3 toxins in addition to botulinum neurotoxins in avian and mammalian cells. C2 and C3 toxins are members of bacterial ADP-ribosyltransferase superfamily, which modify the eukaryotic cell surface proteins by ADP-ribosylation reaction. Herein, the mutant proteins with lack of catalytic and pore forming function derived from C2 (C2I and C2II) and C3 toxins were computationally evaluated to understand their structure-function integrity. We have chosen many structural constraints including local structural environment, folding process, backbone conformation, conformational dynamic sub-space, NAD-binding specificity and antigenic determinants for screening of suitable avirulent toxins. A total of 20 avirulent mutants were identified out of 23 mutants, which were experimentally produced by site-directed mutagenesis. No changes in secondary structural elements in particular to α-helices and ß-sheets and also in fold rate of all-ß classes. Structural stability was maintained by reordered hydrophobic and hydrogen bonding patterns. Molecular dynamic studies suggested that coupled mutations may restrain the binding affinity to NAD(+) or protein substrate upon structural destabilization. Avirulent toxins of this study have stable energetic backbone conformation with a common blue print of folding process. Molecular docking studies revealed that avirulent mutants formed more favorable hydrogen bonding with the side-chain of amino acids near to conserved NAD-binding core, despite of restraining NAD-binding specificity. Thus, structural constraints in the avirulent toxins would determine their immunogenic nature for the prioritization of protein-based subunit vaccine/immunogens to avian and veterinary animals infected with C. botulinum.

Structure-function discrepancy in Clostridium botulinum C3 toxin for its rational prioritization as a subunit vaccine.

Clostridium botulinum is anaerobic pathogenic bacterium causing food-born botulism in human and animals by producing botulinum neurotoxins A-H, C2, and C3 cytotoxins. Physiological group III strains (type C and D) of this bacterium are capable of producing C2 and C3 toxins in cattle and avian. Herein, we have revealed the structure-function disparity of C3 toxins from two different C. botulinum type C phage (CboC) and type D phage (CboD) to design avirulent toxins rationally. Structure-function discrepancy of the both toxins was computationally evaluated from their homology models based on the conservation in sequence-structure-function relationships upon covariation and point mutations. It has shown that 8 avirulent mutants were generated from CboC of 34 mutants while 27 avirulent mutants resulted from CboD mutants. No major changes were found in tertiary structure of these toxins; however, some structural variations appeared in the coiled and loop regions. Correlated mutation on the first residue would disorder or revolutionize the hydrogen bonding pattern of the coevolved pairs. It suggested that the residues coupling in the local structural environments were compensated with coevolved pairs so as to preserve a pseudocatalytic function in the avirulent mutants. Avirulent mutants of C3 toxins have shown a stable structure with a common blue print of folding process and also attained a near-native backrub ensemble. Thus, we concluded that selecting the site-directed mutagenesis sites are very important criteria for designing avirulent toxins, in development of rational subunit vaccines, to cattle and avian, but the vaccine specificity can be determined by the C3 toxins of C. botulinum harboring phages.