Engineering of Cytolethal Distending Toxin B by Its Reducing Immunogenicity and Maintaining Stability as a New Drug Candidate for Tumor Therapy; an In Silico Study.

The cytolethal distending toxin (CDT), Haemophilus ducreyi, is one of the bacterial toxins that have recently been considered for targeted therapies, especially in cancer therapies. CDT is an A-B2 exotoxin. Its catalytic subunit (CdtB) is capable of inducing DNA double strand breaks, cell cycle arrest and apoptosis in host eukaryotic cells. The sequence alignment indicates that the CdtB is structurally homologyr to phosphatases and deoxyribonucleases I (DNase I). Recently, it has been found that CdtB toxicity is mainly related to its nuclease activity. The immunogenicity of CDT can reduce its effectiveness in targeted therapies. However, the toxin can be very useful if its immunogenicity is significantly reduced. Detecting hotspot ectopic residues by computational servers and then mutating them to eliminate B-cell epitopes is a promising approach to reduce the immunogenicity of foreign protein-based therapeutics. By the mentioned method, in this study, we try to reduce the immunogenicity of the CdtB- protein sequence. This study initially screened residue of the CdtB is B-cell epitopes both linearly and conformationally. By overlapping the B-cell epitopes with the excluded conserve residues, and active and enzymatic sites, four residues were allowed to be mutated. There were two mutein options that show reduced antigenicity probability. Option one was N19F, G74I, and S161F with a VaxiJen score of 0.45 and the immune epitope database (IEDB) score of 1.80, and option two was N19F, G74I, and S161W with a VaxiJen score of 0.45 and IEDB score of 1.88. The 3D structure of the proposed sequences was evaluated and refined. The structural stability of native and mutant proteins was accessed through molecular dynamic simulation. The results showed that the mutations in the mutants caused no considerable changes in their structural stability. However, mutant 1 reveals more thermodynamic stability during the simulation. The applied approaches in this study can be used as rough guidelines for finding hot spot immunogen regions in the therapeutic proteins. Our results provide a new version of CdtB that, due to reduced immunogenicity and increased stability, can be used in toxin-based drugs such as immunotoxins.

Identification of cholix toxin gene in Vibrio cholerae non-O1/non-O139 isolated from diarrhea patients in Bushehr, Iran.

BACKGROUND AND OBJECTIVES: Cholixin (cholix toxin) is a novel exotoxin in Vibrio cholerae identified as an elongation factor II specific ADP-ribosyltransferase which inhibits protein synthesis in the eukaryotic cell. Previous researches have suggested that cholixin probably is an important virulence factor in non-O1/non-O139 V. cholerae (NAG) serotypes that could be related to extra-intestinal rather than intestinal infections. This study was aimed to investigate the frequency and genetic diversity of colixin gene (chxA) in clinical V. cholerae NAG isolates. MATERIALS AND METHODS: The presence of chxA gene in 44 clinical V. cholerae NAG isolates were screened using PCR through specific primers designed for the receptor-binding domain (RBD) of chxA gene. The five PCR products of chxA gene were sequenced. RESULTS: This study showed that chxA gene presented in 19 V. cholerae NAG isolates. The sequences analysis of 5 out of 19 the partial chxA genes amplicon showed that 4 of them belonged to chxA I and the other one belonged to chxA II subtypes. Two distinct clusters were revealed for these isolates by phylogenic analysis, too. CONCLUSION: The chxA gene contained high frequency among V. cholerae NAG isolates in Bushehr, Iran. The polymorphism study on RBD of cholixin gene is suggested as an appropriate method for phylogenic characterization of the various chxA gene subtypes.