Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A.
Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1-411), middle (residues 296-701), and C-terminal portions (residues 524-911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan; Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan.
Jui-Hsin
JH
Huang
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Zhe-Qing
ZQ
Shen
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Shu-Pei
SP
Lien
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Kuang-Nan
KN
Hsiao
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Chih-Hsiang
CH
Leng
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Chi-Chang
CC
Chen
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Leung-Kei
LK
Siu
Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan; Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan; Graduate Institute of Immunology, China Medical University, Taichung, Taiwan.
Pele Choi-Sing
PC
Chong
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D001428:Bacterial Vaccines / Q000276:immunology / Q000378:metabolism; D018938:Caco-2 Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile; D004768:Enterotoxins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000276:immunology; D014709:Vero Cells
D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D010446:Peptide Fragments; C041348:tcdA protein, Clostridium difficile