Functional Differences in Pertussis Toxins from Bordetella pertussis Clinical Isolates as Determined by in vitro and in vivo Assays Catpagavalli Asokanathan 1 , Chun-Ting Yuen 1* , Dorota Kmiec 1 , Jun X Wheeler 1 , Kevin Markey 1 , Imogen Kelso 1 , Barbara Bolgiano 1 , Qiushui He 2, Frits R Mooi 3 and Dorothy Xing 1 1 National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK 2 Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland 3 Laboratory for Infectious Disease and Screening, Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, Netherlands * Corresponding Author: Yuen CT, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK, Tel: 4401707 641000; Fax: 4401707 641054; E-mail: ct.yuen@nibsc.org Received date: June 29, 2015; Accepted date: July 28, 2015; Published date: July 30, 2015 Copyright: © 2015 Asokanathan C et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Whooping cough caused by Bordetella pertussis is a serious disease especially for infants and young children. Detoxified pertussis toxin is a key component of vaccines used in campaigns worldwide for the prevention of the disease. A biochemical assay system, which measures pertussis toxin enzymatic and carbohydrate binding activities, has been developed to measure the residual toxin activities in vaccine matrix. We report here that B. pertussis clinical isolates show differences in pertussis toxin (PTx) activities in the in vitro biochemical assays and these in vitro activities were also positively related to the in vivo toxic activities. In addition, interesting information on the genetic and possible post-translational changes of PTx produced by the strains included in this study is tentatively discussed. Of the six strains studied in details, three low- and three high-activity strains, all the DNA sequences of the ptx gene clusters were found to be identical, except that in the high-activity strains, there was a silent mutation with a single nucleotide change at base 681 in ptxC which coded for Cys199 of PTx subunit S3. Mass spectrometric analysis of tryptic peptides from PTx produced by these strains detected two peptides in subunits S1 and S3, which were present in the low-activity strains but not in the high-activity strains. This suggests that PTx might differ in structure between these strains and this is possibly due to post-translational modification. Keywords: B. pertussis; Strains; Pertussis toxin; Binding; Enzyme; Histamine-sensitisation; Lymphocytosis Abbreviations: aP: Acellular Pertussis; ACV: Acellular Pertussis Vaccines; AEBSF: 4-(2-Aminoethyl) Benzenesulfonyl Fluoride Hydrochloride; AGP: Human Plasma Alpha-1 Acid Glycoprotein; EDQM: European Directorate for the Quality of Medicines and Healthcare; Fet: Fetuin; FHA: Filamentous Haemagglutinin; Fim2 and Fim3: Fimbriae type 2 and type 3; HIST: Histamine-Sensitisation Test; hTf: Human Transferrin; i.p: Intra-Peritoneally; NIBSC: National Institute for Biological Standards and Control; Prn: Pertactin; ptx: Pertussis Toxin Allele; PTx: Pertussis Toxin; ptxA: PTx Subunit S1 Allele; ptxC: PTx Subunit S3 Allele; PTd: Pertussis Toxoid; PBSG: Phosphate Buffered Saline Containing 0.2% (w/v) Gelatine; PFGE: Pulsed Field Gel Electrophoresis; RNaseB; Bovine Pancreas Ribonuclease B; WCV: Whole-Cell Pertussis Vaccines Introduction Pertussis (whooping cough) is still a major cause of morbidity and mortality, especially in infants and vaccination against Bordetella pertussis is a key component of immunisation campaigns worldwide for the prevention of the disease. Pertussis toxin (PTx) is a protein toxin which has a wide range of biological activities in vivo including induction of lymphocytosis, histamine-sensitisation, increased insulin production with consequent hypoglycaemia, potentiation of anaphylaxis and lethality in mice and playing a possible role in neuropathology [1-3]. Excessive residual PTx activity in acellular pertussis (aP) combination vaccines that contain diphtheria and tetanus toxoids (DTaP) could also cause intensified sensitisation to components with subsequent severe local reaction to a booster dose in an animal model [4]. Although these biological effects of PTx have been under extensive research, its mechanism(s) of toxicity is still unclear. Two in vivo assay procedures are commonly used for assessing toxicity of PTx: (1) mouse lymphocytosis test which is based on the fact that the severity of pertussis infection in infants was found to accompany with extremely high lymphocyte counts and this characteristic has been attributed to the PTx [5]; (2) mouse histamine sensitisation test (HIST) where PTx, when administered in vivo, enhances vascular permeability and therefore is capable of inducing vasoactive amine-sensitising activities which can result in death due to hypotensive and hypovolemic shock following vasoactive amine (e.g. histamine) challenge of PTx-treated animals. HIST is currently the accepted WHO and European Pharmacopoeia safety test for determination of PTx activity in pertussis vaccines [6,7]. PTx has the A-B 5 type structure typical of many other bacterial toxins, having an enzymatically active A-protomer, the monomeric subunit S1, and a host cell binding B-oligomer which is composed of subunits S2 through to S5 [8,9]. Intact B-oligomer is required for the binding of the holotoxin to receptor sites on the cell surface and enables entry of the A-protomer into the cells [9,10]. The translocated PTx A-protomer catalyses ADP-ribosylation of the α-subunit of eukaryote GTP-binding regulatory proteins, which results in the prevention of the hormonal inhibition of adenylate cyclase and subsequently, an increase in the intracellular levels of cAMP and cell Asokanathan et al., J Clin Exp Pathol 2015, 5:5 DOI: 10.4172/2161-0681.1000243 Research Article open access J Clin Exp Pathol ISSN:2161-0681 JCEP, an open access journal Volume 5 • Issue 5 • 1000243 J o u r n a l o f C l i n i c a l & E x p e r i m e n t a l P a t h o l o g y ISSN: 2161-0681 Journal of Clinical & Experimental Pathology