PTx is an AB-type bacterial toxin composed of an A-protomer made of the S1 subunit, and a B-oligomer comprised of subunit proteins S2 to S5. The B-oligomer complex is responsible for binding of the toxin to glycosylated proteins on cell membranes. Upon binding, the toxin is internalized and transported in a retrograde manner through the Golgi complex and endoplasmatic reticulum. Once in the endoplasmic reticulum, the S1 subunit dissociates from the B-oligomer and is released into the cytosol [6], where it hydrolyses NAD and transfers the released ADP-ribose to the α subunit of inhibitory/other/transducin G (Gα_i/o/t) protein-coupled receptors [7,8]. This prevents GDP to GTP exchange and fixes the Gα_i/o/t subunits in a dissociated and inactive GDP-loaded state. Consequently, the inactive Gα_i/o/t subunits cannot inhibit their target enzymesadenylate cyclases (AC), or affect other target proteins. This potentially causes an increase in the second messenger cAMP, and subsequent downstream effects (Figure 1). This mechanism of PTx action is considered to be the cause of the majority of its biological effects, including exocrine secretion, lipolysis, stimulation of pancreatic islet cells, histamine sensitization, leukocytosis, and inhibition of lymphocyte and neutrophil migration [9,10]. However, PTx-dependent ADP ribosylation might also affect various other cellular mechanisms such as the expression of α_i and α_o subunits [11,12,13], regulation of PI3K/Akt pathway [11], tuberin phosphorylation, and RSG expression [12], as well as ERK/MAP kinase pathways [13,14]. In addition, various ADP ribosylation independent effects of PTx on cell signalling have been described and include the ERK/MAP kinase pathway [15,16,17], and pathways involving tyrosine kinase [18], NF-κB [19] and Rac [20]. Other non-cAMP mechanisms related to the binding of PTx to receptors include the proliferation of T cells [15,16], glucose oxidation in adipocytes [16], and activation of TLR4 [19].

There is some debate regarding the role of PTx in the pathology of pertussis disease. Several lines of evidence underline the importance of PTx for some aspects of the disease such as leukocytosis and the paroxysmal cough [9]. However, despite its name as a toxin—and unlike many other bacterial toxins—PTx is not considered to be particularly noxious or harmful to humans and animals (Table 1). Indeed, considering its high potency to ADP-ribosylate Gα_i proteins, the in vivo response to PTx is rarely one of distress, morbidity or mortality. For example, in the mouse histamine sensitization test described below, the apparent health and behaviour of the animals is normal for several days after PTx administration. In that test, it is not the PTx to which the animals succumb, but rather the histamine administration at a dose which would otherwise be safe in mice not exposed to PTx. In humans, the acute LD₅₀ of PTx has never been estimated and it is impossible to extrapolate an unsafe amount of PTx based on animal data due to the inter-species differences. Nevertheless, a large dose of 0.5 or 1.0 μg/kg of purified PTx has been administered intravenously to adult volunteers in order to study its effect on insulin and blood glucose levels and these volunteers did not experience any reported adverse outcomes [21]. Although the prior immune status or history of vaccination to pertussis was not provided, this study suggests PTx doses in the ranges administered in the study are not toxic for adults. However, it should be kept in mind that the disease caused by B. pertussis can be extremely different in infants under three months of age and is associated with complications such as pulmonary dysfunction and hypertension, leukocytosis, and encephalopathy [22]. As the immune status and the function of the immune system of these extremely young individuals differ from adults [23], the interpretation and extrapolation of the data from studies with adult subjects should be done with care.

The diverse properties of PTx require different approaches and methods, as is reflected in the particularly diverse in vivo models and in vitro assays that are addressed in this review. All of the methods have their own characteristics and limitations, and the methods cannot easily be compared due to their diverse nature, and as a result of the different PTx preparations used across laboratories. A comparison is further compounded by the specific activity of each PTx preparation which, like any purified enzyme, is not directly related to their stated protein content (see Table 2). While early studies utilized PTx purified in house, subsequent studies often included an international reference preparation. The JNIH-5 international reference standard was prepared in 1984 and assigned an activity of 10,000 IU (International Units) per vial for the in vivo histamine sensitization test and the in vitro CHO (Chinese hamster ovary) cell clustering assay (both discussed in more detail below) [21]. However, at the time of calibration, the CHO cell clustering assay was not standardized between laboratories, thereby resulting in a high variability of reported PTx activities. Subsequent calibration studies using standardized protocols caused a diversification between the CHO and HIST unitage. Due to shortage on both the JNIH-5 and the Biological Reference Preparation 1 (BRP1), international collaborative studies were performed to determine the suitability of the 2nd WHO International Standard (IS) for Pertussis Toxin (coded as 15/126) and the replacement preparation BRP2. The 2nd IS reference preparation was assigned 1881 IU/ampoule in the HIST and 680 IU/ampoule in the CHO cell clustering assay [29]. The BRP2 was only calibrated in the CHO cell clustering assay and assigned 130 IU/vial [30]. This highlights the importance of calibrating PTx preparations, and also shows that the units of activity might differ between methods and can thereby influence the interpretation of results.