Tracheal cytotoxin

Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.

Pertussis, caused by Bordetella (B.) pertussis, a Gram-negative bacterium, is a highly contagious airway infection. Especially in infants, pertussis remains a major health concern. Acute infection with B. pertussis can cause severe illness characterized by severe respiratory failure, pulmonary hypertension, leucocytosis, and death. Over the past years, rising incidence rates of intensive care treatment in young infants were described. Due to several virulence factors (pertussis toxin, tracheal cytotoxin, adenylate cyclase toxin, filamentous hemagglutinin, and lipooligosaccharide) that promote bacterial adhesion and invasion, B. pertussis creates a unique niche for colonization within the human respiratory tract. The resulting long-term infection is mainly caused by the ability of B. pertussis to interfere with the host's innate and adaptive immune system. Although pertussis is a vaccine-preventable disease, it has persisted in vaccinated populations. Epidemiological data reported a worldwide increase in pertussis incidence among children during the past years. Either acellular pertussis (aP) vaccines or whole-cell vaccines are worldwide used. Recent studies did not detect any differences according to pertussis incidence when comparing the different vaccines used. Most of the currently used aP vaccines protect against acute infections for a period of 6-8 years. The resurgence of pertussis may be due to the lack of herd immunity caused by missing booster immunizations among adolescents and adults, low vaccine coverages in some geographic areas, and genetic changes of different B. pertussis strains. Due to the rising incidence of pertussis, probable solution strategies are discussed. Cocooning strategies (vaccination of close contact persons) and immunizations during pregnancy appear to be an approach to reduce neonatal contagiousness. During the past years, studies focused on the pathway of the immune modulation done by B. pertussis to provide a basis for the identification of new therapeutic targets to enhance the host's immune response and to probably modulate certain virulence factors.

The most consistent pathological feature of pertussis is the selective colonization and subsequent destruction of ciliated cells in the respiratory epithelium. Phase I B. pertussis can reproduce the human respiratory tract cytopathology during in vitro infection of hamster tracheal organ cultures. However, most isolated biologically active components produced by B. pertussis (lymphocytosis-promoting factor, adenylate cyclase, dermonecrotic toxin, etc.) have no apparent cytopathic effect on the respiratory epithelium. We have purified a glycopeptide from the culture supernatant of virulent B. pertussis that mimics completely the ciliated cell pathology characteristic of pertussis (5). Tracheal cytotoxin (TCT) is released during log phase broth culture and consists of 15 amino acid residues as well as two amino sugars. The selective biological activity of TCT has been studied in tracheal organ cultures by light and electron microscopy. A series of pathological changes precedes the eventual extrusion of ciliated cells, while all other epithelial cell types appear ultrastructurally normal. TCT also causes a dose-dependent inhibition of DNA synthesis in cultured hamster trachea epithelial cells, providing a quantitative bioassay to monitor TCT activity during purification steps. Previously, TCT could be completely purified from oxidized glutathione (a major contaminant from the culture medium) only by high-voltage paper electrophoresis, a procedure not well suited for large scale work. We have now substituted a final column chromatography step that separates TCT from oxidized glutathione and other contaminating peptides. This change and other preparative scale adaptations now allow us to purify 150-250 nmol of biologically active TCT from one liter of culture supernatant (3-4 X 10(13) bacteria), a ten-fold increase over our previous batch size with no increase in processing time.(ABSTRACT TRUNCATED AT 250 WORDS)