The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc-+GalPi4GlcNAc) sequence on surface macromolecules. In

Immunization with the B oligomer of pertussis toxin protected neonatal mice from a lethal respiratory challenge with Bordetella pertussis. All mice immunized with 8 ,ug of B oligomer survived aerosol challenge and had peripheral leukocyte counts and weight gains similar to those of mice immunized with pertussis toxoid before challenge and to those of control mice that were not challenged. Unprotected mice challenged with an aerosol of B. pertussis had an increase in peripheral leukocyte count, failed to gain weight, and died within 21 days of challenge. Protection appeared to be dose dependent, since a dose of 1 ,ug of B oligomer per mouse prevented death in 100% of the mice challenged with B. pertussis, whereas 0.4 ,ug of B oligomer protected 50% of the challenged mice. Mice immunized with the B oligomer had increases in immunoglobulin G (IgG) anti-B oligomer in sera and in IgG and IgA anti-B oligomer in bronchoalveolar lavage fluids 1 to 3 weeks after respiratory challenge. Specific anti-B oligomer antibodies could not be detected in unimmunized, infected mice at the same time after challenge. Intravenous administration of the monoclonal antibody 170C4, which binds to the S3 subunit of the B oligomer, protected neonatal mice from B. pertussis respiratory challenge, while administration of an IgGl anti-tetanus toxin monoclonal antibody, 18.1.7, was not protective. We conclude that anti-B-oligomer-mediated neutralization of pertussis toxin is one mechanism of protection in the mouse model of B. pertussis aerosol challenge.

No abstract

A minitransposon mutant of Salmonella enterica serovar Typhimurium SR-11, SR-11 Fad−, is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129–134, 1998). Here, evidence is presented that the mutation in SR-11 Fad− that renders the strain avirulent is in the cra gene, which encodes the Cra protein, a regulator of central carbon metabolism.

The binding of pertussis toxin (PT) to the human T-cell line Jurkat was examined by using flow cytometry. Fluorescein isothiocyanate (FITC)-labeled PT bound rapidly to the cells in a specific manner as determined by blocking experiments with unlabeled toxin, B oligomer, and the S2-S4 and S3-S4 dimers. Monoclonal antibodies against the S3 subunit of the toxin also significantly inhibited the binding of FITC-PT. Sialidase treatment of the cells resulted in decreased binding of FITC-PT, indicating that sialic acid residues are involved in the binding process. In addition, we studied the effect of PT binding on the expression of cell surface molecules. On binding of PT to the cell surface, a rapid down-regulation of the T-cell receptor (TCR)-CD3 complex was observed. The modulation of the TCR-CD3 complex was independent of the toxin's enzymatic activity, as the B oligomer and a nonenzymatic toxin mutant induced modulation comparable to that caused by the native holotoxin. Isolated dimers did not cause down-regulation. Stimulation of the TCR-CD3 complex, leading to reduced cell surface expression of this complex, provides a possible explanation for the second messenger production associated with the interaction of PT or B oligomer with T lymphocytes. We therefore conclude that PT activates T cells by divalent binding to the TCR-CD3 complex itself or by binding a structure closely associated with it.Pertussis toxin (PT), one of several toxins produced by Bordetella pertussis, the etiological agent of whooping cough, has the A-B structure characteristic of other bacterial toxins (25). The toxin comprises an enzymatically active A subunit (S1) and a B oligomer, made up of five subunits (S2-S4 and S3-S4 dimers connected by S5), which is responsible for binding to the target cell (25, 26). Both dimers have been implicated in the binding process (26), and there is evidence of different binding specificities (12,31).PT shows a variety of biological effects which results from binding to cells, internalization, and subsequent ADP-ribosylation of a family of GTP-binding regulatory proteins (G proteins). The effects of the toxin on cells of the immune system are multiple and include induction of lymphocytosis, inhibition of macrophage migration, adjuvant activity, and T-cell mitogenicity (10). The mitogenic action of the toxin appears to be independent of the enzymatic subunit, as the B oligomer is able to mimic the effects of the holotoxin (26).The interaction of PT or B oligomer with T lymphocytes results in the rapid intracellular accumulation of Ca2+, an increase in the levels of inositol triphosphate and diacylglycerol, and activation of protein kinase C and a tyrosine protein kinase (6,17,24,27). In this study, we have examined the interaction of PT with human T lymphocytes, using the transformed human T-cell line Jurkat and flow cytometry in order to better understand which parts of the toxin are involved in binding. We also studied the effect of PT binding on the expression of T-cell surface markers that are involved in ...