Adenylate cyclase toxin-hemolysin (ACT, AC-Hly, or CyaA) is an important virulence factor for Bordetella pertussis.  Adenylate cyclase toxin is a large (178 kDa), 1,706-residue-long, toxin consisting of an amino-terminal adenylate cyclase (AC) domain of 400 residues and a repeat toxin (RTX) moiety of 1,306 residues.  Sequences within the RTX domain are responsible for target binding, pore-formation and calcium binding.  Also located within this domain are two lysine residues which undergo posttranslational modified by acylation (1).

Adenylate cyclase toxin targets sentinel cells of the host’s innate immune defense. It penetrates a variety of immune cells and, when activated by calmodulin, catalyzes conversion of cellular ATP to cyclic AMP (cAMP), interfering with cell signaling and with the anti-bacterial activity of phagocytes. ACT acts on phagocytes limiting the phagocytes ability to produce oxidative burst and kill bacterial through complement or antibodies (2, 3).  The entire AC-Hly protein is necessary for adenylate cyclase delivery into cells (4).

In the intoxication process, the Hly portion of the toxin binds to CR3 receptor on target cells (CD11b⁺) and allows translocation of the AC enzyme into the cell.  Within the target cell, adenylate cyclase rapidly produces extremely high levels of cAMP, disabling the immune cell (5, 6).  At a lower efficiency, adenylate cyclase-hemolysin can penetrate cells lacking the CR3 receptor and create cAMP (7). In addition to binding target cells, CyaA is able to form small cation-selective pores in cytoplasm membranes, causing hemolysis in erythrocytes (8).

A non-enzymatic, genetically detoxified CyaA-AC^– toxoid has been produced (9). Although the catalytic activity in this AC toxoid is destroyed, it is still cell invasive and able to induce an immune response to co-administered pertussis antigens (10, 11, 12).  This toxoid has been shown to be capable of delivering vaccine antigens into the cytosol of major Histocompatibility complex (MHC) class I antigen-presenting cells (13).CyaA-AC^– toxoid has been used as a tool to deliver antigens to T cells in anti-cancer immunotherapeutic vaccines. (9, 14, 15)

List Labs provides several variations of the B. pertussis Adenylate Cyclase Toxin.  Most recently we have prepared a new formulation of the lyophilized Adenylate Cyclase Toxin Product #188.  This product may be obtained, upon request, as a liquid (Product #188L).  Additionally we have samples available of the Genetically Detoxified CyaA-AC Toxoid, request Product #188X, and samples of an especially low endotoxin preparation of Adenylate Cyclase Toxin Product #188U.  Finally, you will find in our product offering Product #189, Adenylate Cyclase Antigen, Native from Bordetella pertussis.

1. Sebo P., Osicka R., Masin J., (2014) Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines. Expert Review of Vaccines 13(10): 1215-1227. PMID: 25090574
2. Confer DL. and Eaton JW., (1982) Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217:948–950. PMID: 6287574
3. Mock M. and Ullmann A., (1993) Calmodulin-activated bacterial adenylate cyclases as virulence factors. Trends Microbiol 1:187–192. PMID: 8143137
4. Sakamoto H., Bellalou J., Sebo P., Ladant D., (1992) Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalyticand hemolytic activities. J Biol Chem 267:13598–13602. PMID: 1618862
5. Wolff J., Cook GH., Goldhammer AR., Berkowitz SA., (1980) Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci USA 77(7):3841-4. PMID: 6253992
6. Hanski E. and Farfel Z., (1985) Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration. J Biol Chem 260(9):5526-32. PMID: 2859287
7. Vojtova J., Kamanova J., Sebo P., (2006) Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr Opin Microbiol 9(1):69-75. PMID: 16406775
8. Szabo G., Gray MC., Hewlett EL., (1994) Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium and polarity-dependent manner. J Biol Chem 269(36):22496–22499. PMID: 8077197
9. Simsova M., Sebo P., Leclerc C., (2004) The adenylate cyclase toxin from Bordetella pertussis–a novel promising vehicle for antigen delivery to dendritic cells. Int J Med Microbiol 293(7-8):571-6. PMID: 15149033
10. Macdonald-Fyall J., Xing D., Corbel M., Baillie S., Parton R., Coote J., (2004) Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussis towards co-administered antigens. Vaccine 22(31-32):4270-81. PMID: 15474718
11. Orr B., Douce G., Baillie S., Parton R., Coote J., (2007) Adjuvant effects of adenylate cyclase toxin of Bordetella pertussis after intranasal immunisation of mice. Vaccine 25(1):64-71. PMID: 16916566
12. Cheung GY., Xing D., Prior S., Corbel MJ., Parton R., Coote JG., (2006) Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model. Infect Immun 74(12):6797-805. PMID: 16982827
13. Osicka R., Osicková A., Basar T., Guermonprez P., Rojas M., Leclerc C., Sebo P., (2000) Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infection Immunity 68: 247-256. PMID: 10603395
14. Dadaglio G., Morel S., Bauche C.,  Moukrim Z., Lemonnier FA., Van Den Eynde BJ., Ladant D., Leclerc C., (2003) Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes. Int Immunol 15(12):1423-30. PMID: 14645151
15. Fayolle C., Ladant D., Karimova G., Ullmann A., Leclerc C., (1999) Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J Immunol 162(7):4157-62. PMID: 10201941

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

Pertussis Infections (aka Whooping Cough) Increasing

Rates of pertussis infection, commonly referred to as whooping cough, are on the upswing. Whooping cough is a highly contagious respiratory infection with flu-like symptoms including a blocked or runny nose, sneezing, mild fever and the distinctive, hack-like cough. Prior to the availability of effective whole-cell vaccines, before the mid-1940’s, whooping cough was almost entirely a childhood disease. However, up to half of all cases in the USA are among older children, 7 to 19 years old (CDC Pertussis Surveillance and Reporting, 2013).

Why Are There More Instances of Whooping Cough?

Factors in the resurgence of whooping cough include the level of immunity in a given population, the switch from a whole-cell B. pertussis vaccine to an acellular vaccine (which offered reduced side effects), trends in antigenic variation (e.g.,  pertactin expression), and the cyclical nature of B. pertussis infections. “Herd immunity” occurs when more than 95% of a given population has up-to-date vaccinations; without this substantial immunized group, outbreaks become more likely, which affect young infants most severely.

Analysis of the genotype profile in strains isolated in Korea in 2011-2012 indicate that genotypic changes in currently circulating strains are strongly associated with the recent increase of pertussis cases (Kim, 2014). One factor in the resurgence may be the rise of pertactin deficient strains of B. pertussis, possibly as an adaptation (Lam 2014).

Whole Cell Vaccines vs. Acellular Vaccines

Previously whole cell vaccines containing chemically-inactivated pertussis cells were the norm, though fever was a known side-effect. Acellular vaccines were introduced in the late 1990’s and have become the standard of care.  Acellular vaccines are derived from either three or five key virulence factors from the B. pertussis organism, including pertussis toxin and pertacin. While use of the whole-cell pertussis vaccine resulted in a historic low in 1976 of 1,010 cases reported in the USA, 25,616 new cases of pertussis were reported in 2005, with cyclical incidence rates peaking every 3 to 4 years.

Bordetella Pertussis Used in Research

Bordetella pertussis produces a number of proteins that not only contribute to its pathogenicity, but also are used in research as well (Carbonetti, 2010).  The adenylate cyclase toxin (ACT), which is believed to directly penetrate human phagocytes, causes a disruption their normal function by direct production of intracellular cyclic AMP (Kamanova 2008). Pertussis toxin has been used in development of a pertussis diagnosis assay. The recommended pertussis diagnostic tests, culture and real-time PCR assays, lack sensitivity at later stages of the disease. An IgG anti-pertussis toxin ELISA (PT-ELISA) has been introduced as an immunoassay to be used for sero-diagnosis or vaccine evaluation (Kapasi, 2012). Culture and RT-PCR assays detected more cases of pertussis in infants, whereas PT-ELISA detected more cases in adolescents and adults. Serology involving the pertussis toxin is a cost-effective and complementary diagnostic, especially among older children, adolescents, and adults during the late disease phase. More research is required in understanding the long-lasting protective response resulting from vaccination.  Pertussis virulence factors from List Biological Laboratories are effective antigens for serology assays.

Pertussis toxin is a protein-based AB₅-type exotoxin with unique qualities that plays a key role in B. pertussis pathogenesis. The exotoxin comprises six subunits (named S1 through S5, where each complex contains two copies of S4) (Kaslow, 1994; Locht, 1995). The subunits are arranged in A-B structure: the A component is enzymatically active and is formed from the S1 subunit, while the B component is the receptor-binding portion and is made up of subunits S2–S5 (Locht, 1995). The subunits are encoded by ptx genes encoded on a large PT operon that also includes additional genes that encode Ptl proteins. Together, these proteins form the PT secretion complex (Weiss, 1993). PT has also become widely used as a biochemical tool to ADP-ribosylate GTP-binding proteins in the study of signal transduction.

List Labs Provides B. Pertussis for Research

List Biological Laboratories provides B. pertussis virulence factors: Pertussis Toxin, Pertussis Toxin Subunits, Filamentous Hemagglutinin (FHA), Fimbriae 2/3, Pertactin (69 kDa protein), Adenylate Cyclase Antigen and B. pertussis Lipopolysaccharide (LPS), all derived from the native B. pertussis source for research and diagnostic purposes.  Recombinant Adenylate Cyclase from B. pertussis is now offered in a new, highly purified form, ideal for studies with an active enzyme.  Pertussis toxin mutant is a relatively non-toxic protein which may be used in place of the toxin for serology. Additionally, pertussis toxin mutant is a vaccine carrier.  These toxins are purified by a tried-and-true method which ensures their activity to high quality standards.

 

References

CDC Pertussis (Whooping Cough) Surveillance and Reporting: http://www.cdc.gov/pertussis/surv-reporting.html

Carbonetti NH (2010). “Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools”. Future Microbiol 5(3): 455–69.

Kamanova J, Kofronova O, Masin J, Genth H, Vojtova J, Linhartova I, Benada O, Just I, Sebo P. (2008) Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol 181(8):5587-97.

Kapasi A, Meade BD, Plikaytis, B, Pawloski L, et al. (2012) Comparative study of different sources of pertussis toxin (PT) as coating antigens in IgG Anti-PT Enzyme-linked immunsobent assays.  Clin Vaccine Immunol 19(1):64.

Kaslow HR, Burns DL (June 1992) Pertussis toxin and target eukaryotic cells: binding, entry, and activation  FASEB J 6 (9): 2684–90.

Kim SH, Lee J, Sung HY, Yu JY, Kim SH, Park MS, Jung SO (2014) Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea. J Korean Med Sci 29(3):328-33.

Lam C, Octavia S, Ricafort L, Sintchenko V, Gilbert GL, Wood N, et al (2014) Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia. Emerg Infect Dis 20(4):626-33.

Locht C, Antoine R (1995) A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit. Biochimie 77 (5): 333–40.

Weiss A, Johnson F, Burns D (1993) Molecular characterization of an operon required for pertussis toxin secretion  Proc Natl Acad Sci USA 90 (7): 2970–4.