TIBS 14 - November 1989 459 Invasive adenylate cyclase toxin of Borde tella pertussis Emanuel Hanski dependent AC which penetrates cells and generates cAMP from endogenous ATP 11. The penetration of this AC, however, requires the presence of a separate polypeptide 11 and occurs by means of receptor-mediated endocyto- sis into acidic vesicles 12. Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP," as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation. Bordetella pertussis, the causative bac- terium of whooping cough, produces several compounds which have been implicated in the pathogenesis of the diseasel. These include: pertussis toxin, filamentous hemagglutinin dermo- necrotic toxin, lipopolysaccharide, agglutinogens, hemolysin and aden- ylate cyclase (AC). The ,3. pertussis AC exhibits unusual properties. Its location in the bacterium is extracyto- plasmic and several B. pert,xssis strains release considerable amounts of AC activity to the culture medium 2'3. The activity of the bacterial enzyme is greatly stimulated by the eukaryotic Ca2+-binding protein calmodulin (CAM), which is absent in B. pertussis 4. The bacterial enzyme is a toxin respon- sible for some of the manifestations of the disease. It penetrates mammalian cells, interacts with CaM and generates uncontrolled levels of cAMP 5"6. These high levels of cAMP impair the ability of polymorphonuclear leukocytes and macrophages for chemotaxis, phago- cytosis, superoxide generation and microbial killing 1. Whooping cough is characterized by the absence of fever, lack of adequate neutrophi[ic response and a high incidence of secondary bac- terial pneumonia 7, all manifestations of poor immune responses by the host. Recently, it was established that B. pertussis AC is an essential virulence factor; B. pertussis insertion mutant deficient in AC has been shown to be avirulent in an animal model of pertus- sis infection 8, and introduction of a cloned AC gene into tais mutant restored its virulence capacity 9. Several bacterial toxins elicit their action by alteration of intracellular concentrations of cAMP in animal cells. Cholera toxin, pertussis toxin and E. coli heat-labile enterotoxin, are all A-B type toxins which enter host cells from endocytic vesicles. Within the cells these toxins act by means of ADP- ribosylation of the GTP-binding pro- teins that regulate the host membrane- bound AC system ~°. The B. pertussis AC toxin is different in its structure, mechanism of penetration into target cells and duration of its toxic signal within the host. It is composed of a single polypeptide that possesses all the functions necessary for the toxin action. The B. pertussis AC toxin pen- etrates the cell directly from the plasma membrane, is short-lived in the host but generates high levels of cAMP due to its high turnover number. Bacillus anthracis, a Gram-positive bacillus that causes anthrax, also produces extracytoplasmic CaM- E. Hanski is at the Department' of Hormone Research, The Weizmann Institute of Science, Rehovot 76100, Israel. The structure ofB. pertussis AC and functional relationships The AC activity of B. pertussis has been associated with several polypep- tides of different molecular mass rang- ing from 43 to over 660 kDa 13. Until recently it was not clear whether the larger forms of the enzyme represent aggregates of the catalytic subunit with itself or with other polypeptides, or the presence of multiple molecular mass forms of the enzyme. Recently, two laboratories reported purification of B. pertussis AC. Ladant et al. 14 purified the enzyme from bacterial culture medium. Three species (of molecular mass 43, 45 and 50 kDa) having specific activities of 1600 p.mol rain -1 mg -l were isolated, and found to be struc- turally related 1~. We purified AC from an extract of concentrated bacterial cells and isolated two forms (of mol- ecular mass 200 and 47 kDa) having specific activities of 460 and 1680 lamol min i mg-l, respectivelyl6. These forms were shown to have immunologicaily related domains and it was concluded that the smaller form was probably derived from the 200 kDa protein by proteolytic cleavage 16. The B. pertussis AC (cya) gene has ATP- and CaM- F homology with E. coli <x-hemolysin q 1 ATP CaM 450 I ] 1000 1600 deletion J 1 4 ~>, 0 . 5 ! 23 I-o 5 ~ V r, ! 200 400 600 800 1000 1200 1400 1600 Residuenumber Fig. I. Schematic representation of B. pertussis cya gene structure. The location of the A TP- and CaM-binding domains is discussed in detail in the text. The first 450 N-terminal amino acids represent the form of the enzyme which is detected in B. pertussis culture medium. The area of the horizontal bars is highly homologous with internal parts of E. coli a-hemolysin and P. hemolitica leucotoxin z~. A C-terminal domain (residues 1000-1600) contains repeats of nine amino acids, having a typical sequence of GGDGDDTLX. The hydrophobicity plot was produced, as described in Ref. 35, by using a 'window' length of 2l residues. © 1989, Elsevier Science Publishers Ltd, (UK) I)376 51167/89/$02.00