Internal Lysine Palmitoylation in Adenylate Cyclase Toxin from Bordetella pertussis Murray Hackett, Lin Guo, Jeffrey Shabanowitz, Donald F. Hunt, Erik L. Hewlett* A number of bacterial protein toxins, including adenylate cyclase (AC) toxin from Bor- detella pertussis, require the product of an accessory gene in order to express their biological activities. In this study, mass spectrometry was used to demonstrate that activated, wild-type AC toxin was modified by amide-linked palmitoylation on the E-amino group of lysine 983. This modification was absent from a mutant in which the accessory gene had been disrupted. A synthetic palmitoylated peptide corresponding to the tryptic fragment (glutamine 972 to arginine 984) that contained the acylation blocked AC toxin-induced accumulation of adenosine 3',5'-monophosphate, whereas the non- acylated peptide had no effect. Adenylate cyclase toxin is a virulence fac- tor from Bordetella pertussis, the bacterium that causes pertussis or "whooping cough." This disease is responsible for an estimated 340,000 deaths per year, largely among chil- dren in developing countries. It is also a cause of morbidity for children and adults in Europe and the United States (1). AC toxin is so named because it is an adenylate cyclase enzyme of bacterial origin that enters eukaryotic cells and catalyzes the production of supraphysiologic amounts of adenosine 3',5'-monophosphate (cAMP) from adeno- sine triphosphate in the host cell (2). As a consequence of its insertion into target cell membranes, the toxin is also hemolytic for sheep red blood cells (RBCs) (3). AC toxin is expressed from its structural gene, cyaA (4), as a 177.5-kD protein consisting of a single peptide chain that contains no cys- teines. Its toxin and hemolytic activities, however, are dependent on the product of an accessory gene, cyaC (5), which is be- lieved to be involved in a posttranslational modification of AC toxin. AC toxin is required as a virulence fac- tor, and B. pertussis organisms that are de- fective in its production have been shown to be unable to establish a lethal infection in suckling mice (6). The increased intra- cellular concentrations of cAMP caused by the toxin inhibit the normal physiologic processes of phagocytic leukocytes, and it is hypothesized that impairment of immune effector cell function is the major contribu- tion of AC toxin to the pathogenesis of pertussis (2). Activated AC toxin is also a M. Hackett and J. Shabanowitz, Department of Chemis- try, University of Virginia, Charlottesville, VA 22901, USA. L. Guo, Department of Medicine, University of Virginia School of Medicine, Box 419, Charlottesville, VA 22908, USA. D. F. Hunt, Departments of Chemistry and Pathology, University of Virginia, Charlottesville, VA 22901, USA. E. L. Hewlett, Departments of Medicine and Pharmacol- ogy, University of Virginia School of Medicine, Box 419, Charlottesville, VA 22908, USA. *To whom correspondence should be addressed. protective antigen that is a candidate for inclusion in acellular pertussis vaccines (7). AC toxin is a member of a rapidly grow- ing family of Gram-negative bacterial cyto- lysins called "repeat in toxin" (RTX) tox- ins. This name refers to a series of glycine- and aspartic acid-rich nonameric repeats found in the COOH-terminal third of each toxin protein and thought to be involved in calcium binding (8). Many of these toxins 0 Fraction 2C * ~~~~~~~~~~containing acylated pE 0 10 20 30 41 Time (min) 100- 1621 90- 4284 80- Acylated peptide 70. C~~~~~~~~~~~~~~~~~~~~~~~~~~ 6: 40- ; i L 1 42345Interns .0 ~ ~ ~~2 2694 6 ~~~208 4Q ~~~~2756 >60 3264 40 share with AC toxin the dependence on an ancillary gene, such as cyaC, for their bio- logical activities. Activation of Escherichia coli hemolysin, HIyA, can be achieved in vitro by transfer of a fatty acyl group from acyl carrier protein to prohemolysin (9), which leads to the hypothesis that HlyC is an acyltransferase. However, neither the type nor the site of acylation that occurs in vivo has been reported. To investigate the posttranslational modification of AC toxin, we used purified AC toxins from both wild-type B. pertussis (BP338) and a cyaC-deficient mutant strain (BPDE386) (10) and analyzed the toxin structure by mass spectrometry. The toxins were digested with trypsin, and the resulting peptides were separated by high-perfor- mance liquid chromatography (HPLC). The profiles of the peptides generated from BP338 (Fig. 1A) and BPDE386 were simi- lar. Each HPLC fraction was analyzed by matrix-assisted laser desorption-ionization- time-of-flight mass spectrometry (MALDI- TOF MS) (1 1). The MALDI-TOF MS data (Fig. iB) from the HPLC fractions revealed that approximately 180 fragments were de- rived from each AC toxin, whereas 137 fragments were expected on the basis of the Fig. 1. Preliminary screening of A tryptic fragments from wild-type AC toxin. (A) HPLC trace, moni- tored at 214 nm (1.0 absorbance unit full scale), of the tryptic digest from BP338. The palmitoylated peptide was identified in fraction 20 of the 24 collected and ana- lyzed by MALDI-TOF MS. A 2.1- mm by 3-cm C4 Bu 300 HPLC column (ABI) was used with a 60- eptide min binary gradient at 200 ,il/min with 1 0-min delay, 500-,ul injec- tion, and eluted with a gradient of 10% to 100% solvent B. Solvent A, H20 and 0.1 % trifluoracetic acid (TFA); solvent B, 60% aceto- nitrile, 30% N-propanol, 10% -'------------ H20, and 0.085% TFA. (B) MALDI o0 50 mass spectrum of HPLC fraction 20 containing palmitoylated pep- tide at m/z 1621. Several nonacyl- ated peptides were also present. ac-Cyano-4 - hydroxycinnamic B acid (20) matrix was used with 100 fmol of bovine ubiquitin ([M + 2H]--+ = 4284) as an internal standard for mass calibration. ialstandard The instrument used an N2 laser with excitation at 337 nm and was constructed in-house (21). nYz SCIENCE * VOL. 266 * 21 OCTOBER 1994 5 433 on June 6, 2014 www.sciencemag.org Downloaded from on June 6, 2014 www.sciencemag.org Downloaded from on June 6, 2014 www.sciencemag.org Downloaded from