The Conserved Lysine 860 in the Additional Fatty-acylation Site of Bordetella pertussis Adenylate Cyclase Is Crucial for Toxin Function Independently of Its Acylation Status

Abstract

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the ⑀-amino group of lysine 983 (Lys 983 ) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys 860 ), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys 860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of The adenylate cyclase toxin-hemolysin (ACT, 1 AC-Hly, or CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis and a promising protective antigen candidate for acellular pertussis vaccines (1-5). ACT belongs to the RTX (repeats in toxin) protein family (6) and has the capacity to form small cation-selective membrane channels, which account for its weak hemolytic activity (8 -11). The major cytotoxic activity of the 1706-residue-long protein, however, consists in its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane (12-14) and to deliver into cells a catalytic adenylate cyclase (AC) domain. This intoxicates cells by unregulated conversion of ATP to cAMP (15-18) and causes impairment of microbicidal functions of immune effector cells and apoptosis of lung macrophages (19).The capacity of ACT to penetrate into target cell membranes and to intoxicate cells depends on a posttranslational activation by the accessory protein, CyaC (20, 21). It was first established for the Escherichia coli ␣-hemolysin (HlyA) that the activation of RTX toxins consists in amide linked fatty-acylation (22), and Hackett et al. (23) have demonstrated by mass spectrometric analysis that native ACT produced by Bordetella (Bp-ACT) is mono-acylated by a palmitoyl residue at the ⑀-amino group of lysine 983. In contrast, the HlyA from E. coli was found to be acylated at two lysines, both in vitro and in vivo (24,25). Moreover, two highly conserved RTX acylation sites (25), corresponding to lysine 983 (Lys 983 ) and lysine 860 (Lys 860 ) are also found in ACT, and for an unknown reason, the CyaCactivated recombinant ACT produced in E. coli (r-Ec-ACT) is palmitoylated also at Lys 860 , in addition to acylation of Lys 983 (26). When compared with the native mono-acylated Bp-ACT, the doubly acylated r-Ec-ACT exhibits about four times lower specific hemolytic activity on sheep erythrocytes (20) and about 10 times lower specific channel-forming activity in artificial planar lipid bilayers (10). At the same time, however, the characteristics of the channels formed by both proteins are identical, and both proteins have identical capacity to insert ...

Acylation of Lysine 983 Is Sufficient for Toxin Activity of Bordetella pertussis Adenylate Cyclase

“…Native ACT produced by the Bordetella pertussis strain 338 (Bp-ACT) was initially found to be acylated exclusively by palmitoyl residues and only on the lysine 983 (16). In contrast, acylation by a mixture of palmitoleil (cis ⌬9 C16:1), palmitoyl (C16:0), and myristoyl (C14:0) fatty-acyl residues was found for the recombinant r-Ec-ACT activated by CyaC in E. coli (17,28). Moreover, in addition to acylation of…”

“…Bacteria were grown at 37°C in LB medium supplemented with 150 g/ml ampicillin. pT7CACT1 is a construct derived from pCACT3 (29), which was designed for enhanced production of recombinant CyaC-activated ACT in E. coli (r-Ec-ACT) under control of the IPTG-inducible lacZ p promoter (28). To construct pT7NFLAG-CACT1, a synthetic oligonucleotide, 5Ј-TATGGATTATAAAGATGACGATGACAAATCACG, encoding the FLAG epitope (DYKDDDDK), was inserted in-frame into the unique NdeI site encompassing the ATG start codon of cyaC.…”

“…Native ACT produced by the Bordetella pertussis strain 338 (Bp-ACT) was initially found to be acylated exclusively by palmitoyl residues and only on the lysine 983 (16). In contrast, acylation by a mixture of palmitoleil (cis ⌬9 C16:1), palmitoyl (C16:0), and myristoyl (C14:0) fatty-acyl residues was found for the recombinant r-Ec-ACT activated by CyaC in E. coli (17,28). Moreover, in addition to acylation of * This work was supported by Grant QLK2-CT-1999-00556 from the 5th Framework Program of the European Union, Grants 310/95/0432 and 310/96/K102 from the Grant Agency of the Czech Republic, Grant A5020907 of the Grant Agency of the Czech Academy of Sciences, and Grants ME167 and VS96149 of the Ministry of Education Youth and Sports of the Czech Republic (to P. S.).…”

“…In a previous study, we have shown that Lys-860 is by itself important for ACT function and that a conservative substitution of Lys-860 by an arginine residue drastically reduced the capacity of ACT to insert into and translocate across target cell membrane (28). This substitution, however, also prevented the acylation of Lys-860 and it could not be rigorously excluded that this acylation itself, independent of the K860R substitution, was important for cell-invasive activity of ACT.…”

“…: 42-02-475-2762; Fax: 42-02-475-2152; E-mail: sebo@biomed.cas.cz. 1 The abbreviations used are: RTX, Repeat in ToXin family protein; AC, adenylate cyclase; ACT, adenylate cyclase toxin; CID, collisionally induced dissociation; Hly, hemolysin; IPTG, isopropyl-␤-D-thiogalactopyranoside; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; IEF, isoelectric focusing; PAGE, Lys-983, an incomplete (60%) acylation was found at Lys-860 of r-Ec-ACT (17,28). r-Ec-ACT exhibited also a lower capacity to induce protective immune response against Bordetella infection in mice and a significantly lower channel-forming and hemolytic activity, as compared with Bp-ACT (13,15,17,29,30).…”