Acylation of Lysine 860 Allows Tight Binding and Cytotoxicity ofBordetellaAdenylate Cyclase on CD11b-Expressing Cells

Abstract

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.

Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin

“…2), it could be estimated that exposure of cells to 3 g/ml CyaA resulted in a [Ca 2ϩ ] i increase from a basal level of ϳ80 nM to a persistent final level of about 2 M. As further shown in Fig. 1, no elevation of [Ca 2ϩ ] i was observed upon exposure of cells to 3 g/ml of the comparably pure but nonacylated pro-CyaA, which binds CD11/CD18 in a nonproductive manner and exhibits only a residual biological activity (53), and neither did an appropriate lipopolysaccharide control (45 EU/ml) that caused no elevation of [Ca 2ϩ ] i (supplemental Fig. 3).…”

“…The reaction was stopped by addition of 100 mM HCl solution containing 0.2% Tween 20, and the samples were boiled for 15 min at 100°C to denature cellular proteins (cAMP is heat-and acid-resistant). The samples were neutralized by addition of 150 mM unbuffered imidazole, and cAMP concentration was determined by an antibody competition immunoassay as described elsewhere (53).…”

“…CyaA-E509P induced a rather slow calcium influx and promoted only a , or were incubated in DMEM containing 100 M La 3ϩ and were washed once prior to toxin addition, respectively. A, various concentrations of CyaA were added, and incubation of cells with toxin was continued for additional 30 min before levels of cAMP accumulated in cells were determined as described elsewhere (53). B, CyaA was added to 3 g/ml at the indicated time point (arrow), and the effect of La 3ϩ on CyaA-mediated calcium influx was followed in time.…”

Antigen Targeting to CD11b+ Dendritic Cells in Association with TLR4/TRIF Signaling Promotes Strong CD8+ T Cell Responses

“…CyaA is produced as an inactive protoxin, which is converted to an active toxin upon posttranslational modification by a dedicated acyltransferase, the product of the CyaC gene (1). This covalent posttranslational fatty acylation, consisting of palmitoylation at the ε-amino groups of Lys 983 and Lys 860 , is required for CD11b/CD18 binding (35) and the insertion of CyaA into target cell membranes and translocation of the catalytic domain into cytosol of cells, as well as for the formation of a cation-selective oligomeric CyaA pore in target cell membranes. We previously showed that CyaA acylation is necessary to strengthen the interaction of the toxin with CD11b cells (35,36), and it remains to be determined whether these fatty acids can interact with TLR4.…”

Acylation Enhances, but Is Not Required for, the Cytotoxic Activity of Mannheimia haemolytica Leukotoxin in Bighorn Sheep

“…Mass spectrometric analysis indicated that the toxin produced by the wild-type SH1217 strain of M. haemolytica has only one lysine residue (lysine 554 ) modified by C 14 and C 14 OH fatty acid. This is in contrast to other members of the RTX toxin family, including A. actinomycetemcomitans leukotoxin, E. coli hemolysin (HlyA), and Bordetella pertussis adenylate cyclase toxin-hemolysin (CyaA), where two lysine residues are acylated (32)(33)(34). The fact that peptide 550 -560 resulted from tryptic cleavage of the parental strain, SH1217, but not the mutant SH2099 confirmed that lysine 554 was acylated by C 14 and C 14 OH fatty acid in Lkt from the parent, but not from the mutant strain SH2099.…”