Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain

Adenylate cyclase toxin (CyaA) is one of the major virulence factors produced by Bordetella pertussis, the whooping cough agent. CyaA belongs to the repeat in toxin protein family and requires a post-translational fatty acylation to form cation-selective channels in target cell membranes and to penetrate into cytosol. We have demonstrated recently that CyaA uses the alphaMbeta2 integrin (CD11b/CD18) as a specific cellular receptor. Here we show that the acylation of CyaA is required for a productive and tight interaction of the toxin with cells expressing CD11b. In addition, we demonstrate that the catalytic domain is not required for binding of CyaA to CD11b and that the main integrin interacting domain of CyaA is located in its glycine/aspartate-rich repeat region. These data decipher, for the first time, the interaction of CyaA with CD11b-positive cells and open new prospects for understanding the interaction of Bordetella pertussis with innate and adaptive immune systems.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.     

Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (alpha+beta) insertion between the B7 beta-strand and A7 alpha-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (alpha+beta) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (alpha+beta) domain, the thermal stability of the enzyme is substantially reduced, with an apparent T(m) of 42.0+/-1.0 degrees C, compared to the apparent T(m) of 58.1+/-1.0 degrees C of ChiA at pH 9.0. The specific activity of ChiADelta(alpha+beta) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.