Figure 5

Y940A and Y940P substitutions substantially reduce binding of CyaA to cells. (A) ClustalW sequence alignment of a partial sequence of the acylated domain of CyaA and corresponding sequences of related RTX toxins. ApxIA, Actinobacillus pleuropneumoniae (uniprot P55128); HlyA, Escherichia coli (uniprot Q8G9Z4); LtxA, Aggregatibacter actinomycetemcomitans (uniprot P16462); PaxA, Pasteurella aerogenes (uniprot Q9RCG8); AqxA, Actinobacillus equuli (uniprot Q8KWZ9); AppA, Kingella kingae (uniprot F5S905); and CyaA, Bordetella pertussis (uniprot code P0DKX7). The highly conserved tyrosine residues (Y940 in CyaA) are highlighted by a black frame, * identity,: strongly similar,. weakly similar. (B,C) Biological activities of intact CyaA or its mutant variants were analyzed using CD11b⁻ sheep erythrocytes (B) and CD11b⁺ J774A.1 mouse macrophages (C). Preparations and analyses of samples were performed as in the legend to Fig. 1. Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations. Significant differences are indicated by asterisks (****, p < 0.0001). (D) To block the CR3 receptor of CyaA, J774A.1 cells were preincubated for 30 minutes on ice with 5 µg/ml of the CD11b-specific monoclonal antibody M1/70 (Pharmingen) prior to addition of the CyaA variants (1 µg/ml). J774A.1 binding activities are expressed as percentages of intact CyaA binding activity and represent average values ± standard deviations from three independent determinations.