Nuanced differences in adenylate cyclase toxin production, acylation, and secretion may contribute to the evolution of virulence in <i>Bordetella</i> species

<i>Bordetella pertussis</i>, which causes the acute human disease whooping cough, evolved from <i>Bordetella bronchiseptica</i>, which causes long-term, chronic infections in a broad range of mammals. Both <i>B. pertussis</i> and <i>B. bronchiseptica</i> produce adenylate cyclase toxin (ACT), a bifunctional protein with adenylate cyclase-mediated cell intoxication and pore-forming activities. CyaC-mediated acylation of ACT is important for cell intoxication and required for pore-forming activity <i>in vitro</i>, but its role <i>in vivo</i> was unknown. Our comparative analysis showed that although ACT secreted by <i>B. pertussis</i> is fully acylated at residues K860 and K983, only a fraction of ACT secreted by <i>B. bronchiseptica</i> is modified. We showed that <i>B. bronchiseptica</i> produces more ACT than <i>B. pertussis</i> and is more efficient at ACT-dependent intoxication of macrophages <i>in vitro</i> than <i>B. pertussis</i>, but for both organisms, acylation of ACT greatly enhances intoxication. Acylation also enhances ACT secretion. Using a natural-host model, we determined that non-acylated ACT is functional during the initial stage of <i>B. bronchiseptica</i> infection, but not at later time points, and that acylation of K860, but not K983, is required for persistence in the lower respiratory tract. These data indicate a role for both acylated and non-acylated ACT during infection. Acylation of ACT was similarly not absolutely required for <i>B. pertussis</i> persistence in the murine lower respiratory tract. Overall, our data revealed nuanced differences in production, acylation, and secretion of ACT between <i>B. pertussis</i> and <i>B. bronchiseptica</i> that may correlate with their different virulence strategies.IMPORTANCE<i>Bordetella pertussis</i> causes the acute disease whooping cough and survives only in the human respiratory tract, while <i>Bordetella bronchiseptica</i> causes long-term, chronic infections in a broad range of mammals and can also survive in extra-host environments. These bacteria produce a nearly identical set of virulence factors, including adenylate cyclase toxin (ACT), a protein that is modified by the addition of acyl chains. Acylation is required for ACT to cause hemolysis and for efficient intoxication of host cells <i>in vitro</i>. We found that ACT acylation is also important, but not absolutely required, during infection. We also discovered differences in ACT production, acylation, and secretion between <i>B. bronchiseptica</i> and <i>B. pertussis</i> that may contribute to the different virulence strategies of these species. This study highlights the advantage of conducting comparative analyses between closely related species to better understand the evolution of virulence.