Structural basis for receptor binding and antibody-mediated neutralization by Bordetella adenylate cyclase toxin
Creators: name: Goldsmith, Jory A.
name: 0000-0003-2917-0620
Publisher: name: The University of Texas at Austin
Year: 2023
Subjects: Host-pathogen interactions
Toxins
Integrins
Antibodies
Bordetella

description: Bordetella pertussis, the gram-negative pathogen that causes whooping cough, caused 89,000 deaths in 2018 globally. In the United States, introduction of killed whole-cell B. pertussis (wP) vaccines in the 1940s reduced the number of yearly cases from over 100,000 to less than 10,000 in 1965. However, public fears about the reactogenicity of wP vaccine led many countries to switch to less effective acellular B. pertussis (aP) vaccines, containing specific purified antigens. An improved version of aP vaccine is desired, which could be achieved by including better or more optimized antigens. Notably, the adenylate cyclase toxin (ACT) of B. pertussis has been hypothesized to be an effective antigen to include in the aP vaccine as it is essential for lung colonization in mouse intranasal models and has elicited protective immunity in mice. However, ACT was never included in human vaccines as it was too difficult to produce. ACT is a virulence factor secreted by B. pertussis that inserts into host leukocytes and translocates an adenylate cyclase enzyme into the target cell cytosol. The rapid formation of cAMP in host leukocytes inhibits their bactericidal activities, thus promoting survival of B. pertussis in the respiratory epithelium. As a ‘pore-forming RTX toxin’, ACT has a C-terminal calcium-binding ‘RTX’ domain that mediates secretion. However, biochemical data had previously shown that the RTX domain of ACT contains the binding site for the ACT receptor, integrin αMβ₂. The RTX domain was also shown to harbor the epitopes for neutralizing antibodies that prevent αMβ₂ binding, suggesting that this interaction could be targeted with an RTX domain immunogen. However, the structural determinants of the ACT RTX domain’s interaction with αMβ₂ were not well understood prior to this work. Specifically, it was not known how the ACT RTX domain had been adapted to engage in protein-protein interactions, which regions of the RTX domain contacted the αMβ₂ receptor as well as neutralizing antibodies, and how binding of ACT to αMβ₂ favors membrane insertion of ACT. In addition, ACT preferentially binds to the inactive conformation of αMβ₂, and the structural basis for this preference is not understood. Therefore, this work set out to structurally define the interaction of ACT with αMβ₂ and with neutralizing antibodies, to understand key epitopes for mediating protection against B. pertussis. Chapter 1 provides an outline of the epidemiology of B. pertussis and the known details of the ACT intoxication and receptor-binding mechanism. In Chapter 2, a crystal structure of an engineered RTX domain fragment harboring the αMβ₂-binding site bound to neutralizing antibodies is described. This structure showed neutralizing antibodies bound to the variable “linker” regions of the RTX domain, which had a previously unknown fold and connect the segments of the RTX domain containing consensus 9-residue repeats known to form a helical calcium-binding structure. This suggested that the RTX linkers were involved in binding αMβ₂ as these antibodies prevent αMβ₂ binding to the ACT RTX domain. In Chapter 3, a cryo-electron microscopy structure of the ACT RTX domain in complex with the αMβ₂ ectodomain is reported. This structure showed that the RTX linkers, shown in Chapter 2 to harbor the neutralizing epitopes bound by antibodies M2B10 and M1H5, each form a separate binding site on αMβ₂, and can only contact both of these sites when αMβ₂ is in the bent, inactive conformation. This structure also showed that αMβ₂ binding would position lysine residues that require post-translational acylation for ACT activity directly at the host cell membrane, coupling receptor binding to membrane insertion. This work defined RTX linkers as key protein-protein interaction modules of the ACT RTX domain and provides a framework for the design of RTX domain vaccines.