Abstract
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines.
Highlights
Since the 1990s, outbreaks of pertussis have occurred in populations with significant vaccination coverage[1,2,3,4,5,6,7]
Diverse bacterial pathogens use the type 1 secretion system (T1SS) to secrete RTX leukotoxins, which target host leukocytes during infection
The RTX domain of Bordetella pertussis adenylate cyclase toxin (ACT) mediates leukocyte targeting via binding to the αMβ2 integrin receptor, and antibodies that block receptor binding neutralize toxin activity
Summary
Since the 1990s, outbreaks of pertussis have occurred in populations with significant vaccination coverage[1,2,3,4,5,6,7]. ACT belongs to the Repeats-in-ToXin (RTX) family of proteins, which are the substrates of the bacterial type I secretion system (T1SS)[22,23]. The primary function of the RTX domain in proteins of this family is to drive secretion across a Ca2+ gradient, with Ca2+-driven folding on the extracellular side of the T1SS preventing re-entry into the channel and favoring directional transport out of the cell[30]. All RTX proteins contain a conserved C-terminal capping structure that harbors the secretion signal for T1SS recruitment. For ACT, it has been shown that this capping structure is essential for folding of the entire RTX domain, as well as for toxin activity[31]. The cap is likely required to nucleate folding of the β-roll by limiting the conformational entropy of the C-terminus[32]
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