Immunomodulatory Amacstatins facilitate feeding and Rickettsia parkeri transmission by Amblyomma maculatum ticks

Ticks rely on salivary immunomodulators to sustain prolonged blood feeding and transmit pathogens, yet the protein effectors that mediate these processes remain incompletely defined. Here, we characterize three divergent cysteine protease inhibitors—Amacstatins—from the Gulf Coast tick Amblyomma maculatum and delineate their roles in feeding success and Rickettsia parkeri transmission. Integrated transcriptomic, structural, and biochemical analyses reveal that Amacstatin-1, -2, and -3 possess distinct reactive centers, inhibitory selectivity, and tissue-specific expression patterns that are dynamically regulated by blood feeding and rickettsial infection. Silencing all three Amacstatins via RNA interference significantly impaired tick feeding, reducing engorgement, fecundity, and R. parkeri burden in both midgut and salivary glands. Functional assays demonstrated that Amacstatins potently suppress host inflammatory responses, including pro-inflammatory cytokine production, nitric oxide release, and leukocyte recruitment. Together, these findings identify Amacstatins as critical immunomodulatory effectors that promote tick feeding and rickettsial transmission. This work provides a protein-level mechanistic insight into the A. maculatum – R. parkeri interaction and highlights Amacstatins as promising molecular targets for anti-tick and anti-rickettsial strategies.