Ehrlichia chaffeensis and its retropepsin-type protease engage the complement inhibitor C4b-binding protein

ABSTRACT Ehrlichia chaffeensis is a tick-borne obligate intracellular bacterium that causes human monocytic ehrlichiosis. Significant advances have been made in understanding immune evasion strategies in Ehrlichia ; however, the role of the complement system has been largely overlooked. This study provides evidence for a novel interaction between E. chaffeensis and the complement inhibitor C4b-binding protein (C4BP). We confirmed that E. chaffeensis recruits and binds C4BP at its surface, demonstrating binding of C4BP in human serum. This interaction is concentration-dependent, and C4BP retains its regulatory cofactor activity when bound to the bacteria. Furthermore, we identified the ehrlichial retropepsin-type protease—named APEhc—as a new C4BP ligand. We show that the soluble domain of APEhc targets multiple serum components, directly binding factor H, forming complexes with C4BP, and cleaving two major complement components, C3b and C4b. Quantitative binding analysis reveals a strong interaction between APEhc and C4BP, with apparent dissociation constants in the nanomolar range. Analysis of C4BP CCP deletion mutants supports a contribution of CCP3, CCP6, and CCP7 to APEhc-C4BP binding. Additionally, we demonstrate that C4BP retains its factor I cofactor activity in the presence of APEhc, and that APEhc independently cleaves C4b. Consistent with the targeting and modulation of serum components, our results show that APEhc significantly inhibits activation of the classical, lectin, and alternative pathways. Our findings reveal the unprecedented interaction between E. chaffeensis , its retropepsin protease, and complement inhibitors, raising intriguing questions regarding complement targeting by E. chaffeensis and its role in immune evasion. IMPORTANCE Human monocytic ehrlichiosis is a tick-borne disease caused by Ehrlichia chaffeensis , which can be severe or fatal if untreated. Understanding how E. chaffeensis evades the immune system is crucial for identifying new virulence factors and developing new treatments. The complement system, an essential part of the body’s first line of defense, is often targeted by pathogens to avoid immune attacks. We show that E. chaffeensis binds to C4b-binding protein (C4BP), a major inhibitor of the classical pathway (CP) and lectin pathway (LP), and identify the ehrlichial retropepsin-type protease, APEhc, as a novel C4BP ligand. Additionally, we demonstrate that APEhc targets other serum components, inhibiting complement CP, LP, and AP activation, suggesting an unprecedented dual-inhibitory activity for APEhc through complex formation with C4BP (and factor H) and cleavage of C3b and C4b. Our findings reveal a multifunctional role for APEhc in immune evasion, raising intriguing questions about the mechanisms used by intracellular pathogens like E. chaffeensis to bypass host defenses.