Brucella abortus is a facultative intracellular pathogen that survives and replicates within host macrophages by remodeling the Brucella-containing vacuole (BCV) and manipulating host signaling pathways. Proteomic analysis during intracellular infection revealed the overexpression of two homologous cyclophilins, CypA and CypB, both with peptidyl-prolyl cis/trans isomerase (PPIase) activity, classically linked to protein folding and stress adaptation.
Despite their high sequence identity, shared PPIase/chaperone activity, and common role in intracellular survival, mediated by stress response mechanisms, CypA and CypB follow divergent fates. CypA remains in the bacterial periplasm, likely acting as a housekeeping chaperone. In contrast, CypB is translocated into the host cell via the Type IV secretion system through a two-step process involving periplasmic interaction with VirJ, supporting its role as an effector protein.
Structural comparisons reveal that both proteins share a conserved cyclophilin core, but CypB features structurally distinct loop regions compared to CypA. Among them, an SH3-like domain and a conserved GTP-binding motif are predicted, both absent in CypA. CypB also retains a cyclosporin A-binding tryptophan and a conserved cysteine, reinforcing its eukaryotic-like profile and potential for functional mimicry.
Once in the host cytosol, CypB associates with the BCV and recruits N-WASP, which is required for Brucella intracellular replication, likely by promoting actin remodeling and redirecting vacuolar trafficking. Although N-WASP is canonically activated by Cdc42 via its GTPase-binding domain (GBD), CypB lacks a GBD-interacting motif and instead activates N-WASP through a distinct, non-canonical mechanism.
Mechanistically, CypB engages N-WASP through two interfaces: an SH3-like loop in CypB that likely targets the proline-rich domain of N-WASP, and a catalytic site that may recognize a cyclophilin-binding motif within N-WASP. Replacing CypB’s SH3-like loop with that of CypA abrogates its ability to activate N-WASP. Additionally, CypB’s PPIase activity is essential for this process, likely by isomerizing key prolines that relieve autoinhibition and expose the VCA domain, thereby initiating Arp2/3-mediated actin polymerization.
Although GTP binding remains undemonstrated, the conserved GTP-binding motif resides within a loop whose functional relevance is supported by experimental evidence: swapping this region with its counterpart from CypA significantly impairs N-WASP activation. This suggests that the loop is essential for effector function and may mediate allosteric regulation of catalytic activity, adding a new layer of mechanistic complexity.
These findings uncover a novel effector mechanism in Brucella abortus and illustrate how subtle structural divergence, particularly in loop architecture enriched with unique functional motifs, can convert a conserved bacterial chaperone into a highly specialized effector protein.