112 - Microbiología de las Interacciones

THE Brucella abortus T4SS EFFECTOR PROTEIN CYPB MODULATES HOST ACTIN DYNAMICS BY RECRUITING N-WASP TO THE Brucella-CONTAINING VACUOLE.

Hernández Oliva, Daleina-Pepe, María Victoria-Fernández, Agostina B.-Arias, Paula-Briones, Gabriel-Roset, Mara S.

Instituto de Investigaciones Biotecnológicas IIBIO-UNSAM-CONICET - San Martín - Buenos Aires - Argentina
Contacto: dhernandezoliva@iib.unsam.edu.ar

Brucella spp. is an intracellular pathogen responsible for brucellosis, a zoonotic disease with significant impacts on both human and animal health globally. The virulence of Brucella is closely associated with its ability to establish and maintain an intracellular niche within host cells. Previous research has demonstrated that the cyclophilins CypA and CypB are upregulated within Brucella’s intraphagosomal replicative niche, playing essential roles in stress adaptation, intracellular survival, and virulence. Cyclophilins, which are conserved protein-folding enzymes with peptidyl-prolyl cis-trans isomerase (PPIase) activity, are found in nearly all studied organisms. While CypA exhibits characteristics typical of Gram-negative bacterial cyclophilins, our findings reveal that CypB possesses features more closely resembling those of eukaryotic cyclophilins and is translocated into the host cell cytoplasm in a Type IV Secretion System (T4SS)-dependent manner upon Brucella internalization. This study identifies CypB as a critical bacterial effector protein for modulating host cell processes during infection. Through confocal microscopy and pull-down assays, we demonstrated that CypB interacts with N-WASP, a key regulator of the actin cytoskeleton. Furthermore, immunofluorescence analysis revealed that N-WASP is recruited to the Brucella-containing vacuole (BCV) upon infection in both bone-marrow-derived macrophages and J774.A1. This process was significantly impaired in the absence of a functional CypB, as shown in the B. abortus ΔcypAB mutant strain. Our data also indicate that PPIase activity and CypB dimer formation are required for N-WASP recruitment. In contrast, CypA, which is not translocated into the eukaryotic cytoplasm, is not required for N-WASP recruitment to the BCV. To further elucidate the functional role of N-WASP during Brucella infection, we utilized the N-WASP inhibitor Wiskostatin, and a dominant-negative approach using a VCA-GFP fusion protein to sequester the Arp2/3 complex, thereby preventing actin polymerization. Both strategies effectively inhibited Brucella infection, highlighting the essential role of N-WASP in the bacterium’s intracellular lifecycle. In summary, our results demonstrate that the Brucella T4SS effector protein CypB is necessary for the recruitment of N-WASP to the BCV, a process indispensable for the pathogen’s successful infection and intracellular survival.

Palabras clave: Brucella-Cyclophilin B (CypB)-Effector protein-N-WASP-Intracellular infection