Brucella abortus egresses from host cells in infective clusters through an actin-dependent mechanism

Brucella abortus is an intracellular pathogen whose cell cycle encompasses attachment, internalization, trafficking, replication, and egress from host cells. Although the intracellular life of Brucella has been extensively studied, the mechanisms underlying its exit from host cells remain unclear. In this work, we expand the knowledge of this intracellular step by observing a significant increase in the formation of Brucella -containing vacuoles with autophagic features (aBCVs) and abundant B. abortus extracellular clusters (BECs) after 72 h of infection. Membrane extensions protruding through actin polymerization were evident in cells at later stages of infection. Purified aBCVs and BECs were similar in size and predominantly acidic. These vacuoles exhibited a compact arrangement of well-ordered bacteria, comprising a heterogeneous population of dead and live bacteria and host components, including LAMP-1 and actin filaments. A proportion of BECs were enclosed within an impermeable host membrane, while others were not. The actin cytoskeleton was implicated in the protrusion of BECs, since modulation of Rho GTPases affected intracellular aBCV formation and the egress of BECs. BECs protruding from cells invaded uninfected cells, initiating a new cycle of infection. The topological structure and function of BECs underscore their significance in the Brucella life cycle as vehicles for bacterial dissemination to host cells and organs.