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.IMPORTANCEB. abortus is an intracellular pathogen that traffics to the endoplasmic reticulum, where it replicates. However, the mechanisms by which Brucella exits host cells and infects new ones remain poorly understood. Our findings reveal that protruding acidic autophagic-like vesicles containing compact, well-organized Brucella clusters are shed from cells via the GTPase-dependent recruitment of actin filaments. Through this process, the vesicles are impermeable to antibodies and other substances, like antibiotics, and are highly infectious to neighboring cells. This mechanism enhances the understanding of the well-known Brucella stealth strategy to evade the immune system and establish harmful, long-lasting infections.