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

Abstract

Brucella abortus is an intracellular pathogen whose cell cycle encompasses attachment, internalization, trafficking, replication, and egress from cells. While the intracellular life of Brucella has been extensively studied, the mechanisms underlying its exit from host cells are still unclear. In this work, we observed a significant increase in the formation of B. abortus containing vacuoles intracellular clumps (BCVIC) and abundant B. abortus containing vacuoles-derived extracellular clumps (BCVEC) after 72 h of infection. Membrane extensions protruding through the polymerization of actin filaments were evident in cells at later stages of infection. Purified BCVIC and BCVEC were of similar sizes and predominantly acidic. These vacuoles exhibited a compact arrangement of well-ordered bacteria, comprised of a heterogeneous population of dead and live bacteria and host components LAMP-1 and actin filaments. A proportion of BCVEC were naked, while others were enclosed within an impermeable host membrane. The actin cytoskeleton was implicated in the protrusion of BCVEC since the modulation of Rho GTPases affected the egress of BCVEC. The BCVEC protruding from cells were capable of invading non-infected cells, initiating a new cycle of infection. The topological structure and function of BCVEC 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 are not fully understood. We found that protruding acidic autophagic-like vesicles containing compact, well-organized Brucella clusters are shed from cells via GTPase and actin filament recruitment. Through this process, the vesicles are impermeable to antibodies and other substances, like antibiotics, and are highly infectious to neighboring cells. This previously unreported mechanism enhances understanding of the well-known Brucella stealth strategy to evade the immune system and establish harmful, long-lasting infections.