A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

A Quantitative Single-cell Flow Cytometry Assay for Retrograde MembraneTrafficking Using Engineered Cholera Toxin.

The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.