Abstract Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.
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