Abstract
During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.
Highlights
Protein and membrane trafficking events during eukaryotic growth are regulated at many steps, such as the selection of the appropriate cargo, the proper construction, budding, and targeting of secretory vesicles to the appropriate membranebound compartments, and the final fusion of those transport vesicles with their target membranes
As an essential step in all trafficking events including neurotransmission, exocytosis, and hormone secretion, intracellular membrane fusion events are catalyzed by a highly conserved set of core machinery consisting of soluble NSF attachment protein receptors (SNAREs) [1,2], Rab-family GTPases [3,4], Sec1-Munc18 (SM) family member proteins [5,6,7,8], membrane tethering factors [9,10], and the SNARE protein chaperones, N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (a-SNAP) [11,12,13,14]
Yeast strains harboring this plasmid show distinct fragmentation of the yeast vacuolar membrane when grown on media containing 2% galactose, but not under glucose growth conditions (Figure 1A). This vacuole morphology difference is in striking contrast to vector controls, and is in agreement with this previous report describing the effects of LegC3 in yeast. This vacuolar fragmentation is similar to that seen in yeast strains lacking the Vps33p subunit of the two normal endosomal/vacuolar membrane tethering complexes, CORVET and HOPS, which is required for normal vacuolar morphology, vacuolar protein sorting, and homotypic vacuole fusion in vivo and in vitro [5,59]
Summary
Protein and membrane trafficking events during eukaryotic growth are regulated at many steps, such as the selection of the appropriate cargo, the proper construction, budding, and targeting of secretory vesicles to the appropriate membranebound compartments, and the final fusion of those transport vesicles with their target membranes. As a prerequisite for intracellular survival, L. pneumophila requires a functional Dot/Icm Type IV secretion system to inject more than 250 protein effectors into the host cell [25,26,27,28,29,30]. These effector proteins quickly hijack and manipulate normal host cell processes, allowing the bacterium to evade lysosomal degradation through the manipulation of normal phagocytic trafficking pathways [31,32]. To further modulate Rab activity, Legionella produces a secreted Rab GTPase-activating protein (GAP), LepB, allowing Legionella to fully control a Rab GTPase-dependent trafficking pathway within the host [40]
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