LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

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.

Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

“…The chlamydial T3SS substrate IncA harbors two SNARElike coiled-coil motifs (58) and inhibits membrane fusion mediated by endocytic, but not exocytic, SNARE complexes (59). Expression of the L. pneumophila effector LegC3 produces a vacuolar protein sorting defect in yeast (40) linked to the disruption of trans-SNARE complexes by coiled-coil domains within the effector (60). In contrast to L. pneumophila and Chlamydia spp.…”

Space: A Final Frontier for Vacuolar Pathogens

“…The net result is that EEA1, which binds to PI(3)P, no longer associates with the endosome. Legionella also expresses LegC3, a protein with a ‘SNARE-like’ N-terminal domain that blocks homotypic vacuole fusion events in yeast (48). It is not difficult to imagine that Brucella likewise expresses proteins that inhibit membrane fusion in order to maintain its preferred replicative niche and evade destruction within lysosomes.…”

Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution ofLegionellaeffectors

“…Indeed, LegC3 was shown previously to inhibit SNARE-mediated homotypic fusion of yeast vacuoles in vitro, but a direct interaction between LegC3 and SNAREs was not observed (21). We found that all three LegC proteins selectively interact with the R-SNARE vesicle-associated membrane protein 4 (VAMP4), and furthermore, that the availability of VAMP4 is rate limiting for the intracellular proliferation of Legionella.…”