VipD is a Rab5-activated phospholipase A ₁ that protects Legionella pneumophila from endosomal fusion

Abstract

A crucial step in the elimination of invading microbes by macrophages is phagosomal maturation through heterotypic endosomal fusion. This process is controlled by the guanine nucleotide binding protein Rab5, which assembles protein microdomains that include the tethering protein early endosomal antigen (EEA) 1 and the phosphatidylinositol (PI) 3-kinase hVps34, which generates PI(3)P, a phospholipid required for membrane association of EEA1 and other fusion factors. During infection of macrophages, the pathogen Legionella pneumophila bypasses the microbicidal endosomal compartment by an unknown mechanism. Here, we show that the effector protein VipD from L. pneumophila exhibits phospholipase A 1 activity that is activated only upon binding to endosomal Rab5 or Rab22. Within mammalian cells, VipD localizes to endosomes and catalyzes the removal of PI(3)P from endosomal membranes. EEA1 and other transport and fusion factors are consequently depleted from endosomes, rendering them fusion-incompetent. During host cell infection, VipD reduces exposure of L. pneumophila to the endosomal compartment and protects their surrounding vacuoles from acquiring Rab5. Thus, by catalyzing PI(3)P depletion in a Rab5-dependent manner, VipD alters the protein composition of endosomes thereby blocking fusion with Legionella-containing vacuoles.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

“…To identify more clearly whether the ACT-PLA activity cleaves specifically the sn-1 or the sn-2 ester bond of the lipid substrate, we used highly sensitive fluorogenic phospholipid substrates called PED-A1 [N-((6-(2,4-DNP)Amino)Hexanoyl)-1-(BODIPY FL C5)-2-Hexyl-Sn-Glycero-3-Phosphoethanolamine] (which measures PLA 1 activity) and PED6 [N-((6-(2,4-Dinitrophenyl)amino)hexanoyl)-2-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoyl)-1-Hexadecanoyl-snGlycero-3-Phosphoethanolamine] (which measures PLA 2 activity) (34). Both substrates are modified glycerophosphoethanolamines with BODIPY FL dye-labeled sn-1 or sn-2 acyl chains, respectively, and a dinitrophenyl group conjugated to the polar head group of the lipid to provide intramolecular quenching.…”

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

“…VipD is a T4SS-translocated substrate of Legionella pneumophila, the causative agent of a potentially fatal pneumonia known as Legionnaires' disease, and another example of an effector whose catalytic activity depends on the presence of a host factor (11)(12)(13)(14). Following uptake by human alveolar macrophages, L. pneumophila translocates VipD together with more than 250 other effector proteins through its Dot/Icm T4SS into the host cell cytoplasm (15).…”

“…Although the precise biological role of most L. pneumophila effectors remains unclear, we recently showed that VipD is important for endosomal avoidance by LCVs. The protein localizes to endosomes presumably by binding to the small GTPases Rab5 or Rab22, key regulators of endosomal function (13,14). Rab GTPase binding to the C-terminal domain of VipD triggers robust phospholipase A1 (PLA 1 ) activity within the Nterminal domain, resulting in the removal of phosphatidylinositol 3-phosphate [PI(3)P] and potentially other lipids from endosomal membranes (14).…”

“…The protein localizes to endosomes presumably by binding to the small GTPases Rab5 or Rab22, key regulators of endosomal function (13,14). Rab GTPase binding to the C-terminal domain of VipD triggers robust phospholipase A1 (PLA 1 ) activity within the Nterminal domain, resulting in the removal of phosphatidylinositol 3-phosphate [PI(3)P] and potentially other lipids from endosomal membranes (14). Without PI(3)P, endosomal markers such as early endosomal antigen 1 (EEA1) are lost from these membranes, most likely rendering the endosomal compartment fusion incompetent (17).…”

“…Without PI(3)P, endosomal markers such as early endosomal antigen 1 (EEA1) are lost from these membranes, most likely rendering the endosomal compartment fusion incompetent (17). L. pneumophila mutants lacking vipD are attenuated in avoiding endosomal fusion, and their LCVs acquire the endosomal marker Rab5 more frequently than LCVs containing the parental strain producing VipD (14). Thus, by coupling PLA 1 activity to Rab5 binding, the catalytic activity of VipD is directed specifically against the endosomal compartment without visibly affecting neighboring cell organelles.…”