Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3‐kinase

Abstract

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4‐phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella‐containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3‐kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3‐phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P‐binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.

Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis

“…To gain insights into the mechanism of how C. burnetii interferes with host immune response, we performed a screen to identify Dot/Icm proteins capable of inhibiting NF-κB activation. Genes coding for established Dot/Icm effectors or for proteins without known roles in virulence were individually inserted into a vector that allows the expression of 4xFlag-tagged proteins in mammalian cells ( 26 ). Each construct of this library was cotransfected into HEK293T (human embryonic kidney) cells with a plasmid-based luciferase reporter that responds to NF-κB activity ( 27 ).…”

“…To measure NF-κB activation, HEK293T cells seeded (5 × 10 4 cells/well) in 24-well plates were transfected with 300 ng plasmid DNA derived from pCMV4Flag ( 26 ) that harbor the candidate C. burnetii effector genes together with 100 ng NF-κB reporter plasmid pGL4.32 (Promega, E8491) ( 27 , 46 ). To normalize transfection efficiency, 100 ng pRL-TK Renilla luciferase reporter plasmid was added to each transfection.…”

Dynamic remodeling of host membranes by self-organizing bacterial effectors

“…Sequential action of the Legionella PI 4-kinase LepB and PI 3-phosphatase SidF may then mediate the PI3P-to-PI4P conversion in the LCV membrane (2,4). A recent report suggests that MavQ acts in tandem with LepB and SidF to supply PI4P in the LCV membrane (15). While it is possible that MavQ indirectly contributes to PI4P generation in the LCV membrane and thus intracellular Legionella growth, we favor the idea that the membrane remodeling capabilities of MavQ play more important roles based on the following observations: 1) coevolution of MavQ and SidP during Legionella speciation (7) and 2) the requirement of biochemical synergy between MavQ and SidP for effective, dynamic membrane remodeling.…”

Exploitation of the Host Ubiquitin System: Means by Legionella pneumophila

“…One prominent feature of the LCV is that this organelle dynamically recruits and retains an array of proteins of either host or L. pneumophila origin. In some cases, the anchoring of bacterial proteins on the LCV is mediated by their binding to phosphatidylinositol-4-phosphate (PI4P), an important signaling lipid whose enrichment on the bacterial phagosome is achieved in part by sequential action of three independent effectors that coordinate to biosynthesize PI4P from phosphatidylinositol ( Hsu et al, 2012 ; Dong et al, 2016 ; Li et al, 2021 ). SidC (Lpg2511) and its ortholog SdcA anchor on the LCV by binding to PI4P via a lipid-interacting domain localized in the carboxyl portion of these proteins ( Weber et al, 2006 ; Ragaz et al, 2008 ; Luo et al, 2015 ).…”