Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages

Abstract

Host membranes are inherently critical for niche homeostasis of vacuolar pathogens. Thus, intracellular bacteria frequently encode the capacity to regulate host lipogenesis as well as to modulate the lipid composition of host membranes. One membrane component that is often subverted by vacuolar bacteria is cholesterol – an abundant lipid that mammalian cells produce de novo at the endoplasmic reticulum (ER) or acquire exogenously from serum-derived lipoprotein carriers. Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. From within a unique ER-derived vacuole L. pneumophila promotes host lipogenesis and experimental evidence indicates that cholesterol production might be one facet of this response. Here we investigated the link between cellular cholesterol and L. pneumophila intracellular replication and discovered that disruption of cholesterol biosynthesis or cholesterol trafficking lowered bacterial replication in infected cells. These growth defects were rescued by addition of exogenous cholesterol. Conversely, bacterial growth within cholesterol-leaden macrophages was enhanced. Importantly, the growth benefit of cholesterol was observed strictly in cellular infections and L. pneumophila growth kinetics in axenic cultures did not change in the presence of cholesterol. Microscopy analyses indicate that cholesterol regulates a step in L. pneumophila intracellular lifecycle that occurs after bacteria begin to replicate within an established intracellular niche. Collectively, we provide experimental evidence that cellular cholesterol promotes L. pneumophila replication within a membrane bound organelle in infected macrophages.

A nutritional immunity blockade controls extracellular bacterial replication inLegionella pneumophilainfections

“…1b-c). 79,80 The Lp growth kinetics with human U937 macrophages peaked at 48hrs for both JR32 and Lp01 and the curves were comparable regardless of serum availability (Fig. 1 a-b).…”

“…To avoid a pyroptotic cell death response triggered by cytosolic delivery of flagellin, 78 which can convolute data interpretation in infection-based growth assays, we used a flagellin clean deletion mutant. We engineered two distinct L. pneumophila Philadelphia-1 derived strains – Lp01 and JR32 – to bioluminescence by inserting the LuxR operon ( luxC luxD luxA luxB luxE ) from Photorhabdus luminescens on the bacterial chromosome under the constitutive IcmR promoter using two different genetic approaches 79,80 (SFig. 1a).…”

“…The L. pneumophila JR32 strains used in the study encoded the constitutive Lp icmRp-luxR regulon, which has been previously described in detail. 79 The L. jordanis strains encoded the IPTG-inducible Lj pTAC-luxR regulon, which has been previously described in detail. 80 The L. pneumophila Lp01 strains used in the study encoded a new constitutive Lp icmR-luxR locus regulon.…”

Characterization ofNeisseria gonorrhoeaecolonization of macrophages under distinct polarization states and nutrients environment

“…Bioluminescence has emerged as a reliable approach for quantitative analysis of bacterial growth under various conditions, including in cellular infections (44, 45). Measurements of light output from bioluminescent bacteria allows growth kinetics analysis in a high-throughput manner with great resolution (41). Because light production requires ATP, bioluminescence also reflects the metabolic state and viability of the producers (46).…”

“…Inocula for infections were prepared in PBSG, RPMI or RPMI containing inactivated human serum (10%) and the number of bacteria was confirmed by plating dilutions from the inoculum on GCB agar. Legionella pneumophila serogroup 1 strain JR32 Δ flaA luxR was used in this study (41). The Legionella strain was grown on charcoal yeast extract (CYE) plates composed of 1% yeast extract, 1%N-(2-acetamido)-2-aminoethanesulphonic acid (ACES; pH 6.9), 3.3 mM l-cysteine, 0.33 mM Fe(NO3) 3 , 1.5% agar, 0.2% activated charcoal] (42).…”