Francisella Targets Cholesterol-Rich Host Cell Membrane Domains for Entry into Macrophages

Abstract

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains (“lipid rafts”) with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…This novel phenomenon differs from other described bacterial uptake mechanisms, including coiling phagocytosis seen at the surface of phagocytes during Legionella pneumophila infection and conventional phagocytosis observed during infection with multiple bacterial and viral species (102,103,199,200). Furthermore, Francisella LVS can recruit host cell membrane cholesterol-rich lipid domains, or "lipid rafts," with caveolin-1 for successful entry into macrophages (217). Depletion of plasma membrane cholesterol or removal of glycosylphosphatidylinositol (GPI)-anchored proteins results in a severe decrease in Francisella uptake and subsequent intracellular replication (217).…”

“…Furthermore, Francisella LVS can recruit host cell membrane cholesterol-rich lipid domains, or "lipid rafts," with caveolin-1 for successful entry into macrophages (217). Depletion of plasma membrane cholesterol or removal of glycosylphosphatidylinositol (GPI)-anchored proteins results in a severe decrease in Francisella uptake and subsequent intracellular replication (217). Interestingly, in nonphagocytic cells such as hepatocytes, LVS and F. novicida use cholesterol-rich lipid domains dependent on clathrin, not caveolin-1, for entry (123).…”

Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection

“…After recognizing bacteria, the phagocytic receptors trigger cell signaling that results in the rearrangement of cytoskeleton and engulfment of the particle. In the case of Francisella, two pathways have been reported to be activated during the phagocytosis process: phosphoinositol 3-kinase (PI3K)/Akt and the tyrosine kinase Syk/mitogen-activated protein kinase (MAPK) Erk2 pathways (20,21). For the PI3K/Akt pathway, phosphopeptides for PI3K and ribosomal protein S6 kinase beta-1 (Rps6kb1) were identified.…”

“…After being recognized by one of these receptors Francisella triggers a signaling cascade that culminates in the rearrangement of actin filaments and engulfment of the bacteria (17)(18)(19). This signaling cascade is in part regulated by phosphorylation and dependent on kinases such as Akt, Syk and Erk (20,21). More recently, Francisella was also shown to be endocytosed by liver cells in a mechanism that involved cholesterol-rich domains on the plasma membrane and clathrin-coated vesicles (22).…”

“…Also surprising was that ⌬lpcC, ⌬manB, and ⌬manC strains were phagocytosed at a much higher rate than WT F. novicida via a mechanism that does not require actin polymerization (34). Actin polymerization has thus far been demonstrated to be involved in internalization of Francisella by either phagocytic or endocytic pathways (21,22,35); therefore, the finding that ⌬lpcC, ⌬manB, and ⌬manC mutant strains are able to invade host cells independent of actin filaments remains to be understood. Deletion of lpcC from F. tularensis Schu S4 also results in a similar phenotype, suggesting that this phenomenon acts broadly among the different Francisella species rather than specific to F. novicida (Tempel and Heffron, unpublished observations).…”