Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

Abstract

The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-␣ (PKC-␣) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1␣ (HIF-1␣) and the mTORC2 substrate PKC-␣ each control Listeria uptake. Genetic or pharmacological inhibition of PKC-␣ reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-␣ to be host factors exploited by Listeria to promote infection. PKC-␣ controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.KEYWORDS InlB, Listeria monocytogenes, Met receptor, protein kinase C, actin, mTOR L isteria monocytogenes is a foodborne pathogen capable of causing serious infections resulting in meningitis or abortions (1). Critical for disease is the ability of Listeria to induce its internalization into nonphagocytic mammalian cells in the intestine, liver, or placenta (2). One of the major pathways of internalization of Listeria into human cells is mediated by interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met (3). InlB-dependent entry involves remodeling of the host plasma membrane through actin polymerization (4, 5). Substantial progress in characterizing InlB-mediated uptake has been made, resulting in the identification of several host signal transduction pathways that control actin polymerization and perhaps other physiological processes during entry (4). Despite this progress, a detailed understanding of the molecular mechanism of InlB-dependent internalization of Listeria remains incomplete.The human type IA phosphoinositide 3-kinase (PI3K) pathway is one of the major signaling networks controlling InlB-mediated entry (4,(6)(7)(8). Recent RNA interference (RNAi) screens identified 13 components of the type IA PI3K pathway involved in InlB-dependent internalization of Listeria (9, 10). One host protein demonstrated in this work to play an important role in Listeria entry is mTOR (mammalian ...

A1 adenosine receptor signaling reducesStreptococcus pneumoniaeadherence to pulmonary epithelial cells by targeting expression of platelet‐activating factor receptor

“…RNA interference was carried out per manufacturer's protocol and as previously described (Bhalla, Law, Dowd, & Ireton, 2017) using cells were seeded in 6-well plates and 24 hr later washed and transfected with siRNA against the above targets at a final concentration of 100 nmol using Lipofectamine2000 (Thermofisher) in FBS-free H292 media. Twelve to 15 hr following transfection, FBS was added to the media at a final concentration of 10%.…”

The Host Scaffolding Protein Filamin A and the Exocyst Complex Control Exocytosis during InlB-Mediated Entry of Listeria monocytogenes

“…Previous results demonstrated that the human serine/threonine kinases mTOR and protein kinase C-␣ (PKC-␣) are activated downstream of Met and play important roles in InlB-mediated entry of Listeria (13). mTOR and PKC-␣ comprise a signaling pathway involved in several biological events, including cell migration and survival (14,15).…”

“…mTOR forms part of a multicomponent complex called mTORC2 that phosphorylates serine 657 in a hydrophobic motif in PKC-␣, thereby upregulating PKC kinase activity (16). Importantly, InlB-mediated entry of Listeria is accompanied by mTOR-dependent phosphorylation of serine 657 in PKC-␣, indicating that mTOR functions upstream of PKC-␣ during entry (13). How mTOR and PKC-␣ control internalization of Listeria is not well understood.…”

“…How mTOR and PKC-␣ control internalization of Listeria is not well understood. Although PKC-␣ has a minor role in actin polymerization during entry, mTOR has no detectable effect on this process (13). It therefore seems likely that mTOR and PKC-␣ affect uptake of Listeria by regulating host processes apart from the assembly of actin filaments.…”

Targeting of host cell receptor tyrosine kinases by intracellular pathogens

“…After Gab1-mediated or direct interaction with c-Met, activated PI3K mediates Akt activation through phosphorylation of mTORC (67, 68), which, as described above for PRRSV, regulates cytoskeleton dynamics through activation of LIMK-dependent phosphorylation of cofilin (69). PKC- is also activated by mTORC, which is important for L. monocytogenes entry (68). It has been established that some components of the exocytosis machinery play a role in the uptake of L. monocytogenes.…”