Identification of Components of the Host Type IA Phosphoinositide 3-Kinase Pathway That Promote Internalization of Listeria monocytogenes

Abstract

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria promotes its internalization into some human cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. The interaction of InlB with the Met receptor stimulates host signaling pathways that promote cell surface changes driving bacterial uptake. One human signaling protein that plays a critical role in Listeria entry is type IA phosphoinositide 3-kinase (PI 3-kinase). The molecular mechanism by which PI 3-kinase promotes bacterial internalization is not understood. Here we perform an RNA interference (RNAi)-based screen to identify components of the type IA PI 3-kinase pathway that control the entry of Listeria into the human cell line HeLa. The 64 genes targeted encode known upstream regulators or downstream effectors of type IA PI 3-kinase. The results of this screen indicate that at least 9 members of the PI 3-kinase pathway play important roles in Listeria uptake. These 9 human proteins include a Rab5 GTPase, several regulators of Arf or Rac1 GTPases, and the serine/threonine kinases phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTor), and protein kinase C-. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization.

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

“…In a previous study, we obtained genetic evidence indicating an important role for mTOR in InlBmediated uptake into HeLa cells (9). Specifically, four different short interfering RNAs (siRNAs) directed against distinct sequences in mTOR mRNA each reduced the level of target protein expression and also inhibited the entry of Listeria.…”

“…HeLa cells grown in 24-well plates or on 22-by 22-mm coverslips were transfected with siRNAs or plasmid DNA using the Lipofectamine 2000 reagent (Life Technologies) as previously described (8,9).…”

“…We investigated known downstream effectors of mTORC1 or mTORC2 for roles in InlBdependent entry. Previous results indicated that p70S6K or Akt is dispensable for entry of Listeria into HeLa cells (9). We therefore limited our work to the mTORC1 effectors 4E-BP1, HIF-1␣, SREBP-1, and ULK1 and the mTORC2 substrate PKC-␣ (11).…”

“…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 target of rapamycin).…”

Entry of Listeria monocytogenes in Mammalian Epithelial Cells: An Updated View

“…In murine fibroblasts, Cdc42 participates in the activation of Rac1 via PI 3-kinase-dependent and -independent mechanisms (Bosse et al 2007). A recent systems-biology study has identified nine genes encoding known upstream regulators or downstream effectors of the type IA PI 3-kinase required for invasion of target cells by L. monocytogenes including the small GTPase Rab5 and several regulators of Arf or Rac1 small GTPases (Jiwani et al 2011). PI4P, a phosphoinositide usually present at the Golgi and produced by type II PI 4-kinases, is detected at bacterial entry sites and is critical for invasion in LoVo and HeLa cells.…”