RESUMO
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-ζ. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/patogenicidade , Transdução de Sinais , Proteínas de Bactérias/metabolismo , Inativação Gênica , Testes Genéticos , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12). To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.