A number of bacterial pathogens gain intracellular entry into host cell types that are normally non-phagocytic by inducing phagocytosis. Intracellular invasion is crucial to the virulence of these pathogens, enabling them to evade certain immune defenses and proliferate in relatively protected niches. Our long-term efforts are focused on understanding the steps required of microbial pathogens to convert host cells that are dormant phagocytes into active phagocytes in effecting intracellular invasion. Our studies have focused on InIB, a 67 kDa invasion protein produced by the Gram-positive, facultative intracellular pathogen Listeria monocytogenes, a cause of meningitis, abortion, gastroenteritis, and septicemia in humans. InIB exists in both bacterial surface-attached and soluble, released forms, and is responsible for bacterial invasion of a broad variety of host cell types, including epithelial and endothelial cells. InIB acts by binding and activating the host cell receptor tyrosine kinase Met (also called hepatocyte growth factor receptor, HGFR), thereby inducing signaling pathways that lead to actin-dependent uptake of the bacterium. As with a number of other receptor tyrosine kinases, dysfunction of Met is linked to a plethora of malignancies, such as kidney, breast, liver, and gastric carcinomas. In the last grant period, we determined the X-ray crystal structure of InIB and carried out functional studies that support the hypothesis that InIB, once released from the bacterial surface, induces phagocytosis by acting as a close functional mimic of hepatocyte growth factor (HGF). In this proposal, we seek to understand the steps required for InIB to induce phagocytosis and promote intracellular invasion.
Our specific aims are to determine (1) the mode of Met binding and activation by InIB, (2) whether bacterial surface-attachment of InIB is functionally important, and (3) whether InlB-mediated invasion proceeds through localized Met activation at sites of bacterial proximity or contact, or through globally distributed Met activation across the host cell surface. This last aim seeks to understand the cell biological basis by which host cells are made permissive to invasion. Our multidisciplinary approach, which combines biochemistry, genetics, and cell and structural biology, will provide an integrated view of intracellular invasion from molecular to cellular levels. The fundamental knowledge resulting from our studies will be important in guiding antimicrobial strategies directed against intracellular pathogens. We are studying how bacterial pathogens that live within human cells initially gain entry into these cells. An understanding of this process may be applicable to combating a broad variety of intracellular pathogens. In addition, the particular pathogen we are studying, Listeria monocytogenes, affects a protein called Met that is involved in some cancers. Thus, results from our studies may also have an impact on cancer therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI047163-06A1
Application #
7049801
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Mills, Melody
Project Start
2000-05-01
Project End
2010-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
6
Fiscal Year
2006
Total Cost
$218,676
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Pentecost, Mickey; Kumaran, Jyothi; Ghosh, Partho et al. (2010) Listeria monocytogenes internalin B activates junctional endocytosis to accelerate intestinal invasion. PLoS Pathog 6:e1000900
Hrtska, Sybil C Lang; Kemp, Melissa M; Munoz, Eva M et al. (2007) Investigation of the mechanism of binding between internalin B and heparin using surface plasmon resonance. Biochemistry 46:2697-706
Ghosh, Partho (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68:771-95
Banerjee, Manidipa; Copp, Jeremy; Vuga, Danka et al. (2004) GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation. Mol Microbiol 52:257-71
Marino, Michael; Banerjee, Manidipa; Copp, Jeremy et al. (2004) Characterization of the calcium-binding sites of Listeria monocytogenes InlB. Biochem Biophys Res Commun 316:379-86
Lawton, Jeffrey A; Ghosh, Partho (2003) Novel therapeutic strategies based on toll-like receptor signaling. Curr Opin Chem Biol 7:446-51
Copp, Jeremy; Marino, Michael; Banerjee, Manidipa et al. (2003) Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway. J Biol Chem 278:7783-9
Marino, Michael; Banerjee, Manidipa; Jonquieres, Renaud et al. (2002) GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands. EMBO J 21:5623-34