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| Paul Webster, Ph.D. Scientist II and Head Ahmanson Advanced Electron Microscopy and Imaging Center email: pwebster@hei.org |
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Education and Affiliations
Professional Experience: 1990-1998 Yale University School of Medicine, Dept. of Cell Biology Current Projects Cellular and molecular mechanisms of bacterial persistence in recurrent otitis media. Otitis media is a common disease of children that can often lead to either hearing loss or systemic complications of the central nervous system. The development of new anti-infective agents to support and, in some instances replace, antibiotics, requires a better understanding of how bacteria cause disease at both the cellular and molecular levels. In recent years new approaches for studying pathogenesis of bacteria have been applied to a selected group of microorganisms to reveal basic strategies of disease causation. These approaches are being used to examine how non-typeable Hemophilus influenzae (NTHi), a causative agent of otitis media, interacts with epithelial cells at a molecular level. Attachment of NTHi to epithelial cells involves the interaction of bacteria with host cell membranes. Attached NTHi then either remain on the cell surface or are internalized by the host cell into a membrane-bounded vacuole. Surface-bound bacteria may cause damage to host cells by either releasing toxins or by stimulating an inflammatory response that becomes self-destructive. Internalized NTHi are either killed inside the cells or multiply within the vacuole, escaping from the cell either as a result of pathogen-induced cell lysis, by directed sub cellular transport of the phagosome, or by lysis of the phagosome membrane. Using these hypotheses the interaction of bacteria and host cell surface membranes are being examined using high-resolution imaging. The morphology of internalized bacteria and the intracellular environment are being examined and immunocytochemical methods to identify specific intracellular compartments occupied by internalized bacteria are being applied. Autophagic vacuole formation Autophagy, the constitutive process by which cells modulate their protein mass, has been extensively studied. This process involves the identification and sequestration of cytoplasmic components into membrane bound autophagosomes, or autophagic vacuoles (AV’s) for subsequent breakdown in lysosomes. The lack of suitably specific markers for these organelles, as well as the cryptic appearance of their contents, has meant that examination of the very early stages of sequestration into AV’s has not previously been possible. To elucidate early AV formation, we are using the intracellular pathogen Listeria monocytogenes as a cytoplasmic marker for early AV formation. This gram-positive bacterium expresses a membrane disrupting hemolysin (LLO) which it uses to gain access to host cell cytoplasm. When in the cytoplasm, the act a gene product induces host cell actin to polymerize around the bacteria which the bacteria use to move around the cell and pass into non-infected adjacent cells. Preliminary studies have shown that act a deficient forms of L. monocytogenes, killed after gaining access to host cell cytoplasm, are rapidly internalized into autophagic vacuoles. This model system is being used to examine the polypeptide profiles of the membranes involved in the early stages of autophagic vacuole formation. Collaborative Research HEI UCLA Caltech The cell biology of non-viral DNA delivery systems The polymer cyclodextran has been used in synthetic packaging of compounds for targeted drug delivery. More recently, it has been used to package DNA to study the feasibility of using it as a delivery system in gene therapy. A modified polymer of cyclodextran that binds to nucleic acids and protects them from enzyme degradation has been developed. When added to cells the nucleic acid-polymer complexes (polyplexes) are readily taken up. Assays to detect delivery of DNA to the cell nucleus show that this event does occur, but not often. Light microscopic visualization of these internalized polyplexes, achieved by coupling them to fluorescent dyes, shows that they are more likely to be delivered to the perinuclear region of cells where the nucleic acid and cyclodextran appear to separate. This suggests they are being delivered to lysosomes within cells where they presumably undergo some form of enzyme-induced degradation. This has been confirmed at the light microscopy level by colocalizing the polyplex fluorescence with lysotracker. The three aims of this study are 1). To examine the structure of the polyplexes at high resolution; 2). To examine the intracellular fate of the polyplexes using immunocytochemical methods; 3). To modify the polyplexes and alter their intracellular targetting. In this way we aim to produce polymers of modified cyclodextran that will be suitable for use as targetted, non-viral DNA delivery systems. Additional Information National Organization for Hearing Research (NOHR) Foundation: 2001 Research Award. "Cellular and molecular mechanisms of bacterial persistence in recurring otitis media." NASA Space Act Agreement. Awarded unrestricted access to ROSS 3-D reconstruction software. Invited Instructor, European Molecular Biology Organization Member:
Selected Publications 1997-2002:
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