Howard Goldfine
Professor, Dept of Microbiology

Microbiology, Virology and Parasitology Program

Address

425/6 Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104

Office tel.: 215 898-6384
Lab tel.: 215 898-7096
Fax: 215 898-9557
E-mail: goldfinh@mail.med.upenn.edu

Link(s)

Dr. Goldfine's Microbiology Page

Education

City College of the City University of New York (CUNY): BS (Biology/Chemistry), 1953.

University of Chicago: PhD (Biochemistry), 1957.

Research Interests

• Bacterial Pathogenesis and bacterial membrane lipids

Key words: Listeria monocytogenes, Bacillus anthracis, Phospholipase C, Phagocytosis, Intracellular growth of bacteria, phospholipids, plasmalogens.

Description of Research

A genomic approach to discovery of orphan genes involved in plasmalogen biosynthesis
The pathway for the biosynthesis of plasmalogens, alk-1-enyl ether containing polar lipids common to the membranes of clostridia and other anaerobes, is unknown. We have discovered that the strain of Clostridium tetani E88, for which a genome sequence is available, is anomalous in not having plasmalogens.We have obtained the genomic sequences of two strains of C. tetani using the recent 454 technology. We are comparing the sequences of about 60 genes we have identified as potential genes for plasmalogen synthesis with the same genes of strain E88. By eliminating genes that are identical in all three strains or only differ between E88 and one of the other strains, we are narrowing the list down to the point where a set of mutants of these genes could be made in related non-pathogenic clostridia that have the same pathway and genes.

Listeria monocytogenes
Interactions between bacterial pathogens and the mammalian host cell are at the intersection of microbiology and cell biology. Work in Dr.Goldfine's laboratory has been focused on two gram-positive pathogens, Listeria monocytogenes and Bacillus anthracis. Listeria monocytogenes (Lm) is a facultative intracellular pathogen that is a significant cause of human disease. In its initial interactions with a mammalian host cell, two phospholipases and listeriolysin O, a pore forming cytolysin, act together to initiate calcium and PKC signaling in the host cell. One of the phospholipases is a phosphatidylinositol (PI) specific phospholipase C (PI-PLC), which has been shown to play a role in escape of these bacteria from a primary endocytic vacuole. A second broad range phospholipase C (PC-PLC plays a significant role in cell-to-cell spread. Both enzymes release diacylglycerol upon hydrolysis of glycerophospholipids, and the broad range phospholipase C releases ceramide from sphingomyelin in infected cells. Recent work in the lab has shown that mutants in Lm PI-PLC are not complemented by introduction of the gene for PI-PLC from Bacillus cereus (Bc).or B. anthracis. We have previously shown that LmPI-PLC differs from Bacillus PI-PLC in having weak activity on proteins anchored to eukaryotic cell membranes by a glycosyl-PI modification at the C-terminus. It appears that LmPI-PLC has evolved for intracellular growth and cell-to-cell spread by the absence of a specific amino acid sequence needed for binding of the glycan chain of glycosyl-PI-anchored proteins prior to cleavage of the PI anchor.

Intracellular signaling
Recent work in this laboratory has shown that prior to internalization by a macrophage, L. monocytogenes induces a series of elevations of intracellular calcium levels in the host cytoplasm. Using isogenic mutants in the virulence factors, we have shown that listeriolysin O and the two phospholipases are required for the full sequence of calcium changes. These calcium elevations alter the kinetics of association of the bacterium with the host cell and the entry of bacteria into the host by phagocytosis. The current hypothesis is that these signaling events serve to modify the pathway of maturation of the phagosome so that the bacterium is able to escape from the primary vacuole and grow in the cytoplasm. Our more recent work has shown that PI-PLC and LLO cooperate to produce a protein kinase C cascade which is initiated by the generation of diacylglycerol upon hydrolysis of host phospholipids. This cascade leads to translocation of PKC beta I and II to early endosomes. Pharmacological inhibitors of PKC beta significantly diminish the ability of L. monocytogenes to escape from the phagosome. Surprisingly, PI-PLC also plays a role in permeabilizing the primary phagosome after internalization of L. monocytogenes.

Selected Publications

Wei, Z., Zenewicz, L.A., and Goldfine, H., Listeria PI-PLC has evolved for intracellular growth and virulence by greatly reduced activity on GPI-anchors, Proc. Natl. Acad. Sci.USA , 102 (36): 12927-12931, 2005.

Poussin, M. A. and Goldfine, H. Involvement of Listeria monocytogenes phosphatidylinositol-specific phospholipase C and host PKC in permeabilization of the phagosome, Infect. Immun., 73 (7), 4410-4413, 2005.

Zenewicz, L.A., Wei, Z., Goldfine, H., and Shen. H. Phosphatidylinositol-specific phospholipase C of Bacillus anthracis downmodulates the immune response. J. Immunol., 174(12): 8011-8016, 2005.

Wei, Z., Schnupf, P., Poussin, M., Zenewicz, L.A., Shen, H., and Goldfine, H., Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis . Infect. Immun., 73:6639-6646, 2005.

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Lab

Rotation Projects

We are not currently able to support rotation projects

Lab personnel

Norah C. Johnston, Ph.D. - Research Assistant