Description of Research Expertise

Research Interests
Bacterial Pathogenesis.

Key words: Listeria monocytogenes, Bacillus anthracis, Phospholipase C, Phagocytosis, Intracellular growth of bacteria, membrane lipids of clostridia, pathways of lipid biosynthesis.

Description of Research

Listeria monocytogenes
The interactions between bacterial pathogens and the mammalian host cell is at the intersection of microbiology and cell biology. Work in Dr. Goldfine's laboratory is focused on Listeria monocytogenes, 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 hemolysin, act to deliver bacterial secreted proteins into the host. 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 on hydrolysis of glycerophospholipids, and the broad range phospholipase C releases ceramide from sphingomyelin in infected cells.

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. Escape from the primary vacuole is known to require listeriolysin O and to be assisted by the phosphatidylinositol-specific phospholipase C. Our 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 PKCs significantly diminish the ability of L. monocytogenes to escape from the phagosome.

Evolution of Listeria PI-PLC
Previous work in this laboratory has shown that L. monocytogenes (Lm) PI-PLC, unlike the classical PI-PLCs from Bacillus, has very low activity on eukaryotic proteins anchored to cell membranes by way of a C-terminal glycan-PI anchor (GPI-anchors). We have found that all Listeriae lack the Vb beta strand present in the enzymes from Bacillus that recognizes the glycan linkage in GPI-anchors. We constructed an Lm strain expressing B. cereus PI-PLC (BcPIPLC) on its chromosome.. Escape of the new strain from the J774 phagosome was decreased like that of the PI-PLC negative strain DP-L1552, and cell-to-cell spread was impaired. Its growth in the mouse liver was >100 fold lower than that of WT. These results imply that the activity on GPI-anchors by BcPIPLC impairs Lm intracellular growth and virulence. We made further constructs in which the Vb beta strand of BcPI-PLC was either removed or altered. The results obtained provide strong evidence for the hypothesis that LmPI-PLC has evolved to promote Lm intracellular growth and virulence by absence the Vb beta strand and thus the almost complete loss of cleavage activity on GPI-anchors.

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