Drexel University College of Medicine

How Neisseria gonorrhoeae, N. meningitidis and Bacillus anthracis cause disease.

My laboratory personnel have, for years, been investigating two neisseria virulence factors: Opa proteins and sialyltransferase. Opa proteins, a family of outer membrane proteins, are pluripotent virulence factors, allowing Ng and Nm to adhere to, invade and survive within human cells. Using the yeast two hybrid system, we showed that intracellular Opa+ Ng bind at least two human epithelial cell cytosolic proteins, including pyruvate kinase (PK) and thyroid hormone receptor interacting protein 6 (TRIP6). Ng bind PK and appear to require pyruvate for optimal intracellular survival and growth. We hypothesize that Ng bind TRIP6, which is a cytoskeletal-associated protein and a nuclear messenger & transcriptional activator, to signal their presence within the cell, and perhaps alter host responses. We presently focus on structure-function studies to help us better understand the many biological roles of Opa proteins.

We have shown that sialyltransferase (stase) is an outer membrane, surface-exposed glycosyltransferase that transfers sialic acid (N-acetylneuraminic acid, Neu5Ac) from CMP-Neu5Ac to terminal galactose residues of neisseria lipooligosaccharide (LOS). Sialylation of LOS can render Ng and Nm resistant to the bactericidal action of human serum, and can modulate how these pathogens interact with host cells. We study regulation of expression of stase by environmental cues including contact with serum, association with human cells, human immune response to stase during infection, and the role of sialylation in disseminated gonococcal disease (DGI). The regulation of stase expression has turned out to be very complex. Expression mechanisms differ between Nm and Ng, and although in vitro it appears that essentially all Nm and Ng express stase constitutively, association of Ng with epithelial cells completely shuts down expression. We continue to study the mechanisms of stase expression, and we are interested in how the enzyme gets transported and incorporated in the outer membrane.

More recently we have studied the interaction of B. anthracis with human macrophages and neutrophils, focusing on the role of Anthrolysin O (ALO), a cholesterol-dependent cytolysin, and a putative new anthrax toxin. ALO has lethal and potent sublethal effects on human phagocytes, and signals at least partially through the ‘endotoxin receptor’, i.e., Toll Like Receptor 4 (TLR4). ALO, and B. anthracis expressing ALO, have a plethora of ALO-dependent activities, including inducing pro-inflammatory cytokine release and associated intracellular signaling pathways, inducing neutrophil degranulation, and inhibiting phagocyte chemotaxis.  In addition, we have found profound effects of antioxidants, like glutathione and N-acetylcysteine (NAC), on the interaction of ALO and anthrax spores with macrophages.  NAC completely inhibits the sublethal effects of ALO on phagocytes, and dramatically increases the ability of macrophages to kill B. anthracis spores.  Finally, we collaborate with the biomedical engineers at Drexel to build a better anthrax detector for biodefense and homeland security, and to observe the effects of room temperature dielectric barrier discharge plasma on the viability of B. anthracis.

Selected Publications

1. Williams, J.M., L. Zhu, G.-C. Chen and R.F. Rest. Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: Intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth. Molec Microbiol, 27: 171-186, 1998.
2. Hood, D. W., K. Makepeace, M. E. Deadman, R. F. Rest, P. Thibault, A. Martin, J. C. Richards, & E. R. Moxon. Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization. Molec. Microbiol., 33: 679-692, 1999.
3. Liu, S. V., N. J. Saunders, A. Jeffries and R.F. Rest. Genome analysis and strain comparison of Correia repeats and Correia Repeat-Enclosed Elements (CREE) in pathogenic neisseria. J. Bacteriology, 184: 6163-6173, 2002.
4. Shell, D., D. Chiles, R. Judd, S. Seal and R.F. Rest. Outer membrane localization of sialyltransferase in the pathogenic Neisseria. Infect. Immun., 70: 3744-3751, 2002.
5. Shannon, J.G., C. L. Ross, T. M. Koehler and R. F. Rest. 2003. Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin. Infect. Immun., 71: 3183-3189, 2003.
6. Park, J.M., V.H. Ng, S. Maeda, R.F. Rest, and M. Karin. Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. Journal of Experimental Medicine, 200: 1647-1655, 2004.
7. Packiam, M., D. Shell, D., S. V. Liu, Y.B. Liu, D. J. McGee, R. Srivastava, S. Seal and R. F. Rest. Differential expression and transcriptional analysis of the α-2,3-sialyltransferase gene (lst) in pathogenic neisseria. Infect. Immun., 74:2637-2650, 2006.
8. Cocklin, S., M. Jost, N. M. Robertson, S. D. Weeks, H.-W. Weber, E. Young, S. Seal, M. Zhang, P. J. Loll, A. J. Saunders, R. F Rest, and I. M. Chaiken. Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin Anthrolysin O from Bacillus anthracis. J Molec Recognition, 19: 354-362, 2006.
9. Mosser, E. M. and R. F. Rest. Killing of human lymphocytes, monocytes, macrophages and neutrophils by the Bacillus anthracis cytotoxin Anthrolysin O. BMC Microbiology, 6: 56, 2006.
10. Gogotsi, Y., Dash, R. K., Yushin, G., Carroll, B. E., Altork, S. R., Sassi-Gaha, S., and R. F. Rest.  Bactericidal activity of chlorine-loaded carbide-derived carbon against Escherichia coli and Bacillus anthracis. J. Biomed. Materials Res: Part A, 84: 607-613, 2008.
11. McGovern, J. -P., Shih, W. Y., Rest, R. F., Purohit, M., Pandya, Y., and W-H. Shih. Label-Free Flow-Enhanced Specific Detection of Bacillus anthracis Using a Piezoelectric Microcantilever Sensor. The Analyst, in press, 2008.
12. Viator, R. J., Rest, R. F., Hildebrandt, E., and D. J. McGee. Characterization of Bacillus anthracis arginase: effects of pH, temperature, and cell viability on metal preference. Submitted, 2008.
13. Purohit, M., Sassi-Gaha, S., and R. F. Rest. Rapid sporulation of Bacillus anthracis. Submitted, 2008.


14. Mosser, E. M., Rest, R. F., Crasta, O., Mohapatra, S., Evans, C., Sobral, B., and D. J. Simon. Anthrolysin Regulatory Protein (Arp): a unique, global transcription regulator in the Bacillus cereus group. Submitted, 2008.