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, and the human immune response to stase during infection. The regulation of stase expression is very complex. Surprisingly, expression mechanisms differ significantly between Nm and Ng, and although in vitro it appears that Nm and Ng express some level of stase constitutively, association of Ng with epithelial cells dramatically decreases stase expression. We continue to study the mechanisms of stase expression – from transcriptional to post-translational – and we are interested in how the enzyme gets transported to and incorporated into the outer membrane.

In a specially designed BSL2/BSL3 laboratory suite, we study the interaction of B. anthracis with human macrophages and neutrophils, focusing on the role of Anthrolysin O (ALO), a cholesterol-dependent cytolysin, and an anthrax toxin. ALO has lethal and (in the presence of serum or cholesterol) many potent sublethal effects on several cell types, including human phagocytes, and signals 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, priming for the oxidative burst, inducing cell death, 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 biomedical engineers at Drexel to build a better anthrax detector for biodefense and homeland security, to observe the effects of room temperature dielectric barrier discharge plasma on the viability of B. anthracis, and to detect the increasingly serious pathogen, Methicillin Resistant Staphylococcus aureus (MRSA).

Selected Publications

1. Shell, D., Chiles, D., Judd, R., Seal, S., and R. F. Rest.  Outer membrane localization of sialyltransferase in the pathogenic Neisseria.  Infect. Immun., 70: 3744-3751, 2002.Shannon, J. G., Ross, C. L., Koehler, T. M., and R. F. Rest.  Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin.  Infect. Immun., 71: 3183-3189, 2003.
2. Shannon, J. G., Ross, C. L., Koehler, T. M., and R. F. Rest.  Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin.  Infect. Immun., 71: 3183-3189, 2003.
3. Park, J. M., Ng, V. H., Maeda, S., Rest, R. F., and M. Karin.  Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists.  Journal of Experimental Medicine, 200: 1647-1655, 2004.
4. Packiam, M., Shell, D., Liu, S. V., Liu, Y. B., McGee, D. J., Srivastava, R., Seal, S., 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.
5. Cocklin, S., Jost, M., Robertson, N. M., Weeks, S. D., Weber, H. -W., Young, E., Seal, S., Zhang, M., Loll, P. J., Saunders, A. J., Rest, R. F., 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.
6. 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.
7. 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.
8. Nakouzi, A., Rivera, J., Rest. R. F., and A. Casadevall.  Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with Bacillus anthracis.  BMC Microbiology 8: 159, 2008.
9. Bourdeau R. W, Malito, E., Chenal, A., Bishop, B. L., Musch, M. W., Villereal, M. L., Chang, E. B., Mosser, E. M., Rest, R. F., and W. J. Tang.  Cellular functions and X-ray structure of Anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis.  J Biol Chem 284: 14645-14656, 2009.
10. Lehrer, R. I., Jung, G., Ruchala, P., Wang, W., Micewicz, E. D., Waring, A. J., Gillespie, E., J., Bradley, K. A., Ratner, A. J., Rest, R. F., and W. Lu.  Human alpha Defensins Inhibit Hemolysis Mediated by Cholesterol-Dependent Cytolysins.  Infect. Immun. 77: 4028-4040, 2009.
11. McGovern, J.-P., Shih, W. Y., Mattiucci, M., Res, R. F., Purohit, M., Pourrezaei, K., Onaral, B., and W-H Shih.  Array PZT/Glass Piezoelectric Microcantilevers for real-time detection of Bacillus anthracis with 10 spores/ml sensitivity and 1/1000 selectivity in bacterial mixtures.  Rev. Sci. Instrum. 80: 125104-1 – 125104-7, 2009. [also in December 15, 2009 issue of Virtual Journal of Biological Physics Research]
12. Mosser, E. M., Pandya, Y., Bernui, M., and R. F. Rest.  Anthrolysin O, the Bacillus anthracis cholesterol dependent cytolysin, modulates human neutrophil and macrophage function.  Infect. Immun., In revision, 2010.
13. 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
14. Bernui, M. E, Smith, M., and R. F. Rest.  N-Acetylcysteine Inhibits Bacillus anthracis Spore Germination by Altering Specific Germination-pathway Components.  Submitted, 2010.
15. Bernui, M. E., Stone, W., Smith, M., and R. F. Rest.  N-acetylcysteine (NAC) and STIMAL® (liposome-encapsulated NAC) Increase the Ability of Human Macrophages to Kill Bacillus anthracis.  Submitted, 2010