Hansel Fletcher, PhDProfessor & Associate Chair
Graduate Program Coordinator
Division of Microbiology & Molecular Genetics
School of Medicine
Loma Linda University
Loma Linda, CA 92350
Phone: (909) 558-8497
Fax: (909) 558-4035
Email: hfletcher@llu.edu
It appears that only a few Gram-negative anaerobic bacteria, among the more than 700 microbial species known to exist in the human mouth, are associated with periodontal diseases. In addition, some of these organisms are also associated with cardiovascular and other systemic diseases. Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is an important etiological agent of periodontal disease and is also linked to cardiovascular disease. It produces several virulence factors (e.g., capsule, adhesion, membrane vesicles, and hydrolytic enzymes) that can contribute to its pathogenicity. The overall objective of our research program is to elucidate the molecular mechanism(s) for virulence regulation in P. gingivalis and examine the effects of these virulence factors on the host-microbe interaction. Our current research efforts are the following:
1. Studies on the mechanisms of virulence regulation in Porphyromonas gingivalis. The expression of extracellular proteolytic activities is highly regulated in both prokaryotic and eukaryotic systems. This regulation can occur at multiple levels including expression of the protease genes, secretion, processing of an inactive secreted precursor to its active form, and/or the posttranslational glycosylation of the proteins.
These regulatory mechanisms are vital to ensure that expression is tightly controlled in the appropriate temporal and spatial patterns. While several virulence factors have been implicated in the pathogenicity of P. gingivalis, the major proteases called gingipains, are considered to play the most significant role in virulence. There is a gap in our understanding of the regulation/activation of the gingipains. We have identified and partially characterized three novel genes, vimA, vimE, and vimF, which are involved in the posttranslational regulation of the gingipains. These genes have affected the glycosylation of the gingipains further confirming a role for carbohydrate modification in gingipain activation and anchorage to bacterial cell surface. The vimA ,vimE, or vimF gene product could be a regulator or structural protein involved in a pathway(s) to facilitate correct folding conformation of the gingipains, excretion, processing, and/or anchoring of the proteases on the cell surface. Further, this product could also play a role in facilitating interaction with other factors associated directly or indirectly with the proteases. We aim to elucidate the molecular mechanism of virulence modulation in P. gingivalis by first examining the direct effects of these genes on gingipain activation.
2. Oxidative stress resistance mechanisms in Porphyromonas gingivalis. The inflammatory condition of the periodontal pocket resulting in reactive oxygen species indicates that P. gingivalis have properties that will allow its survival in this oxidative environment. We wish to understand how P. gingivalis becomes resistant to this environment and evaluate whether this contributes to its survival. We explored the ability for AhpC to protect against oxidative damage and recently published our findings in a manuscript suggesting that the ahpC gene may play an important role in peroxide resistance in P. gingivalis in vitro but does not contribute significantly to virulence in vivo. The sensitivity of a P. gingivalisbcp-defective mutant also suggested the bcp gene may play a role in oxidative stress resistance but does not function significantly in virulence in the mouse model tested. In collaboration with Dr. Lawrence Sowers (Professor and Chair, Department of Biochemistry and Microbiology, Loma Linda University) we began to explore other mechanisms of oxidative stress resistance in P. gingivalis. DNA damage, a major consequence of oxidative stress, is repaired by several DNA repair mechanisms. Our previous reports suggest a role for a nucleotide excision repair (NER)-like mechanism in the repair of oxidatively damaged DNA. Because the uvrB gene is known to be important in NER, the role of the P. gingivalisuvrB in DNA repair under oxidative stress was investigated. Our results suggest that the uvrB gene in P. gingivalis may not be involved in the removal of 8-oxoG and that another yet unidentified mechanism may be employed in its removal. In response to H 2O 2, a preliminary DNA microarray-mediated transcriptional analysis of isogenic strains of P. gingivalis identified the induced expression of several genes including some of those known to be involved in oxidative stress resistance. Further, the level of oxidative stress was shown to differentially modulate transcription. To further clarify a comprehensive mechanism for oxidative stress resistance we will evaluate the effect of specific mutations of oxidative stress-induced genes on the survival of P. gingivalis. In addition we will characterize the DNA damage and mechanism(s) of repair in isogenic mutants of P. gingivalis under conditions of oxidative stress.
3. P. gingivalis - host interaction: gingipain-induced apoptosis. In collaboration with Dr. Carlos A. Casiano (Division of Microbiology and Molecular Genetics, Loma Linda University) we began to explore the effects of the gingipains on endothelial cells. Several reports have established that apoptosis is involved in the pathogenesis of periodontal diseases. While host factors including cytokines may be involved in much of the tissue destruction, emerging evidence indicates that bacterium-modulated apoptosis may play an important role in this process. The periodontal pocket is a unique microenvironment where a zone of highly vascular tissue is situated in close proximity to the microbial biofilm. Further, endothelial cell death is prominent in periodontitis. The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. There is a significant gap in our knowledge on the mechanism(s) by which the gingipains contribute to tissue damage including the microvasculature. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis. Collectively, our results indicate that the gingipains, at concentrations naturally occurring in the periodontal pocket, can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells with HRgpA and RgpB triggering caspase-dependent and caspase-independent apoptosis. Taken together, these results suggest that P. gingivalis protease-induced cell death may compromise tissue integrity and play an important role in tissue damage. To further clarify this first demonstration of gingipain-induced caspase-independent apoptosis we will determine the involvement of specific cell death pathway(s) in this process. Understanding the possible role of P. gingivalis proteases in the induction of damage to the highly vascularized periodontal tissue could have profound implications for the development of new therapeutic strategies that could reduce the devastating impact of periodontitis in the Western world.
Abaibou, H., Z. Chen, Y. Liu, G. J. Olango, J. Edwards and H. M. Fletcher. 2001. The vimA gene downstream of recA is involved in virulence modulation in Porphyromonas gingivalis W83. Infect. Immun. 69: 325-335.
Y. Liu and H. M. Fletcher. 2001. Environmental regulation of recA gene expression in Porphyromonas gingivalis W83. Oral Microbiol. Immunol. 16:136-43.
Liu, Y. and H. M. Fletcher. 2001. The recA gene in Porphyromonas gingivalis W83 is expressed during infection of the murine host. Oral Microbiol. Immunol. 16:218-223.
Chen, Z. C. A. Casiano and H. M. Fletcher. 2001. Protease-active extracellular protein preparations from Porphyromonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells. J. Periodontol. 72:641-650.
Olango, G. J, F. Roy, S. M. Sheets, M. K. Young and H M. Fletcher. 2003. Gingipain RgpB is excreted as a proenzyme in the vimA-defective mutant Porphyromonas gingivalis FLL92. Infect. Immun. 71:3740-3747.
Johnson, N. A., Y. Liu and H. M. Fletcher. 2004. Alkyl Hydroperoxide peroxidase subunit C(AhpC) protects against organic peroxides but does not affect virulence of Porphyromonas gingivalis W83. Oral Microbiol. Immunol. 19:233-239.
Vanterpool, E., F. Roy and H. M. Fletcher. 2004. vimE gene downstream of vimA is independently expressed and is involved in modulating proteolytic activity in Porphyromonas gingivalis W83. Infect. Immun. 72:5555-5564.
Johnson, N. A., R. McKenzie, L. McClean, L. Sowers and H. M. Fletcher. 2004. 8-Oxo-7,8-dihydroguanine is removed by NER in Porphyromonas gingivalis W83. J. Bacteriol. 186:7697-703.
Sheets, S. M., J. Potempa, J. Travis, C. A. Casiano and H. M. Fletcher. 2005. Porphyromonas gingivalis protease-induce cadherin proteolysis, loss of cell adhesion, and apoptosis in endothelial cells. Infect. Immu. 73: 1543-1552.
Vanterpool, E, F. Roy, L. Sandberg and H.M. Fletcher. 2005. Altered gingipain maturation in the vimA and vimE-defective isogenic mutants of Porphyromonas gingivalis. Infect. Immu. 73: 1357-1366.
Y. Asai, M. Hashimoto, H. M. Fletcher, K. Miyake, S. Akira T. Ogawa. 2005. Lipopolysaccharide Preparation Extracted from Porphyromonas gingivalis Lipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling. Infect. Immu. 73: 2157-2163.
Vanterpool, E, F. Roy and H.M. Fletcher. 2005. Inactivation of vimF, a putative glycosyl transferase gene, which is downstream of vimE, alters the glycosylation and activation of the gingipains in Porphyromonas gingivalis W83 . Infect. Immu. 73: 3971-3982.
Sheets, S. M., Potempa, J., Travis, J. and H. M. Fletcher and C. A. Casiano. 2006. Gingipains from Porphyromonas gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-independent apoptosis. Infect. Immu. 74: 5667- 5678.
Vanterpool, E., F. Roy, S.M. Sheets, L. Sandberg and H. M. Fletcher. 2006. VimA is part of the maturation pathway for the major gingipains of P. gingivalis W83. Microbiology. 152: 3383-3389.
Roy F., E. Vanterpool and H. M. Fletcher. 2006. HtrA of Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions. Microbiology. 152: 3391-3398
Last Revised: Tue, Nov 21, 2006
