Department of microbiology and molecular genetics
Faculty profile
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Hansel M. Fletcher 11021 Campus Street Office of minority student development in the biomedical professions |
- PhD, Temple University 1990
- Current research interests
- Recent publications
The research in our laboratory has focused on elucidating the molecular mechanisms of pathogenesis of periodontal pathogens. It appears that only a few Gram negative anaerobic bacteria, among the more than 350 bacterial 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 widely implicated as an important etiological agent of periodontal disease and is also linked to cardiovascular disease. It produces several potential virulence factors (e.g., capsule, adhesin, membrane vesicles, and hydrolytic enzymes) that may contribute to its pathogenicity.
An ability for DNA repair should also be important in the inflammatory periodontal pocket microenvironment where bactericidal oxygen reactive derivatives generated from neutrophils and occasional exposure to air can cause DNA damage. Thus, the recA gene product, a key protein in DNA repair, should play a role in the virulence of P. gingivalis. We previously have shown the recA gene in P. gingivalis plays the expected role of DNA repair. However inactivation of this gene also caused increased autoaggregation, reduced hemolytic and proteolytic activities in addition to reduced virulence. Our laboratory has now identified a gene called vimA (virulence modulating gene) that is downstream of the recA gene and is part of the same transcriptional unit. The cloned P. gingivalis vimA gene was insertionally inactivated using the ermF-ermAM antibiotic resistance cassette to create a defective mutant by allelic exchange. The mutant strain, designated FLL92, was non-black-pigmented and showed increased autoaggregration in addition to a significant reduction in proteolytic, hemolytic and hemagglutinating activities. In in vivo experiments using a mouse model, FLL92 was dramatically reduced in virulence when compared with wild-type W83 strain. Taken together, these results suggest an important role for the P. gingivalis W83 vimA gene in virulence modulation.
In another project, we have developed a non-invasive gene expression reporter system for P. gingivalis. This system is being used to clone and characterize virulence genes which are expressed only in vivo (in the host ) and not in vitro (in culture). Proteases produced by P. gingivalis are widely accepted as an important virulence factor. In collaboration with Dr. Carlos Casiano we are involved in understanding the effects of proteases on host tissue integrity. To explore the effects of these proteases on epithelial cells, we exposed human KB cells to extracellular protease preparations from isogenic mutants of P. gingivalis. Treated cells exhibited rounding and detachment from the underlying surface and from each other, while maintaining viability. This effect was correlated with the different levels of cysteine-dependent proteolytic activity of the isogenic mutants tested. We tested the possibility that proteases from P. gingivalis induced cell rounding and detachment via proteolysis of KB proteins involved in cell adhesion. Cleavage of N-cadherin, a cell surface protein involved in cell adhesion and signal transduction, was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin and cell detachment. In contrast, no cleavage of integrin beta1, a protein involved mainly in cell-matrix adhesion, was observed. Cell rounding and detachment, and N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis extracellular protease preparations with the cysteine protease inhibitor TLCK. Finally, in control experiments, the cleavage of N-cadherin was detected after treatment of KB with trypsin but not after cell dissociation by a non-enzymatic method. Taken together, these results indicate that proteases from P. gingvalis can alter N-cadherin, which could have implications in the pathogenicity of this organism.
Current research topics
* Identification and characterization of virulence-associated genes of P. gingivalis which are expressed exclusively in the host (in vivo).
* Characterization of the vimA (virulence modulating) gene in P. gingivalis.
* Characterization of protective immunogenic proteins in P. gingivalis.
* Environmental regulation of recA gene expression in Porphyromonas gingivalis W83
* Understanding the mechanism(s) for oxidative stress response in periodontal pathogens.
* Studies of the effect of extracellular proteases from Porphyromonas gingivalis on host cell surface proteins involved in cell adhesion and signal transduction.
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Z. Chen, C. A. Casiano and H. M. Fletcher. Cleavage of N-cadherin by extracellular proteases from Porphyromonas gingivalis. In preparation.
Abaibou, H., Z. Chen, Y. Liu, G. J. Olango, J. Edwards and H. M. Fletcher. A gene in the recA locus is involved in virulence modulation in Porphyromonas gingivalis W83. Submitted.
Abaibou, H., Q. Ma, G. J. Olango, J. Potempa, J. Travis and H. M. Fletcher. 1999. Unaltered expression of the major protease genes in a non-virulence recA-defective mutant of Porphyromonas gingivalis W83 . In press.
H. M. Fletcher, R. M. Morgan and F. L. Macrina. 1997. Nucleotide sequence of Porphyromonas gingivalis W83 recA homolog and construction of a recA-defective mutant. Infect. Immun. 65: 4592-4597.
H. A. Schenkein, H. M. Fletcher, M. Bodnar and F. L. Macrina. 1995. Opsonization of a prtH-defective mutant of Porphyromonas gingivalis W83. J. Immunol. 154:5331-5337.
H. M. Fletcher, H. A. Schenkein, K. A. Bailey, R. M. Morgan, C. R. Berry and F. L. Macrina. 1995. Virulence of a mutant of Porphyromonas gingivalis W83 that is defective in the prtH gene. Infect. Immun. 63: 1521-1528.
School of Medicine - Graduate School - Loma Linda University
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