Loma Linda University

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Hansel Fletcher, PhD
Assistant Dean, Graduate Student Affairs, School of Medicine
School of Medicine
Vice Chair, Basic Sciences, Microbiology Division
School of Medicine
Professor, Basic Sciences
School of Medicine
Professor, Periodontics
School of Dentistry
Member, Microbiology&Molecular Gen, SM, Faculty of Graduate Studies
Publications    Scholarly Journals--Published
  • Aruni, W., E. Vanterpool, D. Osbourne, F. Roy, A. Muthiah, Y. Dou and H. M. Fletcher. 2011. Sialidase and sialoglycoproteases can modulate virulence in Porphyromonas gingivalis. Infect. Immu. 79:2779-91. (Journal cover highlight). ( 7/2011 )
  • Robles-Price, A. K. Reid, R. Roy and H. M. Fletcher. 2011. Elucidating the role of Porphyromonas gingivalis MutY in repairing DNA damaged by oxidative stress. Mol Oral Microbiol. 26:175-86. ( 6/2011 )
  • Dou, Y., D. O. Osbourne, R. McKenzie, and H. M. Fletcher. 2010. Involvement of extracytoplasmic function sigma factor in virulence regulation in Porphyromonas gingivalis W83. FEMS Microbiol. Lett. 312:24-32. ( 11/2010 )
  • Vanterpool, E, A.W. Aruni, F. Roy, and H.M. Fletcher. 2010. regT can modulate gingipain activity and response to oxidative stress in Porphyromonas gingivalisMicrobiology. 156:3065-72. ( 10/2010 )
  • Osbourne, D. O., W. Aruni, F. Roy, C. Perry, L. Sandberg, A. Muthiah And H. M. Fletcher. 2010.  The role of vimA in cell surface biogenesis in Porphyromonas gingivalis. Microbiology. Microbiology. 156:2180-93. (Featured Journal article) ( 7/2010 )
  • Henry, L, L. Sandberg, K. Zhang and H.M. Fletcher. 2008. DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis. J. Bacteriol. 190:7985-93.  ( 12/2008 )
  • S. M. Sheets, A. Robles-Price, R. M. E. Mckenzie, C. A. Casiano, and H. M. Fletcher. 2008. Gingipain-dependent interactions with the host are important for survivalof Porphyromonas gingivalis. Frontiers in Bioscience. 13:3215-3238. ( 1/2008 - 12/2008 )
  • Roy F., E. Vanterpool and H. M. Fletcher. "HtrA of Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions." Microbiology 152. (2006): 3391-3398. ( 11/2006 ) Link...
  • Sheets, S. M., Potempa, J., Travis, J. and H. M. Fletcher and C. A. Casiano. "Gingipains from Porphyromonas gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-independent apoptosis." Infection & Immunity 74. (2006): 5667-5678. ( 10/2006 ) Link...
  • Vanterpool, E., F. Roy, S.M. Sheets, L. Sandberg and H. M. Fletcher. "VimA is part of the maturation pathway for the major gingipains of P. gingivalis W83." Microbiology 152. (2006): 3383-3389. ( 10/2006 ) Link...
  • Vanterpool E., Roy F., Fletcher H.M.. "Inactivation of vimF, a putative glycosyltransferase gene downstream of vimE, alters glycosylation and activation of the gingipains in Porphyromonas gingivalis W83.." Infect. Immun. 73.7 (2005): 3971-3982. ( 7/2005 ) Link...
    Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A 1.2-kb open reading frame, a putative glycosyltransferase, downstream of vimE, was cloned, insertionally inactivated using the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, this mutant, designated P. gingivalis FLL95, was nonpigmented and nonhemolytic when plated on Brucella blood agar. Arginine- and lysine-specific gingipain activities were reduced by approximately 97% and 96%, respectively, relative to that of the parent strain. These activities were unaffected by the growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of the rgpA, rgpB, and kgp gingipain genes was unaffected in P. gingivalis FLL95 in comparison to the wild-type strain. In nonactive gingipain extracellular protein fractions, multiple high-molecular-weight proteins immunoreacted with gingipain-specific antibodies. The specific gingipain-associated sugar moiety recognized by monoclonal antibody 1B5 was absent in FLL95. Taken together, these results suggest that the vimE downstream gene, designated vimF (virulence modulating gene F), which is a putative glycosyltransferase group 1, is involved in the regulation of the major virulence factors of P. gingivalis.
  • Yasuyuki Asai,1 Masahito Hashimoto,1 Hansel M. Fletcher,2 Kensuke Miyake,3 Shizuo Akira,4 and Tomohiko Ogawa1*. "Lipopolysaccharide Preparation Extracted from Porphyromonas gingivalis Lipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling ." Infect Immun. 73.4 (2005): 2157-2163. ( 4/2005 ) Link...
    We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a Porphyromonas gingivalis lipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that of Escherichia coli LPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis using an ermF-ermAM antibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (PG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well as Limulus amoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the PG1828-LPS preparation to activate NF-κB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the PG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A of P. gingivalis by TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the PG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor of P. gingivalis.
  • Shaun M. Sheets,1* Jan Potempa,2,3 James Travis,3 Carlos A. Casiano,1,4 and Hansel M. Fletcher1. "Gingipains from Porphyromonas gingivalis W83 Induce Cell Adhesion Molecule Cleavage and Apoptosis in Endothelial Cells ." Infect Immun 73.3 (2005): 1543-1552. ( 3/2005 ) Link...
    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. 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. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin β1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin β1 cleavage, could be inhibited or significantly delayed by preincubation of P. gingivalis W83 gingipain-active extracellular extracts with the cysteine protease inhibitor Nα-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains from P. gingivalis can alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism.
  • Elaine Vanterpool,1* Francis Roy,1 Lawrence Sandberg,1 and Hansel M. Fletcher1. "Altered Gingipain Maturation in vimA- and vimE-Defective Isogenic Mutants of Porphyromonas gingivalis ." Infect Immun 73.3 (2005): 1357-1366. ( 3/2005 ) Link...
    We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.
  • Sheets, S. M., J. Potempa, J. Travis, C. A. Casiano and H. M. Fletcher. "Porphyromonas gingivalis protease-induce cadherin proteolysis, loss of cell adhesion, and apoptosis in endothelial cells." Infect. Immu 73. (2005): 1543-1552. ( 1/2005 )
  • Y. Asai, M. Hashimoto, H. M. Fletcher, K. Miyake, S. Akira T. Ogawa. "Lipopolysaccharide Preparation Extracted from Porphyromonas gingivalis Lipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling." Infect. Immu 73. (2005): 2157-2163. ( 1/2005 )
  • Vanterpool, E, F. Roy and H.M. Fletcher. "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. (2005): 3971-3982. ( 1/2005 )
  • Sheets, S. M., Potempa, J., Travis, J. and H. M. Fletcher and C. A. Casiano. "Gingipains from Porphyromonas gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-independent apoptosis. ." Submitted to Infection and Immunity . (2005): -. ( 1/2005 )
  • Vanterpool, E., F. Roy and H. M. Fletcher. "vimE gene downstream of vimA is independently expressed and is involved in modulating proteolytic activity in Porphyromonas gingivalis W83." Infect. Immun 72. (2004): 5555-5564. ( 1/2004 )
  • Johnson, N. A., R. McKenzie, L. McClean, L. Sowers and H. M. Fletcher. "8-Oxo-7,8-dihydroguanine is removed by NER in Porphyromonas gingivalis W83." J. Bacteriol. 186. (2004): 7697-7703. ( 1/2004 )
  • Vanterpool, E, F. Roy, L. Sandberg and H.M. Fletcher. "Altered gingipain maturation in the vimA and vimE-defective isogenic mutants of Porphyromonas gingivalis." Infect. Immu 73. (): 1357-1366. (*)
  Scholarly Journals--Accepted
  • Aruni, A. W., F. Roy   and    H. M. Fletcher. 2011. Filifactor alocis has virulence attributes that can enhance its persistence under oxidative stress conditions and mediate invasion of epithelial cells by Porphyromonas gingivalis. Infect. Immu. Published ahead of print on 8 August 2011, doi:10.1128/IAI.05631-11 ( 8/2011 ) Link...
  Books and Chapters
  • J. D. Kettering and H. M. Fletcher. Microbiology, Pre-Test? Self-Assessment and Review. USA: McGraw Hill , 2007. ( 1/2007 )
  • Fletcher, H.M., A Progulske-Fox and J.D. Hillman. Applied molecular biology and the oral microbes. In Oral Microbiology and Immunology. USA: ASM Publications , 2006. ( 10/2006 )
  • Fletcher, H.M., A Progulske-Fox and J.D. Hillman. Applied molecular biology and the oral microbes. In Oral Microbiology and Immunology. USA: ASM Publications , 2006. 169 - 188 ( 1/2006 )
  • J. D. Kettering and H. M. Fletcher. Microbiology, Pre-Test? Self-Assessment Review. USA: McGraw Hill , . (*)