Publications

Scholarly Journals--Published

  • Li X, X. Wang, R. Huang, A. Stucky, X. Chen, L. Sun, Q. Wen, Y. Zeng, H. Fletcher, C. Wang, Y. Xu, H. Cao, F. Sun, S.C. Li, X. Zhang, J.F. Zhong. 2022. The Machine-Learning-Mediated Interface of Microbiome and Genetic Risk Stratification in Neuroblastoma Reveals Molecular Pathways Related to Patient Survival. Cancers (Basel). 2022 Jun 10;14(12):2874. doi: 10.3390/cancers14122874. PMID: 35740540; PMCID: MC9220810. (06/2022)
  • Natto Z.S., M. Afeef, M.A. Bakhrebah, H. Ashi, K. A. Alzahrani, A. F. Alhetheel and H. M. Fletcher. 2022.  Can periodontal pockets and caries lesions act as reservoirs for coronavirus? Mol Oral Microbiol. 2022 Jan 21. doi: 10.1111/omi.12362. Online ahead of print. PMID: 35060684.  (01/2022)
  • Aja E, A. Mishra, Y. Dou Y and H. M. Fletcher. 2021.  Role of the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance. Microbiol Spectr. 2021 Dec 22;9(3):e0121221. doi: 10.1128/Spectrum.01212-21. Epub 2021 Nov 10.PMID: 34756068 (12/2021)
  • Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.  (04/2021) (link)
  • Filifactor alocis, a fastidious Gram-positive obligate anaerobic bacterium, is a newly appreciated member of the periodontal community that is now proposed to be a diagnostic indicator of periodontal disease. Its pathogenic characteristics are highlighted by its ability to survive in the oxidative stress-rich environment of the periodontal pocket and to significantly alter the microbial community dynamics by forming biofilms and interacting with several oral bacteria. Here, we describe the current understanding of F. alocis virulence attributes, such as its comparative resistance to oxidative stress, production of unique proteases and collagenases that can cause structural damage to host cells, and dysregulation of the immune system, which enable this bacterium to colonize, survive, and outcompete other traditional pathogens in the inflammatory environment of the periodontal pocket. Furthermore, we explore the recent advancements and future directions for F. alocis research, including the potential mechanisms for oxidative stress resistance and our evolving understanding of the interactions and mechanisms of bacterial survival inside neutrophils. We also discuss the current genetic tools and challenges involved in manipulating the F. alocis genome for the functional characterization of the putative virulence genes. Collectively, this information will expedite F. alocis research and should lead to the identification of prime targets for the development of novel therapeutics to aid in the control and prevention of periodontal disease.  (03/2021) (link)
  • Anti-sigma factors play a critical role in regulating the expression of sigma factors in response to environmental stress signals. PG1659 is cotranscribed with an upstream gene PG1660 (rpoE), which encodes for a sigma factor that plays an important role in oxidative stress resistance and the virulence regulatory network of P. gingivalis. PG1659, which is annotated as a hypothetical gene, is evaluated in this study. PG1659, composed of 130 amino acids, is predicted to be transmembrane protein with a single calcium (Ca2+ ) binding site. In P. gingivalis FLL358 (ΔPG1659::ermF), the rpoE gene was highly upregulated compared to the wild-type W83 strain. RpoE-induced genes were also upregulated in the PG1659-defective isogenic mutant. Both protein-protein pull-down and bacterial two-hybrid assays revealed that the PG1659 protein could interact with/bind RpoE. The N-terminal domain of PG1659, representing the cytoplasmic fragment of the protein, is critical for interaction with RpoE. In the presence of PG1659, the initiation of transcription by the RpoE sigma factor was inhibited. Taken together, our data suggest that PG1659 is an anti-sigma factor which plays an important regulatory role in the modulation of the sigma factor RpoE in P. gingivalis.  (02/2021) (link)
  • Dou Y, H. Rutanhira, N. Schormann, C. Deivanayagam and H.M. Fletcher. 2021. PG1659 functions as anti-sigma factor to extracytoplasmic function sigma factor RpoE in Porphyromonas gingivalis W83. Mol Oral Microbiol. 2021 36:80-91. doi: 10.1111/omi.12329. Epub 2021 Jan 13. PMID: 33377315 (01/2021)
  • Porphyromonas gingivalis is a gram-negative anaerobic bacterium and an etiologic agent of adult periodontitis. By inducing a dysbiotic state within the host microbiota it contributes to a chronic inflammatory environment in the oral cavity. Under some circumstances, the oral bacteria may gain access to systemic circulation. While the most widely recognized function of platelets is to reduce hemorrhage in case of vascular damage, it is known that platelets are also involved in the hematologic responses to bacterial infections. Some pathogenic bacteria can interact with platelets, triggering their activation and aggregation. The aim of this study was to assess platelet responses to the presence of P. gingivalis in whole blood. Human whole blood was pretreated with P. gingivalis and then platelet plug formation was measured under high shear conditions using the PFA-100. In the presence of P. gingivalis, time for a platelet plug to occlude the aperture in the collagen/ADP cartridge was shortened in a manner dependent on bacterial concentration and the duration of bacterial preincubation of blood. P. gingivalis enhances thrombus forming potential of platelets in whole blood.  (12/2020) (link)
  • 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). (07/2011)
  • 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. (06/2011)
  • 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) (07/2010)
  • 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. (01/2008 - 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)
  • 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)
  • 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., 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. 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. (07/2005) (link)
  • 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. 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. (04/2005) (link)
  • 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. 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. (03/2005) (link)
  • 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. 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. (03/2005) (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. ." Submitted to Infection and Immunity . (2005): -. (01/2005)
  • 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. (01/2005)
  • 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. (01/2005)
  • 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. (01/2005)
  • 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. (01/2004)
  • 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. (01/2004)
  • 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.

Abstract

  • Filifactor alocis, a Gram-positive anaerobic bacterium, is now a proposed diagnostic indicator of periodontal disease. Because the stress response of this bacterium to the oxidative environment of the periodontal pocket may impact its pathogenicity, an understanding of its oxidative stress resistance strategy is vital. Interrogation of the F. alocis genome identified the HMPREF0389_00796 gene that encodes for a putative superoxide reductase (SOR) enzyme. SORs are non-heme, iron-containing enzymes that can catalyze the reduction of superoxide radicals to hydrogen peroxide and are important in the protection against oxidative stress. In this study, we have functionally characterized the putative SOR (FA796) from F. alocis ATCC 35896. The recombinant FA796 protein, which is predicted to be a homotetramer of the 1Fe-SOR class, can reduce superoxide radicals. F. alocis FLL141 (?FA796::ermF) was significantly more sensitive to oxygen/air exposure compared to the parent strain. Sensitivity correlated with the level of intracellular superoxide radicals. Additionally, the FA796-defective mutant had increased sensitivity to hydrogen peroxide-induced stress, was inhibited in its ability to form biofilm and had reduced survival in epithelial cells. Collectively, these results suggest that the F. alocis SOR protein is a key enzymatic scavenger of superoxide radicals and protects the bacterium from oxidative stress conditions.  (06/2020) (link)
  • (PEER REVIEWED) Osbourne, D., W. Aruni, Y. Dou, A. Muthiah, C. Perry and H. M. Fletcher. Nanoscale organization of peptidoglycan in Porphyromonas gingivalis. Seeing at the Nanoscale Conference, UC Santa Barbara July 2011. (07/2011)
  • (PEER REVIEWED) Aruni, W. and H. M. Fletcher. Epithelial cell invasion by Filifactor alocis is enhanced by Porphyromonas. Rochester Conference on Oral Biology: Post-Genomics for the Oral Microbiome. Rochester, NY, June 2011. (Award winning). (06/2011)
  • (PEER REVIEWED) National Institute of Health, National Institute for Dental Research. "Mechanisms for adaptation to oxidative stress in Porphyromonas gingivalis", H. M. Fletcher, Principal Investigator. Total budget $1,806,625. 06/08/2009 – 03/31/2014 (1 R01 DE019730). (04/2011 - 03/2012)
  • (PEER REVIEWED) Aruni, W., J. Lee, D. Osbourne, F. Roy, A. Muthiah, H. M. Fletcher. Elucidating the role of VimA in Porphyromonas gingivalis through in silico and proteomic studies.   International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (PEER REVIEWED) Robles, A., and H. M. Fletcher. VimA‘s effect on the Glycosylation of RgpB from Porphyromonas gingivalis. International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (PEER REVIEWED) Muthiah, A., W. Aruni, D. Osbourne and H. M. Fletcher. VimF, a putative glycosyltranferase, is localized on outer membrane and membrane vesicles in Porphyromonas gingivalis W83. International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (PEER REVIEWED) Muthiah, A., W. Aruni, D. Osbourne and H. M. Fletcher. VimF, a putative glycosyltranferase, is localized on outer membrane and membrane vesicles in Porphyromonas gingivalis W83. International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (NON-PEER REVIEWED) Boutrin, M-C and H. M. Fletcher. Studies on the involvement of PG0893 and PG2213 in Nitric Oxide stress resistance in Porphyromonas gingivalis. International Association for Dental Research, San Diego, March 2011 (03/2011)
  • (PEER REVIEWED) Henry, L. G. and H. M. Fletcher. Protective role of the uvrA-pg1037-uvrD operon in oxidative stress defense in Porphyromonas gingivalis W83. International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (PEER REVIEWED) Osbourne, D.,  W.Aruni, Y. Dou, A. Muthiah, F.Roy and H. M. Fletcher. The VimA of Porphyromonas gingivalis is a putative acyl transferase. International Association for Dental Research, San Diego, March 2011. (03/2011)
  • (PEER REVIEWED) Yuetan D., F. Roy, D. Osbourne, W. Aruni and H. M. Fletcher. The role of RgpB in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83. International Association for Dental Research, San Diego, March 2011. (03/2011)

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 (08/2011)

Books and Chapters

  • J. D. Kettering and H. M. Fletcher. Microbiology, Pre-Test? Self-Assessment and Review. USA: McGraw Hill , 2007. (01/2007)
  • 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 (01/2006)
  • J. D. Kettering and H. M. Fletcher. Microbiology, Pre-Test? Self-Assessment Review. USA: McGraw Hill , .