Oral Flora I

1. Oral Flora I Karen Ross 2007

2. Anaerobic Bacteria of Clinical Importance

5. Porphrymonas gingivalis • Gram -ve coccobacilli • Anaerobic, dark pigmentation on media containing lysed blood. • Black pigmentation due to accumulated hemin used as an iron source for growth • The diseases collectively termed periodontitis are bacterial infections which begin with inflammation of the periodontium, and can progress to loss of teeth. Untreated infections lead to destruction of the periodontal ligament and alveolar bone. It is estimated that over 49,000,000 people in the United States have some form of periodontitis (Cutler et al., 1995). These diseases are infectious conditions which may progress over several years with episodes of exacerbation and remission. Periodontitis occurs with higher frequency in patients with systemic diseases such as diabetes mellitus, AIDS, leukemia, neutropenia, Crohn's disease, and Down's syndrome (Neville et al., 1995)

6. Virulence Factors of P.gMolecules and Organelles Proteases -arginine and lysine specific cysteine proteases Hemagglutinins -mediate the binding of Pg to epithelial cells and erythrocytes LPS-very different composition from enteric bacteria lacks endotoxicity Fimbriae -peritrichous (all directions), neccesary for infection Outer membrane vesicles -outgrowth of outer membrane, platelet aggregation, precise delivery of virulence factors, can enter where Pg cannot Polysaccharide capsule -six serotypes, reduced phagoscytosis Cytotoxic metabolic end products -include butyrate, propionate, have low molecular weights which allows them to easily penetrate periodontal tissue and disrupt the host cell activity Tsute Chen, Department of Molecular Genetics, The Forsyth Institute.

7. Mechanisms Adhesion, colonization and dental plaque (biofilm) formation Epithelial and Endothelial cell invasion Proteolysis - proteases degrade host proteins including fibrinogen, plasma proteins, cytokines, can also activate neutrophils Inflammatory response -can activate and suppress components of the innate immune response. Bone resorption, bone destruction, and bone formation inhibition -LPS, fimbrial and outer membrane consituents play a major role. Eg. LPS causes release of bone resorption mediators (cytokines) from fibroblasts, macrophages and monocytes. These mediators also induce host cell proteases that destroy both bone and connective tissue and inhibit synthesis of collagen by osteoblasts. (Lamont et al., 1995)

8. CCR5 tropic HIV-1 Porphyromonas gingivalis CXCR4 tropic HIV-1 Oral keratinocytes HIV target CD4(+) T cells CCR5 gingipains LPS CCR5 PAR 1-2 TLR 2-4 CCR5 Endosome CD4 CCR5 Other HIV receptors Courtesy of Rodrigo Giacaman-Sarah; Herzberg Lab

9. Morphological changes of epithelial cells after exposure to P. gingivalis Morphological changes were delayed in KB-MRP8/14.

10. wild type fimbriae-deficient mutant Morphological changes of epithelial cells after exposure to P. gingivalis for 24 h

11. P. gingivalis proteases • Cysteine proteases • Arg-gingipain, RgpA, RgpB • Lys-gingipain, Kgp

12. Morphological changes of epithelial cells after exposure to P. gingivalis wild type and protease-deficient mutants for 24 h Cells expressing calprotectin are more resistant to cell detachment mediated by P. gingivalis proteases, Kgp and Rgp.

13. Seok-Woo Lee, School of Dental and Oral Surgery Columbia University Gram -ve, filament shaped, non motile, non pigmented Anaerobe Virulence factors Hydrolases -produces a trypsin-like protease, and an arginine-specific cysteine protease, and a sailase. Hemolytic activity -cyteine protease, iron acquisition from erythrocytes Co-aggregation -P. gingivalis, S. cristatis Adhesion -leucine rich surface protein (BspA), binding to RBCs, fibroblasts, leukocytes, and epithelial cells (see above) Tannerella forsythia

14. Treponema denticola Motile helical rods Irregular (3-8) spirals Gram-ve cell wall Anaerobe, able to be grown in vitro Molecules Major outer membrane protein(Msp) - 53-kDa adhesin with pore forming activity, cytotoxic for epithelial cells and erythrocytes Proteinases -Dentilisin/PrtP/CTLP involved in cell attachment, tissue destruction, tissue invasion Hemin- and lactoferrin-binding proteins -iron acquisition and utilises lactoferrin from the saliva 'TREPONEMA DENTICOLA’ by Joe Dixon, 2007

15. Treponema denticola Mechanisms Motility and chemotaxis - motility mutants fail to infect their hosts. Maneuvering in viscous fluids, gingival crevice is highly viscous. Hemagglutination and hemolytic activity Adhesion - Fibroblasts, extracellular matrix, epithelial cells, endothelial cells coaggregation Invasion-Tissue and cellular invasion, produce lesions in tissue models, penetrate epithelial and endothelial cell monolayers Proteolytic activity Dentilisin/PrtP/CTLP disrupts cell junction and impairs epithelial monolayers Immunosupppressive activity - lymphocyte proliferative responses to antigens and mitogens suppressed T. d induces actin rearrangement and detachment of human gingival fibroblasts. Baehni et al., 1992

16. Fusobacterium nucleatum Gram -ve, anaerobic, cigar-shaped bacilli with pointed ends Recovered mainly from periodontal pockets In combination with oral spirochaetes) causes fusospirochaetal infections -acute necrotizing ulcerative gingivitis, vincents angina, cancrum oris or noma Toxic metabolites -butyrate, proprionate, ammonium ions Coaggregation -anchor, bridge formation Adhesion -binds to fibronectin -binds PMNs, macrophages, lymphocytes, HeLa cells, fibroblasts, both periodontal ligament and gingival fibroblasts and buccal epithelial cells Invasion -epithelial cells

17. Collaborative invasion Tissue culture experiment F. nucleatum invades epithelial cells S. cristatus does not invade cells After coaggregation, S. cristatus is carried inside by F. nucleatum Edwards, Grossman, and Rudney 2006, Infect Immun 74: 654

19. Prevotella intermedia Anaerobic, Non-motile, Short, round-ended, Gram-ve rods Virulence Fimbriae -4 different types Hydrolases Hemolysin and hemagglutinin Coaggregation Adhesion Invasion Induction of inflammatory lymphokines

20. Veillonella spp. • V. parvula, V. dispar, V. atypica • Non-motile, nonsporulating, small, anaerobic Gram-ve cocci • Commensals of the oropharynx, gastrointestinal tract and female genital tract • Unable to use carbohydrates or amino acids, ferment organic acids to propionic and acetic acids, CO2 and H2 • Component of early plaque, may use lactic acid produced by streptococci the early colonisers -metabolic cooperation

21. Capnocytophaga spp. C. gingivalis C. ochracea C. sputingena C. granulosa C. haemolytica Hydrolases Trypsin-like protease

22. Actinomyces • Gram+ve anaerobic and microaerophilic • Filamentous, branching,gram-positive rods. Older microbiologists sometimes referred to actinomyces as fungi because of the were long like fungal hyphae. However, they are only about a tenth as wide. • They normally reside in the human mouth, throat, large intestine, vagina, and the crevices between teeth and gums, especially underneath dental plaque. • Normal flora and is not transmitted from person to person. • Not usually virulent, reside as saprophytes in the body without producing disease. • Problem only if they have the opportunity to grow on a surface away from oxygen. They stick to one another, and eventually can break loose as a mass that is too big to be engulfed by the body's defense cells.

23. Actinomycosis • Actinomyces israelii • Tooth abscess or a tooth extraction and the endogenous organism becomes established in the traumatized tissue and causes a suppurative infection(pus). • These abscesses are not confined to the jaw and may also be found in the thoracic area and abdomen. • The patient usually presents with a pus-draining lesion, so the pus will be the clinical material you send to the laboratory. • Yellow sulfur granules, characteristic of this organism, can be seen with the naked eye. You can also see these granules by running sterile water over the gauze used to cover the lesion. The water washes away the purulent material leaving the golden granules on the gauze.

24. Aggregation • Co-aggregation Fusobacterium nucleatum cells coaggregating with Porphyromonas gingivalis cells.

25. Fusobacterium nucleatum intergeneric coaggregations in the form of corn-cob formations Kolenbrander et al., 2000

26. Advantages of co-aggregation Physical contact -attachment Metabolic exchange Small-signal-molecule-mediated communication Exchange of genetic material Kolenbrander et al., 2006

27. Streptococcus oralis in three-fold excess over partner Prevotella loescheii (arrows) Fusobacterium nucleatum P. loeschii does not coaggregate with F. nucleatum but S. oralis coaggregates with both and acts as a coaggregation bridge Kolenbrander et al., 2006

28. Kolenbrander 2006

30. Reviews: light reading :) Socransky and Haffagee Periodontal microbial ecology Periodontology 2000 Vol 38:135-187, 2005 Kolenbrander et al., Bacterial interactions and sucessions during plaque development Periodontology 2000 Vol 42: 47-79, 2006