Microbiology and Immunology in Periodontology PDF

MICROBIOLOGY AND IMMUNOLOGY IN PERIODONTOLOGY Beatriz Panariello, DDS, MSc, Ph.D. CONTENT 1- Overview of bacterial structures and functions 2- Pathogenesis of bacterial infections ▪ Koch’s Postulates ▪ Overview of innate immune host defenses ▪ Mic...

MICROBIOLOGY AND IMMUNOLOGY IN PERIODONTOLOGY Beatriz Panariello, DDS, MSc, Ph.D. CONTENT 1- Overview of bacterial structures and functions 2- Pathogenesis of bacterial infections ▪ Koch’s Postulates ▪ Overview of innate immune host defenses ▪ Microbial mechanisms of pathogenicity 3- Periodontal pathogens ▪ Virulence factors of periodontal pathogens (LTA) ENDOTOXIN CONTENT 1- Overview of bacterial structures and functions 2- Pathogenesis of bacterial infections ▪ Koch’s Postulates ▪ Overview of innate immune host defenses ▪ Microbial mechanisms of pathogenicity 3- Periodontal pathogens Virulence factors of periodontal pathogens IDENTIFICATION OF A PATHOGEN IN THE LAB  K O C H ’ S P O S T U L AT E S for the identification of pathogens Presence of an infectious agent is associated with the disease. Isolation and growth of infectious agent in pure culture. Inoculation of an infectious agent into an experimental host should cause the same disease. Re-isolation in pure culture from the diseased experimental host. EXCEPTIONS NOT ALL INFECTIOUS DISEASES MEET KOCH’S CRITERIA Asymptomatic carriers Microorganisms that can’t be grown in a culture Lack of animal models Immunocompromised host Acquired or genetic immunity HOST’S DETECTION OF A PATHOGEN The innate immune host defenses OVERVIEW OF INNATE IMMUNE HOST DEFENSES Detection of an infectious agent by recognizing Pathogen-Associated Molecular Patterns (PAMPs) or Microbial-Associated Molecular Patterns (MAMPs) ► Lipopolysaccharides (LPS) ► Peptidoglycan ► Lipoteichoic acids (LTA) ► Mannose-Rich Glycans (glycoproteins/glycolipids) ► Flagellin ► Pilin ► Bacterial DNA  OVERVIEW OF INNATE IMMUNE HOST DEFENSES Host defense cells have PATTERN-RECOGNITION RECEPTORS (PRR), primarily Toll-like receptors (TLR) on leucocytes, macrophages, mucosal epithelial cells, endothelial cells, dendritic cells. Binding of an infectious agent to PRR → Triggers synthesis and secretion of inflammatory cytokines and chemokines, which attract more phagocytes and activate the complement and coagulation pathways.  OVERVIEW OF INNATE IMMUNE HOST DEFENSES BINDING OF BACTERIA TO PHAGOCYTES CAN BE: – UNENHANCED: Direct binding of PAMPs to PRRs – ENHANCED (OPSONIZATION): Via opsonins: Immunoglobulin (IgG), complement (C3b)  OVERVIEW OF INNATE IMMUNE HOST DEFENSES Following attachment to the phagocyte, the infectious agent is engulfed in a vesicle (phagosome). The phagosome fuses with the lysosome, and the infectious agent is killed by hydrolytic enzymes and reactive oxygen species (ROS). HOW DO MICROORGANISMS CAUSE DISEASE?  MICROBIAL MECHANISMS OF PATHOGENICITY Number of Invading Microbes PORTALS OF ENTRY Damage to Host Cells/ Cytopathic Effects Portals of Exit Penetration or Evasion Mucous membranes of Host Defenses Direct damage Respiratory Respiratory tract Gastrointestinal tract Indirect damage Gastrointestinal Capsules Genitourinary Genitourinary tract Toxins: Conjunctiva Cell wall components Skin Exotoxins Skin Enzymes Blood Endotoxins Adherence  NATURE OF VIRULENCE FACTORS CELL STRUCTURES Features unique to a bacterial cell BACTERIAL PHYSIOLOGY Enzymes related to nutrient acquisition and breakdown Exotoxins Metabolic intermediate and end products INFECTION STRATEGIES Biofilm formation Flagellum Tissue invasion Molecular mimicry Antigenic change  VIRULENCE STAGES Attachment/Colonization Exit PATHOGENIC LIFE CYCLE Multiplication and Nutrition Invasion TISSUE DAMAGE Evasion of host defenses AT TACHMENT/COLONIZATION  ATTACHMENT/COLONIZATION ATTACHMENT VIA ADHESINS: Fimbriae Capsules Pili Cell wall components: LTA, surface proteins The attached bacteria must resist physical removal by the host: Coughing Sneezing Shedding of epithelial cells from mucous membranes Vomiting and diarrhea Bodily fluids: saliva, tears, blood, mucus, urine  MULTIPLICATION AND NUTRIENT ACQUISITION EXTRACELLULAR ENZYMES Proteases/Peptidases Lipases Glucosidases (e.g., amylases, cellulases) Nucleases IRON-CAPTURE SYSTEM Heme-containing proteins, such as hemoglobin, lactoferrin, transferrin, ferritin, ferric iron Siderophores: high-affinity iron-chelating compounds to capture Ferric Iron Specific receptors in outer membrane of gram-negative bacteria/cell wall receptors BIOFILM FORMATION Role in adherence Established food chain Traps nutrients in a polysaccharide matrix  EVASION OF HOST DEFENSES PREVENTION OF RECOGNITION MOLECULAR MIMICRY: surface proteins A sequence or structural resemblance of molecules of the host on the microbial surface (can elicit autoimmune reactions). INVASINS: Class of proteins associated with the penetration of pathogens into host cells; trigger significant cytoskeletal changes at the host-cell plasma membrane to promote invasion. ➔ protection from complement, antibodies, phagocytes, and other host defense molecules. ANTIGENIC VARIATION (PHASE VARIATION): Surface proteins Alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system.  EVASION OF HOST DEFENSES PREVENTION OF PHAGOCYTOSIS CAPSULE, LTA, AND CELL WALL SURFACE PROTEINS BIOFILM FORMATION: antiphagocytic, protection from host defense molecules (e.g., antimicrobial peptides). PROTEASES that degrade antibodies (IgA, IgG) and complement.  EVASION OF HOST DEFENSES RESISTANCE TO KILLING BY PHAGOSOME PROTEINS THAT INHIBIT PHAGOSOME-LYSOSOME FUSION allow bacteria to persist and replicate inside the phagosome, shielded from the host’s immune defenses. ESCAPE from the phagosome into cytoplasm prior to fusion with the lysosome. RESISTANCE TO KILLING BY OXYGEN RADICALS after fusion (e.g., catalase, peroxidase). KILLING OF THE PHAGOCYTE VIA EXOTOXINS type II and III targeting neutrophils and lymphocytes.  EVASION OF HOST DEFENSES EXOTOXINS SUPERANTIGENS OR TYPE I TOXINS Cause an intense immune response due to the release of cytokines from host cells Fever, nausea, vomiting, diarrhea, shock, death Examples: toxic shock syndrome toxin, enterotoxin MEMBRANE-DISRUPTING TOXINS OR TYPE II TOXINS Lyse host cells membranes by: - Making protein channels in the plasma membrane - Disrupting phospholipid bilayer Examples: hemolysin, leukotoxin INTRA-CELLULAR TARGETING TOXINS OR TYPE III TOXINS A-B toxins: A (active) and B (binding) Intracellular Targets: The A component of the toxin affects specific intracellular targets within the host cell, disrupting normal cellular function. Examples: cholera toxin, pertussis toxin, diphtheria toxin, tetanus toxin, cytolethal distending toxin (CDT)  INVASION CELL AND TISSUE DAMAGE DIRECT DAMAGE (BACTERIA-MEDIATED) - RELEASE OF EXOTOXINS (e.g., hemolysins, neurotoxins) - RELEASE OF ENZYMES (e.g., hyaluronidase, collagenase, etc.)  INVASION CELL AND TISSUE DAMAGE INDIRECT DAMAGE (HOST-MEDIATED) PAMPs-mediated induction of excessive inflammatory response Organ damage (endotoxin)- SEPTIC SHOCK Activation of matrix metalloproteinases (MMPs) and collagenases. Phagocyte necrosis: release of phagocyte content to the environment, causing damage to mammalian cells. CONTENT 1- Overview of bacterial structures and functions 2- Pathogenesis of bacterial infections ▪ Koch’s Postulates ▪ Overview of innate immune host defenses ▪ Microbial mechanisms of pathogenicity 3- Periodontal pathogens ▪ Virulence factors of periodontal pathogens PERIODONTAL PATHOGEN DEFINITION Bacteria that have been shown to significantly contribute to periodontal disease.  CRITERIA FOR IDENTIFICATION OF PERIODONTAL PATHOGEN SOCRANSKY’S CRITERIA A potential periodontal pathogen must 1. Be associated with disease, as evidenced by increases in the number of organisms at diseased sites 2. Be eliminated or decreased in sites that demonstrate the clinical resolution of disease with treatment 3. Induce a host response in the form of an alteration in the host cellular or humoral immune response 4. Be capable of causing disease in experimental animal models 5. Produce noticeable virulence factors that allow the microorganism to damage periodontal tissues BASED ON SOCRANSKY’S CRITERIA, DATA SUPPORT THE ROLE OF Porphyromonas gingivalis AND Aggregatibacter actinomycetemcomitans (Aa) AS PERIODONTAL PATHOGENS.  PERIODONTAL PATHOGENS Porphyromonas gingivalis Aggregatibacter (Pg) is a Gram-negative, actinomycetemcomitans (Aa) rod-shaped bacterium. It is a Gram-negative, is nonmotile, anaerobic, facultative anaerobic bacillus that causes and pathogenic, periodontal diseases. It is characterized by its capnophilic, meaning it formation of black thrives in environments with colonies on blood agar. high concentrations of CO2.  STAGES OF INFECTION ATTACHMENT/COLONIZATION EXIT PATHOGENIC LIFE CYCLE MULTIPLICATION AND NUTRITION INVASION TISSUE DAMAGE EVASION OF HOST DEFENSES  VIRULENCE FACTORS OF PERIODONTAL PATHOGEN ATTACHMENT TO HOST TISSUE Both P. gingivalis and A. actinomycetemcomitans (Aa) can attach to hydroxyapatite and epithelial cells via FIMBRIAE and other SURFACE PROTEINS. MULTIPLICATION AT SUSCEPTIBLE SITE Both P. gingivalis and A. actinomycetemcomitans (Aa) can form BIOFILMS via fimbriae attachment to surfaces and bacterial cells. P. gingivalis is known to produce extracellular PROTEASES to obtain nutrients. Aa does not produce proteases but produces EXOTOXINS. Aa biofilm  VIRULENCE FACTORS OF PERIODONTAL PATHOGENS EVASION OF HOST DEFENSE Biofilm formation protects bacteria from phagocytosis. P. gingivalis: - Suppression of inflammasomes (inhibition of the release of proinflammatory cytokines) - Extracellular proteases degrade immunoglobulins and complement → prevention of opsonization A. actinomycetemcomitans: - Leukotoxin (apoptosis or necrosis of NEUTROPHILS) Exotoxins - Cytolethal distending toxin (CDT) – Apoptosis of B and T cells  VIRULENCE FACTORS OF PERIODONTAL PATHOGEN Neutrophil lysis by A. actinomycetemcomitans leukotoxin (necrosis) Phagocytosed A. actinomycetemcomitans survive while neutrophils undergo apoptosis. Pictures provided by Dr. Dominique Galli, Indiana University School of Dentistry  VIRULENCE FACTORS OF PERIODONTAL PATHOGENS INVASION: ENTRY INTO EPITHELIAL CELLS Both A. actinomycetemcomitans (Aa) and P. gingivalis can invade, multiply, and spread through tissues while protected from the host's immune system. This allows them to move from epithelial cells to other areas, potentially entering the bloodstream and infecting non-oral sites Oral-systemic disease connection (e.g., Endocarditis)  VIRULENCE FACTORS OF PERIODONTAL PATHOGEN TISSUE DAMAGE Degradation of extracellular matrix and bone either directly or indirectly. Most destruction of the periodontium is host-mediated, driven by a bacterial challenge. direct indirect Bacteria-mediated pathogenesis Host-mediated pathogenesis TISSUE DESTRUCTION BONE RESORPTION  VIRULENCE FACTORS OF PERIODONTAL PATHOGEN TISSUE DAMAGE DIRECT P. gingivalis produces various enzymes (gingipains, trypsin-like protease, collagenase, hyaluronidase, chondroitin sulphatase) INDIRECT (activation and modulation of the local inflammatory response) ACTIVATION OF PHAGOCYTES AND INVASION OF EPITHELIAL CELLS → release of inflammatory mediators → activation of host collagenases and matrix metalloproteinases by fibroblasts NEUTROPHIL NECROSIS via leukotoxin activity → release of lysosomal enzymes and free radicals FRUSTRATED PHAGOCYTOSIS → release of lysosomal enzymes and free radicals LPS and microbial DNA from A. actinomycetemcomitans and P. gingivalis STIMULATE B AND T CELLS (LYMPHOCYTES) to produce RANKL*, which activates osteoclasts and increases bone resorption ( RANKL induce  osteoclast differentiation) *Receptor Activator of Nuclear Factor Kappa-B Ligand SUMMARY: LOCAL INFLAMMATION AND TISSUE DAMAGE Indirect Direct Biofilm Leukotoxin BACTERIUM Enzymes Cell Invasion Indirect Inflammatory mediators TISSUE DAMAGE (cytokines, prostaglandins) Lymphocytes (B and T cells) Phagocyte Fibroblasts, etc. Free radicals Lysosomal enzymes RANKL Collagenases, MMPs (↑ osteoclasts) TISSUE DAMAGE BONE RESORPTION  SUMMARY: STAGES OF INFECTION AND VIRULENCE FACTORS IN PERIODONTAL PATHOGENS ATTACHMENT/COLONIZATION Fimbriae Surface Proteins EXIT PATHOGENIC LIFE Direct: CYCLE MULTIPLICATION AND Biofilm Enzymes (Pg) NUTRITION Proteases (Pg) Indirect: INVASION Exotoxins (Aa) Activation and modulation of the TISSUE DAMAGE local inflammatory response by Pg Biofilm and Aa. EVASION OF HOST DEFENSES Suppression of inflammasomes (Pg) Proteases (Pg) Exotoxins (Aa) THANK YOU! Beatriz Panariello, DDS, MSc, PhD [email protected]