Microbial etiological agents of destructive periodontal diseases

The extracellular secreted mucus and the cell surface glycocalyx prevent infection by the vast numbers of microorganisms that live in the healthy gut. Mucin glycoproteins are the major component of these barriers. In this Review, we describe the components of the secreted and cell surface mucosal barriers and the evidence that they form an effective barricade against potential pathogens. However, successful enteric pathogens have evolved strategies to circumvent these barriers. We discuss the interactions between enteric pathogens and mucins, and the mechanisms that these pathogens use to disrupt and avoid mucosal barriers. In addition, we describe dynamic alterations in the mucin barrier that are driven by host innate and adaptive immune responses to infection.

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Mucin dynamics and enteric pathogens

The interpretation of diagnostic tests for the detection of subgingival bacterial species is dependent on knowledge of the microbial etiology of destructive periodontal diseases. Specific etiologic agents of these diseases have been sought for over 100 years; however, the complexity of the microbiota, an incomplete understanding of the biology of periodontal diseases, and technical problems have handicapped this search. Nonetheless, a number of possible pathogens have been suggested on the basis of their association with disease, animal pathogenicity, and virulence factors. The immunological response of the host to a species and the relation of successful therapy to the elimination of the species have also been used to support or refute suspected periodontal pathogens. Current data suggest that pathogens are necessary but not sufficient for disease activity to occur. Factors which influence activity include susceptibility of the individual host and the presence of interacting bacterial species which facilitate or impede disease progression. Recent studies have attempted to distinguish virulent and avirulent clonal types of suspected pathogenic species and seek transmission of genetic elements needed for pathogenic species to cause disease. Finally, the local environment of the periodontal pocket may be important in the regulation of expression of virulence factors by pathogenic species. Thus, in order that disease result from a pathogen, 1) it must be a virulent clonal type; 2) it must possess the chromosomal and extra-chromosomal genetic factors to initiate disease; 3) the host must be susceptible to this pathogen; 4) the pathogen must be in numbers sufficient to exceed the threshold for that host; 5) it must be located at the right place; 6) other bacterial species must foster, or at least not inhibit, the process; and 7) the local environment must be one which is conducive to the expression of the species' virulence properties.

The bacterial etiology of destructive periodontal disease: current concepts.

In this Review, Schwechheimer and Kuehn describe recent developments in elucidating the mechanisms of biogenesis and cargo selection of the outer-membrane vesicles (OMVs) produced by Gram-negative bacteria. They also discuss the functions of OMVs in bacterial physiology and during pathogenesis. Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

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Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

Mossbauer spectroscopy was used to re-evaluate iron protoporphyrin IX, FePPIX, binding and the chemical nature of the black iron porphyrin pigment of Porphyromonas gingivalis. We demonstrate that FePPIX is bound to the cell in the mu-oxo dimeric form, [Fe(III)PPIX]2O, and that the iron porphyrin pigment is also composed of this material. P. gingivalis also assimilated monomeric Fe(II)- and Fe(III)PPIX into mu-oxo dimers in vitro. Scatchard analysis revealed a greater binding maximum of cells for mu-oxo dimers than for monomeric Fe(III)-or Fe(II)PPIX, although the relative affinity constant for the dimers was lower. Formation of [Fe(III)PPIX]2O via reactions of Fe(II)PPIX with oxygen, and its toxic derivatives, would serve as an oxidative buffer and permit P. gingivalis and other black-pigmenting anaerobes to engender and maintain a local anaerobic environment. Tying up of free oxygen species with iron protoporphyrin IX would also reduce and limit Fe(II)PPIX-mediated oxygen-radical cell damage. More importantly, formation of a cell-surface mu-oxo dimer layer may function as a protective barrier against assault by reactive oxidants generated by neutrophils. Selective interference with these mechanisms would offer the possibility of attenuating the pathogenicity of P. gingivalis and other iron protoporphyrin IX-binding pathogens whose virulence is regulated by this reactive molecule.

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The periodontopathogen Porphyromonas gingivalis binds iron protoporphyrin IX in the mu-oxo dimeric form: an oxidative buffer and possible pathogenic mechanism.

The major haem component in the black pigment of Porphyromonas gingivalis is the μ-oxo bishaem of iron protoporphyrin IX and formation and cell-surface binding of this haem species is proposed as an extracellular buffer against reactive oxidants [Smalley, J.W. et al. (1998) Biochem. J. 331, 681–685]. P. gingivalis cells grown in the presence of the μ-oxo bishaem were protected against H2O2 compared to control cells grown without it. When added to the growth medium, soluble μ-oxo bishaem inactivated H2O2 and supported cell growth. Cells carrying a surface layer of μ-oxo bishaem were less susceptible to peroxidation by H2O2. Cell-surface haems were slowly destroyed during reaction with H2O2. Binding of μ-oxo bishaem by P. gingivalis may aid survival during neutrophil attack through inactivation of hydrogen peroxide.

The periodontal pathogen Porphyromonas gingivalis harnesses the chemistry of the μ-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide

SUMMARY: Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of Nα-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10000 g and 75000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50

The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50

Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore.

John W. Smalley

Papers

The periodontopathogen Porphyromonas gingivalis binds iron protoporphyrin IX in the mu-oxo dimeric form: an oxidative buffer and possible pathogenic mechanism.

The periodontal pathogen Porphyromonas gingivalis harnesses the chemistry of the μ-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50

The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50

Heme acquisition mechanisms of Porphyromonas gingivalis – strategies used in a polymicrobial community in a heme-limited host environment