Diane H. Meyer

Oral pathogens: from dental plaque to cardiac disease

Oral bacteria exhibit highly specific adherence mechanisms and as a result they colonize and cause disease principally in the oral cavity. Oral pathogens, however, can produce systemic disease and are known causative agents of infective endocarditis. Recent studies have revealed that periodontal disease per se is also a statistically significant risk factor for cardiovascular disease. A link between the two diseases is the secretion and systemic appearance in periodontitis of pro-inflammatory cytokines capable of eliciting effects associated with atherosclerosis and coronary heart disease.

The role of Actinobacillus actinomycetemcomitans in the pathogenesis of periodontal disease

Periodontal disease consists of a constellation of complex bacterium-host cell interactions. One example of these oral pathogens, Actinobacillus actinomycetemcomitans, has an arsenal of putative virulence determinants that account for its potent periodontopathogenicity. Of these determinants, invasion of host cells and leukocytotoxicity have been studied extensively.

Characteristics of Actinobacillus Actinomycetemcomitans Invasion of and Adhesion to Cultured Epithelial Cells

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCI]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

Models of Invasion of Enteric and Periodontal Pathogens Into Epithelial Cells: A Comparative Analysis

Bacterial invasion of epithelial cells is associated with the initiation of infection by many bacteria. To carry out this action, bacteria have developed remarkable processes and mechanisms that co-opt host cell function and stimulate their own uptake and adaptation to the environment of the host cell. Two general types of invasion processes have been observed. In one type, the pathogens (e.g., Salmonella and Yersinia spp.) remain in the vacuole in which they are internalized and replicate within the vacuole. In the other type, the organism (e.g., Actinobacillus actinomycetemcomitans, Shigella flexneri, and Listeria monocytogenes) is able to escape from the vacuole, replicate in the host cell cytoplasm, and spread to adjacent host cells. The much-studied enteropathogenic bacteria usurp primarily host cell microfilaments for entry. Those organisms which can escape from the vacuole do so by means of hemolytic factors and C type phospholipases. The cell-to-cell spread of these organisms is mediated by microfilaments. The investigation of invasion by periodontopathogens is in its infancy in comparison with that of the enteric pathogens. However, studies to date on two invasive periodontopathogens. A actinomycetemcomitans and Porphyromonas (Bacteroides) gingivalis, reveal that these bacteria have developed invasion strategies and mechanisms similar to those of the enteropathogens. Entry of A. actinomycetemcomitans is mediated by microfilaments, whereas entry of P. gingivalis is mediated by both microfilaments and microtubules. A. actinomycetemcomitans, like Shigella and Listeria, can escape from the vacuole and spread to adjacent cells. However, the spread of A. actinomycetemcomitans is linked to host cell microtubules, not microfilaments. The paradigms presented establish that bacteria which cause chronic infections, such as periodontitis, and bacteria which cause acute diseases, such as dysentery, have developed similar invasion strategies.

Aae, an Autotransporter Involved in Adhesion of Actinobacillus actinomycetemcomitans to Epithelial Cells

ABSTRACT The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html ) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.

Deposition of bismuth by Yersinia enterocolitica

Yersinia enterocolitica 808lc cultures in exponential growth were incubated for 1 h in 0.1 % microcrystalline bismuth subsalicylate (BSS) suspensions. Scanning electron microscopy (SEM) revealed microcrystals directly bound to BSS-treated bacteria. Energy dispersive spectroscopy (EDS) X-ray microanalysis of the attached microcrystals confirmed that the crystals were the microcrystalline BSS. X-ray spectra positive for bismuth were also obtained by SEM-EDS X-ray microanalysis of whole bacteria, suggesting metal incorporation into the bacteria in regions absent of bound microcrystals. Transmission electron microscopy of thin sections of embedded preparations of BSS-treated exponential-growth-phase bacteria showed electron-dense deposits in the periphery of the bacteria. Y. enterocolitica cultures that were in stationary phase at the time of incubation with microcrystalline BSS showed no evidence of the electron-dense deposits and EDS spectra were negative for bismuth. Bacteria incubated in the absence of microcrystalline BSS also lacked electron-dense deposits. Scanning transmission electron microscopy used in conjunction with EDS X-ray microanalysis to view and analyze semi-thick sections (250–300 nm) of embedded preparations of BSS-treated bacteria in exponential growth confirmed that the electron-dense deposits at the periphery of the bacteria are the sites of bismuth depositions.

Evidence that bismuth salts reduce invasion of epithelial cells by enteroinvasive bacteria

The effects of sublethal concentrations of bismuth salts on bacterial invasion of mammalian cells were investigated. Pepto-Bismol, bismuth subsalicylate, and bismuth oxychloride, produced by interacting bismuth subsalicylate and simulated gastric juice, in suspension at concentrations as low as 1.4 mM significantly interfered with the invasion of RPMI-4788 cells by two different strains of Yersinia enterocolitica. Invasion of the mammalian epithelial cells by other enteric bacteria was also reduced significantly by some of these bismuth salts. Commercially obtained bismuth oxychloride, bismuth sulfide, and sodium salicylate had no affect on invasion by Y. enterocolitica. Exposure of Y. enterocolitica 808lc to Pepto-Bismol for as brief a time as 5 min was sufficient to produce the inhibitory effect. Removal of bismuth bound to bacteria by sodium potassium tartrate did not reverse the inhibition. Electron-dense deposits are observed in Y. enterocolitica 808lc exposed to bismuth subsalicylate, suggesting that interference of invasion may result from bismuth permeation of the bacterial cell wall.

[30] Adhesion of oral bacteria to soft tissue

Publisher Summary This chapter focuses on the adhesion of oral bacteria to soft tissue and describes procedures utilized to study this adhesion. The focus is on Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis . Adhesion is a prerequisite for colonization of oral bacteria as the flow of saliva removes exposed bacteria that are not firmly bound. Bacteria adhere to oral surfaces in a highly specific manner. The physical conditions in the mouth and components derived from the bacterium, from the host, from the diet of the host, and from other bacteria are responsible for the specificity. Most bacteria that colonize the soft tissues of the mouth are slow growing and fastidious. Components on the surface of bacteria that are associated with adhesion include fimbriae, flagella, lipopolysaccharide, polysaccharide, microvesicles, and outer membranes. Adhesion of oral bacteria to soft tissue is likely mediated by specific and nonspecific interactions of one or more of the adhesion determinants. Components of saliva have molecules that mimic those of receptors found on oral epithelial cells. The interaction of bacterial adhesins with receptors in secretions diminishes or precludes interaction with receptors on oral epithelial cells. The ability of saliva and antibodies to inhibit adhesion may represent important antiadhesion mechanisms for oral bacteria.

Properties of polynucleotidase activities of the mealworm, Tenebrio molitor☆

Abstract Two predominant polynucleotidase activities of Tenebrio molitor tissues have been characterized. One activity is Mg-dependent, optimally active near pH 8, and heat and acid labile. It is largely due to an endonuclease yielding nucleotides with 5′-phosphate termini. The second activity is Mg-independent with a pH optimum near 6. This activity appears to be due to a combination of an exonuclease and an endonuclease which yields products containing 3′-phosphate termini. Twenty to 40% of the total Mg-independent activity is present in a latent form indicating the existence of an RNAse—inhibitor system in insects.

Detection of bacteria-microtubule interactions in a cell-free extract

Host cell microtubules, or its primary constituent tubulin, participate in the cell-to-cell spread of some pathogenic bacteria. A cell-free method was devised for study of the interaction between these invasive bacteria and microtubules. The basis of the assay is a cytosolic extract of KB oral epithelial cells lysed by osmotic swelling and mechanical shearing. This technique is used in immunofluorscence micrography for visualization and an ELISA ideal for the screening of mutants deficient in the interaction.