Abstract
BackgroundAggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.ResultsBy employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.ConclusionA. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.
Highlights
Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis
Free-soluble material released by A. actinomycetemcomitans induces proinflammatory responses in whole blood To study the pathogenic potential of free-soluble material released by A. actinomycetemcomitans cells, we implemented our cell culture plate insert model for stimulation of human whole blood as described in Methods
To our previous studies using the insert model [17], the absence of A. actinomycetemcomitans cell lysis in the wells during the experiments was confirmed by immunoblot analysis of samples taken from the wells outside the inserts, using an antiserum raised against the cytoplasmic protein cyclic AMP receptor protein (CRP)
Summary
Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, the underlying mechanisms are poorly understood. The Gram-negative bacterium Aggregatibacter (Actinobacillus) actinomycetemcomitans is implicated in aggressive forms of periodontitis [1,2]. Increasing evidence points to a link between periodontitis and cardiovascular diseases [4,5,6,7]. The pathogenic mechanisms that would render periodontitis patients to increased cardiovascular risk are still poorly understood. Previous experimental studies on the background of the association between periodontitis and cardiovascular diseases have mainly worked on the basis of the infection hypothesis that suggests that chronic lowgrade bacterial and/or viral infections have a causal role in the development of atherosclerosis and its sequels, such as myocardial infarction and stroke [8,9]. It is believed that infections raise systemic inflammatory status, as evidenced by elevated circulating levels of proinflammatory cytokines and acute phase reactants, which in turn may promote endothelial dysfunction and proatherogenic and proinflammatory phenomena in arterial walls [10,11]
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