Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells

Background: Aggregatibacter 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. Results: By 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. Conclusion: A. 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

Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction

Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction

Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles are internalized in human host cells and trigger NOD1- and NOD2-dependent NF-κB activation

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis, and endocarditis. We recently demonstrated that OMVs disseminated by A. actinomycetemcomitans could deliver multiple proteins including biologically active cytolethal distending toxin (CDT) into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work we have used immunoelectron- and confocal microscopy analysis, and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Co-localization analysis revealed that internalized OMVs co-localized with the endoplasmic reticulum, and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active PAMPs.Originally included in thesis in manuscript form.</p

MV-associated α-toxin is biologically active.

<p>Hemolytic activity <i>in vitro</i> of MVs isolated from <i>S. aureus</i> strains 8325-4 (WT), and DU1090 (<i>hla</i>), respectively. Rabbit erythrocytes (100% in PBS) were incubated for 60 min with MVs or with MVs disrupted by sonication (2, 4, and 10 µg protein as indicated). Control treatment (C) erythrocytes incubated with PBS. Shown are the means ± SEM for three independent experiments. *<i>P</i><0.02, 8325-4 MVs vs DU1090 MVs for all tested concentrations; **<i>P</i><0.05, sonicated vs non-sonicated strain 8325-4 MVs for all tested concentrations; ***<i>P</i><0.03, 8325-4 MVs vs DU1090 MVs for all tested concentrations.</p

Tight association of α-toxin with <i>S. aureus</i> MVs.

<p>(A) Immunoblot detection of α-toxin (Hla) and protein A (SPa) in density gradient fractions of MVs from strain 8325-4. Fractions (15 µl applied on the gel) are numbered from left to right (2–10) according to increasing density. A polyclonal antiserum specific for α-toxin was used for immunoblot detection. The sizes (kDa) of the proteins in the prestained molecular weight marker (M) are indicated along the right side. (B) Relative hemolytic activity of density gradient fractions 3, 6, 8, and 15, respectively, as determined using an <i>in vitro</i> assay (20% rabbit erythrocytes). Data were normalized to the activity of fraction 3, having the highest α-toxin/protein A-ratio. Shown are the means ± SEM for three independent experiments. *<i>P</i><0.03, fraction 3 activity vs the activity of either of the other tested fractions. (C) Dissociation assays using MVs isolated from strain 8325-4. An MV preparation in PBS was treated for 60 min on ice in the presence of: PBS (buffer), urea (0.8 M and 8 M), or SDS (1%), respectively. The resulting pellets (P) and supernatants (S) after centrifugation were analyzed by immunoblotting, using a polyclonal anti-α-toxin (Hla) antiserum.</p

Detection of α-toxin in <i>S. aureus</i> MV preparations.

<p>Immunoblot detection of α-toxin (Hla; panel A), and CodY (lysis marker; panel B) in MV preparations, and in whole cell (WC) preparation samples from <i>S. aureus</i> strain 8325-4 (WT), and from the strain 8325-4 <i>hla</i> mutant, DU1090 (<i>hla</i>). Polyclonal antisera specific for <i>S. aureus</i> α-toxin, and <i>B. subtilis</i> CodY, respectively were used for immunoblot detection, and the reactive bands corresponding to these proteins are indicated with an arrowhead. The sizes (kDa) of the proteins in the prestained molecular weight marker (M) are indicated along the left sides. Protein samples equal to 10 µg were applied on the gels. Bar graphs indicate results of densitometric analysis of the immunoblots. Shown are the means ± SEM of relative band density for Hla (A) and CodY (B) from three independent experiments. Data were normalized to the whole cell lysate of the parental strain.</p

Detection of α-toxin in association with <i>S. aureus</i> MVs.

<p>Electron microscopy and immunogold-labeling of α-toxin, using a polyclonal antiserum specific for <i>S. aureus</i> α-toxin (A), or <i>B. subtilis</i> CodY (B). Immunoelectron micrographs of MVs isolated from strain 8325-4 (WT), DU1090 (<i>hla</i>), and WA764 (<i>spa</i>) are shown. Examples of vesicle structures are indicated by arrows, or highlighted by square boundaries and also shown in larger magnification below the corresponding micrograph. Arrows also indicate gold particles surrounding one 8325-4 (WT) vesicle structure in panel A. Gold particles associated with disrupted vesicle structures are indicated by arrowheads. Bars  = 100 nm. (C) Atomic force micrograph of strain 8325-4 (WT) cultivated on agar. Arrows indicate examples of the released MVs. Bar  = 300 nm.</p

Cholesterol-dependent fusion of <i>S. aureus</i> MVs with HeLa cells.

<p>Localization of rhodamine B-R18-labeled MVs (red fluorescence used as a readout for MV fusion with the host cell plasma membrane), and FITC-conjugated lipid raft marker CtxB (green fluorescence) in HeLa cells after 30 min of incubation with membrane-derived vesicles obtained from strain 8325-4 (MV; panel A), and with PBS (buffer; panel B). Treatment was done in the absence (−FIL) and in the presence (+FIL), respectively, of the cholesterol-sequestering agent Filipin III (final concentration 10 µg/ml). Bar graphs show quantitative analysis of red (B-R18) and green (FITC) fluorescence in treated HeLa cell samples. Values represent arbitrary units of pixel intensity for red and green fluorescence determined using ImageJ, and shown are the means ± SEM of data collected from 10 cells. *<i>P</i> = 0.0001, **<i>P</i><0.0001, and ***<i>P</i> = 0.0002, for treatment in the absence vs presence of Filipin III. The merged images show the labeling with both fluorescent dyes. The scattergrams in panel A with red (B-R18) and green (FITC) pixels plotted on graphs were used to obtain the colocalization coefficient (r_p) between MVs and CtxB in the HeLa cells treated with strain 8325-4 MVs for 30 min. (C) B-R18-labeled MVs alone. Magnification: 1000×. Bars  = 10 µm.</p