- Research article
- Open Access
Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form
© Karched et al; licensee BioMed Central Ltd. 2008
- Received: 02 July 2007
- Accepted: 28 January 2008
- Published: 28 January 2008
Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles.
The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL.
Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.
- Cell Culture Insert
- cAMP Receptor Protein
- Extracellular Release
- Vesicle Preparation
- Outer Membrane Vesicle
Aggregatibacter actinomycetemcomitans  is a small Gram-negative rod implicated in aggressive forms of periodontitis . We recently identified and characterized in A. actinomycetemcomitans a 17-kDa peptidoglycan-associated lipoprotein (PAL) [3, 4], which is a widely conserved outer membrane lipoprotein (OMLP) among Gram-negative bacteria . The PALs of several pathogens, such as Haemophilus influenzae, Escherichia coli [6–9], Legionella pneumophila, Bordetella pertussis, and Campylobacter jejuni [10–12] are proinflammatory and/or strongly immunogenic and can contribute to serum resistance of the bacterium . PAL may be an important bacterial mediator in sepsis caused by Gram-negative bacteria  and is crucial for full expression of virulence of Haemophilus ducreyi .
Extracellular release of bacterial virulence factors is a principal mechanism of bacterial pathogenicity . Different strategies of release include specialized secretory systems [15–17], outer membrane vesicles [18, 19], and autolysis . Release of outer membrane vesicles is regarded as the primary mechanism for delivering structural surface components, such as outer membrane proteins (OMP) and lipopolysaccharide (LPS), into the extracellular milieu. Most previous studies on the released bacterial outer membrane components, however, have focused on detecting and isolating them from serum or culture supernatants, or on testing their biological activity [21–25]. Less attention has been directed to elucidating whether these components in the test material were derived from lysed cells, outer membrane vesicles, and/or were released from live bacteria in free-soluble form. Importantly, the bacterial cell components that are released in free-soluble form may be biologically more active than their membrane-bound counterparts associated with intact bacterial cells or outer membrane vesicles .
The present study was designed to investigate the presence and conservation of PAL among clinical A. actinomycetemcomitans strains and whether live A. actinomycetemcomitans cells release PAL (AaPAL) in free-soluble form. To accomplish these objectives, inserts with 0.02-μm pore-size filter were used to separate bacterial cells from cell culture wells containing serum or broth. We present compelling evidence for the release of AaPAL in free-soluble form by demonstrating presence of AaPAL in the filtrates obtained after incubation, by monitoring bacterial viability in the insert, and by checking for the absence of cell lysis and outer membrane vesicles in the filtrate. Proinflammatory effect of AaPAL was shown in an ex vivo model using human whole blood.
The pal gene was conserved and the protein expressed in clonally diverse A. actinomycetemcomitansstrains
The RFLP analysis with two enzymes, DdeI and BspMI, cutting once within the pal sequence  showed that the restriction patterns were similar among all test strains (Fig. 1). The DdeI-digested fragments were 175 and 250 bp, as predicted, whereas the BspMI-digested fragments were 100 and 225 bp, which were not the predicted sizes of 324 and 101 bp, respectively. The reason seems to be a recognition site for BspMI on the opposite strand approximately 100 bp away from the reverse primer binding sequence.
AaPAL was expressed in various culture conditions
Phenotype of the pal-deficient mutants
The strain D7SS-p was constructed to be used as a negative control in the experiments. The strain D7S-p was also constructed to serve as a negative control, but particularly in the experiments aimed to demonstrate with a clinical strain, D7S, that AaPAL release was not restricted to its spontaneous laboratory variant, D7SS.
AaPAL was detected in serum and broth filtrates
Densitometric quantification indicated that for strain D7SS incubated in serum, AaPAL concentration after the first 2 h was 637 ng/ml and at 8 h was 932 ng/ml (a 1.5-fold increase from 2 h). Similarly, for strain D7S incubated in serum, AaPAL concentration after the first 2 h was 437 ng/ml and at 8 h was 750 ng/ml (a 1.7-fold increase from 2 h). For strain D7SS in broth, AaPAL concentration at 24 h was 194 ng/ml (for strain D7S, only serum incubation was performed).
To ensure the viability of the bacteria during the AaPAL release experiments described above, we employed bacterial viable counts for samples taken from serum and broth inside the inserts at the same time points as samples that were taken from filtrates for the immunoblot analyses (data for D7SS and D7SS-p are presented in Fig. 4C). In serum, the mean CFU (per ml) counts of D7SS were 1.1-fold higher at 2 h than at 0 h and 46.0-fold higher at 8 h than at 2 h. Similarly, the mean CFU counts of D7SS-p were 1.3-fold higher at 2 h than at 0 h and 30.0-fold higher at 8 h than at 2 h.
In broth, the mean CFU counts of D7SS were 34.3-fold higher at 24-h than at 0 h. In contrast, the CFU counts of D7SS-p were 62.2-fold lower at 24 h than at 0 h (Fig. 4C).
Since the AaPAL release experiment was performed for strains D7S and D7S-p in serum only, the viable count data are given here: The mean CFU counts of D7S were at 0 h 6.2 × 108, at 2 h 8.7 × 108 (a 1.4-fold increase), and at 8 h 1.16 × 1010 (a 13.3-fold increase from 2 h). The respective mean CFU counts for D7S-p were at 0 h 6.1 × 108, at 2 h 9.5 × 108 (a 1.5-fold increase), and at 8 h 1.4 × 1010 (a 15.0-fold increase from 2 h).
No bacterial growth was found in the samples from the serum or broth filtrates at any time point, ensuring that bacteria growing within the inserts had not passed through the filter and/or that the filtrates had not been contaminated.
We also tested for the release of additional A. actinomycetemcomitans components, apart from PAL and LPS, in the filtrates. Immunoblot analysis of the filtrates after an 8-h serum incubation of D7SS, D7SS-p, and No bacteria was carried out by using an antiserum raised against whole cell antigen of A. actinomycetemcomitans serotype a (both D7SS and D7SS-p are of serotype a). Several immunoreactive bands of unknown identity but representing apparent molecular weights ≈ 10–120 kDa were detected in the filtrates from both D7SS and D7SS-p, but not in the No bacteria controls (data not shown).
Extracellular release of AaPAL was independent of cell lysis
Our findings demonstrate that cAMP receptor protein could not be detected in any of the filtrate samples obtained from the cell-culture insert model (Fig. 6), not even when the filtrates were concentrated with trichloroacetic acid precipitation (data not shown).
To show that soluble cAMP receptor protein could be detected in filtrates from lysed bacteria, deliberately broken (See Methods) A. actinomycetemcomitans cells were incubated in cell culture inserts containing broth for 8 h as described above. As shown in Fig. 6, filtrates from lysed but not from unlysed D7SS cells contained detectable amounts of cAMP receptor protein.
AaPAL was associated with A. actinomycetemcomitansouter membrane vesicles
Extracellular release of AaPAL was independent of outer membrane vesicles
AaPAL induced cytokines in an ex vivo model
The present study demonstrates that A. actinomycetemcomitans releases AaPAL, a structural OMLP, in free-soluble form. The primary route for outer membrane components of bacteria, such as LPS and various proteins including porins [18, 19, 28, 29] to the extracellular space is through the release of outer membrane vesicles. Although not widely studied, there are a few reports on the release of OMPs, such as OmpA, PAL, and major lipoprotein (Braun's lipoprotein), either in soluble form [21, 25, 30] or in the form of complexes [23, 31]. In those studies, however, the methods were not designed to exclude the presence of vesicles in the samples [25, 31, 32] or bacterial death or lysis [21, 25, 30–32]. Our study made an attempt to control those factors and showed that AaPAL is released independent of vesicles. Release of free-soluble bacterial components, such as shown here for a lipoprotein, would be of biological significance, since these components can cross anatomical barriers impermeable to whole bacteria or even vesicles [33–35]. In addition, there is evidence to suggest that in free form bacterial components convey higher biological activity than when they are membrane-bound in intact bacteria or in vesicles . Regardless, the results of the present study accentuate a less studied strategy by which oral bacteria, like bacteria in other localized infections, may express extended pathogenicity.
Our experimental model comprised a cell culture insert. Based on the previous literature on A. actinomycetemcomitans outer membrane vesicles , we chose a membrane filter with a pore size (0.02 μm) that would not allow cells or vesicles to pass through. An electron microscopic examination verified that the vesicle size for the present A. actinomycetemcomitans strains indeed was larger than the pores in the chosen filter. This renders unlikely the possibility that vesicles would have passed through the filter, which was further confirmed by the absence of vesicles in the filtrates. No literature was found about release of material from intact vesicles. Instead, vesicles are known to be considerably stable structures [37, 38] and therefore, the possibility that lysed vesicles would have been the source of free-soluble AaPAL may be low.
Yet another possibility was that A. actinomycetemcomitans cells had died and lysed in the inserts, so we continuously monitored the viability of the bacteria grown in the inserts. The fact the total bacterial numbers increased over time shows that viability was maintained in the experiments. However, despite the increased total numbers, lysis of dying bacteria in the growing A. actinomycetemcomitans population may have presented a source of AaPAL in the filtrate. Therefore, we, like others [39, 40], excluded the possibility of bacterial lysis by confirming the absence of a cytoplasmic marker in the same filtrate samples from which AaPAL was found.
Thus, neither vesicles passing through the filters nor cell death/lysis seemed to be the source of AaPAL in the filtrate samples. Further studies are needed to clarify the mechanism by which free-soluble AaPAL is released into extracellular space and how the release is regulated. Currently, it would be tempting to speculate that the mechanism involves activation of outer membrane phospholipase A [41–43]. By degrading phospholipids of the outer membrane, phospholipase A could liberate AaPAL along with other components of the outer membrane into the extracellular milieu. For other species, release of cell components, such as proteins, LPS and phospholipids has been reported following activation of phospholipase A, without an effect on growth or cell morphology . Due to its strict regulation, phospholipase A activity does not lead to bacterial cell lysis [41, 45].
For the biological activity of the released free-soluble AaPAL it is crucial that it retains the lipid domain, because lipoprotein-mediated cytokine induction is solely attributed to the lipid domain . Our results support the retention of the lipid part of AaPAL, since the immunoblot detection from the filtrate demonstrated a band of the same MW (≈ 17 kDa) as that of purified AaPAL (without lipid domain, the amino acid sequence-based theoretical MW of AaPAL is ≈ 14 kDa). Therefore, the extracellularly released AaPAL seems to be neither proteolytically cleaved nor missing its lipid domain. The activity of free-soluble AaPAL may also be related to its association with other bacterial components. Our present study did not provide data to suggest an association but demonstrated co-release of AaPAL with LPS and other unidentified A. actinomycetemcomitans components. However, an association is possible, since previous studies have shown tight association between LPS and released E. coli PAL , as well as between extracellularly released OmpA, major lipoprotein, PAL and LPS [23, 31].
Since in this study we were unable to obtain the extracellularly released AaPAL in pure form and therefore did not know whether or not it was complexed with other bacterial components, we chose to study the immunostimulatory effect of purified AaPAL in an ex vivo model. Our results showed a dose-dependent enhancement in the production of IL-6, IL-8, and MIP-1β after an 8-h stimulation, using purified AaPAL. A search of the literature revealed no previous studies of cytokine production from whole blood by using AaPAL, or other lipoproteins or OMPs of this species. The only study on other species also reported an increased IL-6 production from blood after incubation with E. coli PAL . In general, IL-6 and IL-8 are among the major cytokines produced by whole blood after stimulation with bacterial components [47–52]. IL-6 has a wide range of biological activities, among them proinflammatory properties and a role as the main stimulus for acute phase protein synthesis and release from liver in infection . One of the two chemokines induced by AaPAL in our study, IL-8, a prototypic CXC-chemokine and classically known as a neutrophil chemoattractant, mediates monocyte trafficking and functions as a macrophage stimulator . The other chemokine, MIP-1β, a CC-subfamily chemokine, is a potent chemoattractant for monocytes and T cells . Thus, even though we currently do not know the bioactivity of released free-soluble AaPAL, in purified form AaPAL has proinflammatory and prochemotactic properties that can lead to stimulation and differentiation of various cell types and further production of cytokines.
Our consistent results from RFLP of the A. actinomycetemcomitans pal gene and the immunoblot detection of the gene product indicate that the gene is conserved and the protein ubiquitously expressed in clonally diverse A. actinomycetemcomitans strains. This is in agreement with the intraspecies conservation of pal in other Pasteurellaceae members, such as Haemophilus influenzae . The intraspecies conservation of AaPAL suggests that its bioactivity is comparable among different A. actinomycetemcomitans strains. The fact that AaPAL was expressed in a variety of different nutritional and atmospheric conditions may underscore its importance for the normal function of the bacterium. In addition, AaPAL was expressed during the exposure to serum and culture in anaerobic atmosphere. Since these conditions bear resemblance to those in pathologically deepened periodontal pockets, the results suggest that AaPAL is also expressed in vivo.
We included the pal-deficient mutant strains of A. actinomycetemcomitans primarily to corroborate our results from the immunoblot and other experiments. Comparisons of the phenotypic characteristics of the mutant and parent may, however, provide preliminary information about the role and function of AaPAL in A. actinomycetemcomitans cell physiology. We found that the mutants had decreased growth rate and filamentous cell morphology, differing partly from the pal-deficient mutant of H. ducreyi, which showed unchanged growth rate . Although there were limitations in the present methods for estimating bacterial numbers, our results are consistent with previous reports demonstrating reduced growth rate of bacteria with mutations in tol-pal genes [13, 57, 58]. Moreover, the antimicrobial susceptibility of the pal-deficient A. actinomycetemcomitans mutant, D7SS-p, selectively changed. The susceptibility to erythromycin, but not to the β-lactams, increased. Previous studies have shown increased antibiotic susceptibility of strains with tol-pal gene mutations . Additionally, mutations in genes encoding certain OMPs, such as ssc and envA of E. coli (encode 36- and 34-kDa OMPs, respectively), have resulted in hypersusceptibility to hydrophobic antibiotics, including erythromycin . Our finding that the susceptibility of the pal-deficient A. actinomycetemcomitans mutant to the tested β-lactams did not increase may result from the inefficiency of the β-lactam antibiotics to kill slowly multiplying bacteria .
Our results suggest that live A. actinomycetemcomitans cells release AaPAL in free-soluble form independent of vesicles. The proinflammatory and prochemotactic properties of AaPAL may contribute to producing a long-term immune challenge in persistent A. actinomycetemcomitans-associated infections. The extracellular release of PAL is also of more general interest because bacterial lipoproteins are increasingly being recognized as playing an important role in bacterial pathogenicity [7, 14, 62].
Bacteria and culture conditions
The test bacteria included 33 A. actinomycetemcomitans strains of 6 serotypes [63–65] and 15 genotypes [66, 67]. A. actinomycetemcomitans strains were of oral (N = 31) and non-oral (N = 2) origin. Unless otherwise stated, strains were cultured on either supplemented blood agar plates , tryptic soy agar plates (TSA), or broth (TSB) (supplemented with 0.6% yeast extract, 0.8% glucose) (Difco, Sparks, MD, USA) and incubated in either CO2-enriched air (5%) or anaerobic atmosphere (85% N2, 10% H2, 5% CO2) at 37°C for 3 d. For biofilm growth , the bacteria were cultured in TSB in a 24-well cell culture plate (Nunc™, Nunc, Denmark) and incubated in either CO2-enriched air or anaerobic atmosphere, as detailed above, for 5 d. V. cholerae strains BML24/pHA 7 (crp+) and BML24/pBR322 (crp-) (Wai, unpublished) were cultured in Luria Bertani broth at 37°C overnight with shaking.
Production of anti-AaPAL peptide antiserum
Antiserum was raised in rabbit against two synthetic peptides of AaPAL (N-terminal: 4–37 and C-terminal: 123–136), as described earlier .
SDS-PAGE and immunoblot
Bacterial outer membrane fraction was extracted as previously described . Bacteria suspended in PBS were used for whole cell protein preparation. SDS-PAGE and immunoblot were performed as previously described . For immunoblots we used anti-AaPAL peptide antiserum , an antiserum against whole cells of A. actinomycetemcomitans serotype a , and V. cholerae cAMP receptor protein . Unless specified otherwise, the antisera were used at final dilutions of 1:100 000, 1:1000, and 1:3000, respectively. Horseradish peroxidase-conjugated anti-rabbit secondary antibodies were used at a final dilution of 1:10000 or 1:20000. Immunoreactive bands were visualized using SuperSignal® (Pierce, Rockford, IL, USA).
pal-deficient mutant strains
D7SS-p, the pal-deficient mutant derivative of strain D7SS (spontaneous smooth-colony variant of wild-type D7S), was constructed by gene replacement technique as described earlier . The pal::Spe allele of D7SS-p was transferred to the wild-type fimbriated strain D7S, using natural transformation , generating D7S-p. The presence of the pal::Spe allele in D7S-p was confirmed by PCR using the primers PAL-Sm 5'-CTTTCCCGGGACATAACGG and PAL-Hd 5'-GCTGAAAGCTTATAGTG TAAGAG. The abolished AaPAL expression in the pal-deficient mutants was confirmed by immunoblot using anti-AaPAL peptide antiserum.
PCR-restriction fragment length polymorphism (PCR-RFLP) of the A. actinomycetemcomitans palsequence
For RFLP, PCR amplicons generated by the primers PAL-SA 5'-GCTGGTTCTGTGGCTGTGT and PAL-MK 5'-ACTGCACGACGGTTTTTAGC were purified (QIAquick® Gel Extraction Kit, Qiagen GmbH, Helden, Germany) and digested with BspMI and DdeI (New England BioLabs, Beverly, MA, USA), respectively, prior to analysis on agarose (1.8%) gel electrophoresis.
Phenotypic characterization of mutants
Phenotypic characteristics, such as colony size and density, cell morphology, growth rate, and antibiotic susceptibility were studied as follows:
10-μl inocula containing 103 cells of strains D7SS, D7SS-p, D7S, and D7S-p were cultured on supplemented blood agar and TSA plates as described above and observed under a stereomicroscope (Nikon SMZ800, Tokyo, Japan). The cell morphology was evaluated using phase contrast microscopy at × 1000 magnification (Leica DMRBE).
Scanning electron microscopy (SEM)
The strains D7SS, D7SS-p, D7S, and D7S-p cultured on TSA plates were fixed (2.5% glutaraldehyde in PBS; RT, 5 min) and prepared according to established methods. The cells were dehydrated using increasing concentrations of ethanol, and thereafter dried from liquid CO2 by the critical-point drying technique  using a Polaron E-3000 CPD apparatus (Polaron Equipment Ltd, Watford, UK). The cover slips with attached bacteria were mounted on aluminum stubs with silver dag (Agar Scientific Ltd, Stansted, UK) and coated (approximately 15 nm of carbon and gold) in an Edwards E12 Vacuum Coating Unit (Edwards High Vacuum Ltd, Crawley, UK). Bacterial cell morphology was examined in a Cambridge S360ixp scanning electron microscope run with a LaB6 electron emittor (Leica Cambridge Ltd, Cambridge, UK) and the micrographs were recorded from randomly selected areas with standardized magnifications.
For determining the growth rates for the strains D7SS and D7SS-p, log-phase cultures were used to inoculate TSB. The growth was measured as turbidity at OD600.
The D7SS and D7SS-p strains were grown in lawn cultures (1 × 108 CFU/plate) on TSA plates on which discs of erythromycin (15 μg/disc), cefotaxime (30 μg/disc), and penicillin (10 U/disc) (BD Biosciences, San Jose, CA, USA) were placed. The plates were incubated at 37°C in CO2-enriched air for 3 d. Inhibition zones were measured in millimeters.
Isolation of outer membrane vesicles
Outer membrane vesicles were isolated as described previously  with some modifications. A. actinomycetemcomitans strains D7SS and D7SS-p were cultured on supplemented blood agar plates for 3 days as described above. Bacteria were harvested and suspended into sterile PBS and centrifuged at 5000 × g (10 min, RT) to pellet the bacteria. The supernatant was filtered (0.22 μm; Millipore, Billerica, MA, USA) to remove any remaining intact bacterial cells. The filtrate was ultracentrifuged at 125000 × g (3 h, 4°C) using a 70 Ti rotor (Beckman Instruments Inc. Palo Alto, CA, USA). The obtained pellet was lyophilized and suspended in sterile PBS. The outer membrane vesicle preparations were examined by electron microscopy as described earlier . Briefly, the vesicle samples were stained with 0.5% uranyl acetate and examined by a JEM 2000 ET electron micoscope (JEDL, Akishima, Tokyo, Japan) at 100 KV.
Cell culture insert model for studying extracellular release of AaPAL in vitro
Cell culture insert experiments were carried out using exposure to serum (2 and 8 h) and culture in broth (24 h) as follows: A. actinomycetemcomitans cells (1 × 109 CFU/ml) were suspended in heat inactivated 50% fetal calf serum (FCS) (Invitrogen Gibco, Carlsbad, CA, USA) in PBS. The cell suspension was transferred into inserts (membrane pore size 0.02 μm), which were placed in cell culture wells (Nunc) containing FCS. The procedure for culturing in broth was similar to that for FCS, except that the wells contained TSB and the bacterial cells were suspended in TSB to a concentration of 1 × 108 CFU/ml. Inserts containing serum or broth without bacteria served as negative controls.
For detection of released AaPAL, following incubation at 37°C in CO2-enriched air, 100 μl samples, referred to as "filtrates" henceforth, were taken from serum (at 0, 2, and 8 h) and broth (at 0 and 24 h) from the wells outside the inserts. Samples from serum and broth filtrates, containing 10 and 30 μg protein, respectively, were subjected to immunoblot analysis. The amount of AaPAL released into serum filtrate was quantified by densitometry (Quantity One® software, Bio-Rad, Hercules, CA, USA) using purified AaPAL (2, 4, 6, and 8 ng) as a reference. As the intensities of AaPAL bands were very similar in all three independent experiments, data from only one experiment was chosen for densitometric quantification.
To monitor the viability of A. actinomycetemcomitans cells in serum and broth during incubation, 10-μl samples of the bacterial suspensions were taken from the inserts at the same time points as the samples were taken from the filtrates for immunoblot. Since bacterial cells might have sedimented to the bottom during incubation, the bacterial suspensions inside the inserts were pipetted in and out repeatedly 4–5 times to make the suspension homogeneous before taking out the samples for culture and viable counts. Appropriate serial dilutions of the samples were plated on supplemented blood agar plates and incubated at 37°C in CO2-enriched air for 3 d. To exclude bacterial contamination of the serum or broth filtrates, 10-μl samples were also taken and cultured on supplemented blood agar plates. Limulus assay (Cape Cod, E. Falmouth, MA, USA) was used for the detection of LPS in the filtrates.
To ensure that the integrity of the insert membrane remained intact (i.e., no leakage of bacterial cells) and that AaPAL found in the filtrate was not due to cell lysis in the insert, immunoblot was used to analyze the presence of a cytoplasmic marker protein, cAMP receptor protein, in the filtrates, as described above for the detection of AaPAL. For concentration, the filtrates were precipitated using ice-cold trichloroacetic acid (final concentration 10%).
To deliberately cause release of cytoplasmic proteins, A. actinomycetemcomitans D7SS cells (2 × 109 cells/ml) were lysed using Lysing matrix E provided in the FastDNA ® SPIN Kit for Soil (BIO 101, Q-Biogen, Montreal, Canada) with Mini-BeadBeater-8 (BioSpec Products Inc., Bartlesville, OK, USA) at highest speed for 3 min. The lysed cells were incubated in broth in the cell culture insert as described above. Unlysed cells were incubated similarly as a negative control. After 8 h incubation, filtrates collected from the wells were TCA-precipitated and the cAMP receptor protein was detected by immunoblotting as above.
To investigate whether outer membrane vesicles passed through the insert membrane filters in our experimental setup, serum filtrates were ultracentrifuged using the same procedure for sample preparation and electron microscopic examination as described above for the isolation of outer membrane vesicles.
Cytokine induction from human whole blood by AaPAL
AaPAL was purified by affinity chromatography as described previously . Each microgram of purified AaPAL had LPS contamination of less than 5 pg, an amount which was shown not to contribute to AaPAL-induced cytokine production . AaPAL TranSignal human cytokine antibody arrays (Panomics, Redwood City, CA, USA) were used to profile cytokines produced by human whole blood. Blood sample from a healthy individual was drawn via venipuncture into heparin-containing tubes (BD Biosciences). Blood was immediately stimulated with AaPAL (final concentration 1 and 5 μg/ml blood; 37°C, 5% CO2, 8 h), followed by centrifugation (5000 × g, 5 min) and cytokine analysis of the supernatants (5-fold dilutions) as instructed by the array manufacturer. Blood stimulated with AaPAL storage buffer served as a reference. Aliquots of blood were taken at 0 h and 8 h and plated on supplemented blood agar plates for bacterial contamination check. The experiment was repeated once.
Unless otherwise indicated all experiments were repeated thrice. Mann-Whitney U test was used to determine the significance of the differences between the parental strain and the isogenic mutant in antibiotic susceptibilities and in the LPS amounts released into serum or broth. A P-value of less than 0.05 was regarded as statistically significant.
We thank Mrs. Elisabeth Granström for excellent technical assistance.
This work was supported by grants from the Swedish Medical Research Council No. 521-2002-6520/S.A. and 2006-06X-14747-04-3/S.A., and County Council of Västerbotten, Sweden/S.A.
- Norskov-Lauritsen N, Kilian M: Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol. 2006, 56 (Pt 9): 2135-2146. 10.1099/ijs.0.64207-0.View ArticlePubMedGoogle Scholar
- Socransky SS, Haffajee AD: The bacterial etiology of destructive periodontal disease: current concepts. J Periodontol. 1992, 63 (4 Suppl): 322-331.View ArticlePubMedGoogle Scholar
- Ihalin R, Karched M, Eneslatt K, Asikainen S: Characterization of immunoaffinity purified peptidoglycan-associated lipoprotein of Actinobacillus actinomycetemcomitans. J Chromatogr B Analyt Technol Biomed Life Sci. 2006, 831 (1-2): 116-125. 10.1016/j.jchromb.2005.11.052.View ArticlePubMedGoogle Scholar
- Paul-Satyaseela M, Karched M, Bian Z, Ihalin R, Boren T, Arnqvist A, Chen C, Asikainen S: Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein. J Med Microbiol. 2006, 55 (Pt 7): 931-942. 10.1099/jmm.0.46470-0.View ArticlePubMedGoogle Scholar
- Sturgis JN: Organisation and evolution of the tol-pal gene cluster. J Mol Microbiol Biotechnol. 2001, 3 (1): 113-122.PubMedGoogle Scholar
- Berenson CS, Murphy TF, Wrona CT, Sethi S: Outer membrane protein P6 of nontypeable Haemophilus influenzae is a potent and selective inducer of human macrophage proinflammatory cytokines. Infect Immun. 2005, 73 (5): 2728-2735. 10.1128/IAI.73.5.2728-2735.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Hellman J, Roberts JD, Tehan MM, Allaire JE, Warren HS: Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice. J Biol Chem. 2002, 277 (16): 14274-14280. 10.1074/jbc.M109696200.View ArticlePubMedGoogle Scholar
- Liang MD, Bagchi A, Warren HS, Tehan MM, Trigilio JA, Beasley-Topliffe LK, Tesini BL, Lazzaroni JC, Fenton MJ, Hellman J: Bacterial peptidoglycan-associated lipoprotein: a naturally occurring toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide. J Infect Dis. 2005, 191 (6): 939-948. 10.1086/427815.View ArticlePubMedGoogle Scholar
- Zhu X, Bagchi A, Zhao H, Kirschning CJ, Hajjar RJ, Chao W, Hellman J, Schmidt U: Toll-like receptor 2 activation by bacterial peptidoglycan-associated lipoprotein activates cardiomyocyte inflammation and contractile dysfunction. Crit Care Med. 2007, 35 (3): 886-892. 10.1097/01.CCM.0000256723.37586.A2.View ArticlePubMedGoogle Scholar
- Burnens A, Stucki U, Nicolet J, Frey J: Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni. J Clin Microbiol. 1995, 33 (11): 2826-2832.PubMed CentralPubMedGoogle Scholar
- Carbonetti NH, Artamonova GV, Andreasen C, Dudley E, Mays RM, Worthington ZE: Suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by Bordetella pertussis and identification of an immunodominant lipoprotein. Infect Immun. 2004, 72 (6): 3350-3358. 10.1128/IAI.72.6.3350-3358.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoon WS, Park SH, Park YK, Park SC, Sin JI, Kim MJ: Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein. DNA Cell Biol. 2002, 21 (2): 99-107. 10.1089/104454902753604970.View ArticlePubMedGoogle Scholar
- Murphy TF, Kirkham C, Lesse AJ: Construction of a mutant and characterization of the role of the vaccine antigen P6 in outer membrane integrity of nontypeable Haemophilus influenzae. Infect Immun. 2006, 74 (9): 5169-5176. 10.1128/IAI.00692-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Fortney KR, Young RS, Bauer ME, Katz BP, Hood AF, Munson RS, Spinola SM: Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection. Infect Immun. 2000, 68 (11): 6441-6448. 10.1128/IAI.68.11.6441-6448.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Stathopoulos C, Hendrixson DR, Thanassi DG, Hultgren SJ, St Geme JW, Curtiss R: Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story. Microbes Infect. 2000, 2 (9): 1061-1072. 10.1016/S1286-4579(00)01260-0.View ArticlePubMedGoogle Scholar
- Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D, Nelson WC, Heidelberg JF, Mekalanos JJ: Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A. 2006, 103 (5): 1528-1533. 10.1073/pnas.0510322103.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandkvist M: Type II secretion and pathogenesis. Infect Immun. 2001, 69 (6): 3523-3535. 10.1128/IAI.69.6.3523-3535.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Beveridge TJ: Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol. 1999, 181 (16): 4725-4733.PubMed CentralPubMedGoogle Scholar
- Kuehn MJ, Kesty NC: Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev. 2005, 19 (22): 2645-2655. 10.1101/gad.1299905.View ArticlePubMedGoogle Scholar
- Fujita Y, Yamaguchi K, Kamegaya T, Sato H, Semura K, Mutoh K, Kashimoto T, Ohori H, Mukai T: A novel mechanism of autolysis in Helicobacter pylori: possible involvement of peptidergic substances. Helicobacter. 2005, 10 (6): 567-576. 10.1111/j.1523-5378.2005.00364.x.View ArticlePubMedGoogle Scholar
- Aliprantis AO, Weiss DS, Radolf JD, Zychlinsky A: Release of Toll-like receptor-2-activating bacterial lipoproteins in Shigella flexneri culture supernatants. Infect Immun. 2001, 69 (10): 6248-6255. 10.1128/IAI.69.10.6248-6255.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Freudenberg MA, Meier-Dieter U, Staehelin T, Galanos C: Analysis of LPS released from Salmonella abortus equi in human serum. Microb Pathog. 1991, 10 (2): 93-104. 10.1016/0882-4010(91)90070-Q.View ArticlePubMedGoogle Scholar
- Hellman J, Zanzot EM, Loiselle PM, Amato SF, Black KM, Ge Y, Kurnick JT, Warren HS: Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria. J Infect Dis. 1997, 176 (5): 1260-1268.View ArticlePubMedGoogle Scholar
- Tesh VL, Duncan RL, Morrison DC: The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing. J Immunol. 1986, 137 (4): 1329-1335.PubMedGoogle Scholar
- Zhang H, Niesel DW, Peterson JW, Klimpel GR: Lipoprotein release by bacteria: potential factor in bacterial pathogenesis. Infect Immun. 1998, 66 (11): 5196-5201.PubMed CentralPubMedGoogle Scholar
- Ricci V, Chiozzi V, Necchi V, Oldani A, Romano M, Solcia E, Ventura U: Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity. Biochem Biophys Res Commun. 2005, 337 (1): 173-178. 10.1016/j.bbrc.2005.09.035.View ArticlePubMedGoogle Scholar
- Kaplan JB, Meyenhofer MF, Fine DH: Biofilm growth and detachment of Actinobacillus actinomycetemcomitans. J Bacteriol. 2003, 185 (4): 1399-1404. 10.1128/JB.185.4.1399-1404.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Skare JT, Champion CI, Mirzabekov TA, Shang ES, Blanco DR, Erdjument-Bromage H, Tempst P, Kagan BL, Miller JN, Lovett MA: Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi. J Bacteriol. 1996, 178 (16): 4909-4918.PubMed CentralPubMedGoogle Scholar
- Vaughan TE, Skipp PJ, O'Connor CD, Hudson MJ, Vipond R, Elmore MJ, Gorringe AR: Proteomic analysis of Neisseria lactamica and Neisseria meningitidis outer membrane vesicle vaccine antigens. Vaccine. 2006, 24 (25): 5277-5293. 10.1016/j.vaccine.2006.03.013.View ArticlePubMedGoogle Scholar
- Gophna U, Ideses D, Rosen R, Grundland A, Ron EZ: OmpA of a septicemic Escherichia coli O78--secretion and convergent evolution. Int J Med Microbiol. 2004, 294 (6): 373-381. 10.1016/j.ijmm.2004.08.004.View ArticlePubMedGoogle Scholar
- Hellman J, Loiselle PM, Tehan MM, Allaire JE, Boyle LA, Kurnick JT, Andrews DM, Sik Kim K, Warren HS: Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum. Infect Immun. 2000, 68 (5): 2566-2572. 10.1128/IAI.68.5.2566-2572.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Hellman J, Loiselle PM, Zanzot EM, Allaire JE, Tehan MM, Boyle LA, Kurnick JT, Warren HS: Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5. J Infect Dis. 2000, 181 (3): 1034-1043. 10.1086/315302.View ArticlePubMedGoogle Scholar
- Lee VT, Schneewind O: Protein secretion and the pathogenesis of bacterial infections. Genes Dev. 2001, 15 (14): 1725-1752. 10.1101/gad.896801.View ArticlePubMedGoogle Scholar
- Mayrand D, Grenier D: Biological activities of outer membrane vesicles. Can J Microbiol. 1989, 35 (6): 607-613.View ArticlePubMedGoogle Scholar
- Smalley JW, Birss AJ, Shuttleworth CA: The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50. Arch Oral Biol. 1988, 33 (5): 323-329. 10.1016/0003-9969(88)90065-9.View ArticlePubMedGoogle Scholar
- Kato S, Kowashi Y, Demuth DR: Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin. Microb Pathog. 2002, 32 (1): 1-13. 10.1006/mpat.2001.0474.View ArticlePubMedGoogle Scholar
- McBroom AJ, Johnson AP, Vemulapalli S, Kuehn MJ: Outer membrane vesicle production by Escherichia coli is independent of membrane instability. J Bacteriol. 2006, 188 (15): 5385-5392. 10.1128/JB.00498-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Renelli M, Matias V, Lo RY, Beveridge TJ: DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology. 2004, 150 (Pt 7): 2161-2169. 10.1099/mic.0.26841-0.View ArticlePubMedGoogle Scholar
- Hoekstra D, van der Laan JW, de Leij L, Witholt B: Release of outer membrane fragments from normally growing Escherichia coli. Biochim Biophys Acta. 1976, 455 (3): 889-899. 10.1016/0005-2736(76)90058-4.View ArticlePubMedGoogle Scholar
- Wai SN, Lindmark B, Soderblom T, Takade A, Westermark M, Oscarsson J, Jass J, Richter-Dahlfors A, Mizunoe Y, Uhlin BE: Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell. 2003, 115 (1): 25-35. 10.1016/S0092-8674(03)00754-2.View ArticlePubMedGoogle Scholar
- Dekker N: Outer-membrane phospholipase A: known structure, unknown biological function. Mol Microbiol. 2000, 35 (4): 711-717. 10.1046/j.1365-2958.2000.01775.x.View ArticlePubMedGoogle Scholar
- Istivan TS, Coloe PJ: Phospholipase A in Gram-negative bacteria and its role in pathogenesis. Microbiology. 2006, 152 (Pt 5): 1263-1274. 10.1099/mic.0.28609-0.View ArticlePubMedGoogle Scholar
- Sitkiewicz I, Stockbauer KE, Musser JM: Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis. Trends Microbiol. 2007, 15 (2): 63-69. 10.1016/j.tim.2006.12.003.View ArticlePubMedGoogle Scholar
- Audet A, Nantel G, Proulx P: Phospholipase A activity in growing Escherichia coli cells. Biochim Biophys Acta. 1974, 348 (3): 334-343.View ArticlePubMedGoogle Scholar
- Snijder HJ, Dijkstra BW: Bacterial phospholipase A: structure and function of an integral membrane phospholipase. Biochim Biophys Acta. 2000, 1488 (1-2): 91-101.View ArticlePubMedGoogle Scholar
- Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, Zychlinsky A: Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science. 1999, 285 (5428): 736-739. 10.1126/science.285.5428.736.View ArticlePubMedGoogle Scholar
- Araya AV, Pavez V, Perez C, Gonzalez F, Columbo A, Aguirre A, Schiattino I, Aguillon JC: Ex vivo lipopolysaccharide (LPS)-induced TNF-alpha, IL-1beta, IL-6 and PGE2 secretion in whole blood from Type 1 diabetes mellitus patients with or without aggressive periodontitis. Eur Cytokine Netw. 2003, 14 (3): 128-133.PubMedGoogle Scholar
- Frieling JT, Mulder JA, Hendriks T, Curfs JH, van der Linden CJ, Sauerwein RW: Differential induction of pro- and anti-inflammatory cytokines in whole blood by bacteria: effects of antibiotic treatment. Antimicrob Agents Chemother. 1997, 41 (7): 1439-1443.PubMed CentralPubMedGoogle Scholar
- Heinzelmann M, Bosshart H: Heparin binds to lipopolysaccharide (LPS)-binding protein, facilitates the transfer of LPS to CD14, and enhances LPS-induced activation of peripheral blood monocytes. J Immunol. 2005, 174 (4): 2280-2287.View ArticlePubMedGoogle Scholar
- Jagger MP, Huo Z, Riches PG: Inflammatory cytokine (interleukin 6 and tumour necrosis factor alpha) release in a human whole blood system in response to Streptococcus pneumoniae serotype 14 and its capsular polysaccharide. Clin Exp Immunol. 2002, 130 (3): 467-474. 10.1046/j.1365-2249.2002.01946.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Matera G, Liberto MC, Quirino A, Barreca GS, Lamberti AG, Iannone M, Mancuso E, Palma E, Cufari FA, Rotiroti D, Foca A: Bartonella quintana lipopolysaccharide effects on leukocytes, CXC chemokines and apoptosis: a study on the human whole blood and a rat model. Int Immunopharmacol. 2003, 3 (6): 853-864.View ArticlePubMedGoogle Scholar
- Mohrschladt MF, Weverling-Rijnsburger AW, de Man FH, Stoeken DJ, Sturk A, Smelt AH, Westendorp RG: Hyperlipoproteinemia affects cytokine production in whole blood samples ex vivo. The influence of lipid-lowering therapy. Atherosclerosis. 2000, 148 (2): 413-419. 10.1016/S0021-9150(99)00316-0.View ArticlePubMedGoogle Scholar
- Curfs JH, Meis JF, Hoogkamp-Korstanje JA: A primer on cytokines: sources, receptors, effects, and inducers. Clin Microbiol Rev. 1997, 10 (4): 742-780.PubMed CentralPubMedGoogle Scholar
- Boisvert WA: Modulation of atherogenesis by chemokines. Trends Cardiovasc Med. 2004, 14 (4): 161-165. 10.1016/j.tcm.2004.02.006.View ArticlePubMedGoogle Scholar
- Menten P, Wuyts A, Van Damme J: Macrophage inflammatory protein-1. Cytokine Growth Factor Rev. 2002, 13 (6): 455-481. 10.1016/S1359-6101(02)00045-X.View ArticlePubMedGoogle Scholar
- Nelson MB, Munson RS, Apicella MA, Sikkema DJ, Molleston JP, Murphy TF: Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms. Infect Immun. 1991, 59 (8): 2658-2663.PubMed CentralPubMedGoogle Scholar
- Llamas MA, Ramos JL, Rodriguez-Herva JJ: Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability. J Bacteriol. 2000, 182 (17): 4764-4772. 10.1128/JB.182.17.4764-4772.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodriguez-Herva JJ, Ramos JL: Characterization of an OprL null mutant of Pseudomonas putida. J Bacteriol. 1996, 178 (19): 5836-5840.PubMed CentralPubMedGoogle Scholar
- Cascales E, Bernadac A, Gavioli M, Lazzaroni JC, Lloubes R: Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity. J Bacteriol. 2002, 184 (3): 754-759.PubMed CentralView ArticlePubMedGoogle Scholar
- Vaara M: Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium. Antimicrob Agents Chemother. 1993, 37 (11): 2255-2260.PubMed CentralView ArticlePubMedGoogle Scholar
- Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A: The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol. 1986, 132 (5): 1297-1304.PubMedGoogle Scholar
- Adu-Bobie J, Lupetti P, Brunelli B, Granoff D, Norais N, Ferrari G, Grandi G, Rappuoli R, Pizza M: GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence. Infect Immun. 2004, 72 (4): 1914-1919. 10.1128/IAI.72.4.1914-1919.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Kaplan JB, Perry MB, MacLean LL, Furgang D, Wilson ME, Fine DH: Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f. Infect Immun. 2001, 69 (9): 5375-5384. 10.1128/IAI.69.9.5375-5384.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Saarela M, Asikainen S, Alaluusua S, Pyhala L, Lai CH, Jousimies-Somer H: Frequency and stability of mono- or poly-infection by Actinobacillus actinomycetemcomitans serotypes a, b, c, d or e. Oral Microbiol Immunol. 1992, 7 (5): 277-279. 10.1111/j.1399-302X.1992.tb00588.x.View ArticlePubMedGoogle Scholar
- Suzuki N, Nakano Y, Yoshida Y, Ikeda D, Koga T: Identification of Actinobacillus actinomycetemcomitans serotypes by multiplex PCR. J Clin Microbiol. 2001, 39 (5): 2002-2005. 10.1128/JCM.39.5.2002-2005.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Asikainen S, Chen C, Slots J: Actinobacillus actinomycetemcomitans genotypes in relation to serotypes and periodontal status. Oral Microbiol Immunol. 1995, 10 (2): 65-68. 10.1111/j.1399-302X.1995.tb00120.x.View ArticlePubMedGoogle Scholar
- Dogan B, Saarela M, Asikainen S: Genotyping of Actinobacillus actinomycetemcomitans serotype d isolates based on polymerase chain reaction. Oral Microbiol Immunol. 1999, 14 (6): 387-390. 10.1034/j.1399-302X.1999.140611.x.View ArticlePubMedGoogle Scholar
- Kouokam JC, Wai SN, Fallman M, Dobrindt U, Hacker J, Uhlin BE: Active cytotoxic necrotizing factor 1 associated with outer membrane vesicles from uropathogenic Escherichia coli. Infect Immun. 2006, 74 (4): 2022-2030. 10.1128/IAI.74.4.2022-2030.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang Y, Chen C: Mutation analysis of the flp operon in Actinobacillus actinomycetemcomitans. Gene. 2005, 351: 61-71. 10.1016/j.gene.2005.02.010.View ArticlePubMedGoogle Scholar
- Wang Y, Goodman SD, Redfield RJ, Chen C: Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans . J Bacteriol. 2002, 184 (13): 3442-3449. 10.1128/JB.184.13.3442-3449.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson TF: Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. 1951, Trans NY Acad Sci, 13: 130-134.Google Scholar
- Ihalin R: Peptidoglycan-associated lipoprotein of Actinobacillus actinomycetemcomitans induces TLR2 mediated production of proinflammatory cytokines from macrophages: September 6-9; Paris.2006Google Scholar
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