The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hasebe, A.
Right arrow Articles by Cole, B. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hasebe, A.
Right arrow Articles by Cole, B. C.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2006, 177: 4826-4832.
Copyright © 2006 by The American Association of Immunologists, Inc.

A Microbial TLR2 Agonist Imparts Macrophage-Activating Ability to Apolipoprotein A-11

Akira Hasebe, Nathan D. Pennock, Hong-Hua Mu, Fok V. Chan, Michael L. Taylor and Barry C. Cole2

Division of Rheumatology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H2O2 oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-{alpha} and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mycoplasmas, members of the Mollicutes, are cell wall-free prokaryotes with limited genomes and, hence, limited biosynthetic abilities. As a result, they parasitize the cells of their hosts, acquiring needed nutrients. They lack a rigid cell wall and instead are bound by a triple-layered membrane that maintains structural integrity by incorporating cholesterol derived from high-density lipoprotein (HDL)3 or low-density lipoprotein (LDL) (1, 2). Thus, optimal in vitro culture requires complex medium with serum added as a source of cholesterol and other nutrients. Mycoplasma arthritidis is a natural pathogen of rodents, which causes acute and chronic arthritis, lethal toxic shock, and a necrotizing fasciitis-like syndrome in genetically predisposed mice and rats (3). Virulence factors include a potent superantigen (SAg), M. arthritidis mitogen (MAM) (3), which uses TLR2 and TLR4 (4). It also possesses two distinct adhesins and a bacteriophage, all of which are thought to contribute to disease (5). Recently, cell-associated, macrophage and dendritic cell-activating, TLR2-dependent moieties were found in M. arthritidis, the most active of which had a molecular mass of 25–30 kDa (6). Subsequent work has characterized a distinct 41-kDa lipoprotein (A. Hasebe, H. Mu, L. Washburn, F. Chan, N. Pennock, M. Taylor, and B. Cole, submitted for publication) that also possesses TLR2-dependent bioactivity and that is related to the Mlp family of lipoproteins of M. arthritidis described by Washburn et al. (7).

A number of TLR2-using, biologically active lipoprotein or lipopeptide agonists have been isolated from a variety of bacterial and mycoplasmal species (8, 9, 10, 11). It has now been established that diverse TLRs not only control the innate immune response recognizing a wide variety of exogenous and endogenous agonists (9, 12), but may also control the subsequent direction of the adaptive immune response (4, 13, 14, 15). These interactions may be beneficial to the host by promoting a rapid immune response to invading organisms or may trigger an acute inflammatory reaction as seen in toxic shock syndromes (4). TLR/endogenous interactions may also lead to autoimmunity (16, 17), and chronic infections may promote asthma (18); in addition, there is increasing evidence that agonists from multiple microbial agents may be a contributing factor to the development of atherosclerosis (19, 20).

In view of our findings on multiple TLR usage by several agonists present in the arthritogenic and toxigenic M. arthritidis species (4, 6), the present work was initiated to more fully characterize these moieties. In this study, we purify and characterize the 28-kDa bioactive component described previously (6) and show, unexpectedly, that it is an apolipoprotein A-1 (apoA-1)-like molecule that acquires bioactivity by associating with mycoplasma cells. The TLR2-dependent bioactive apoA-1 could also be generated by the direct binding of bioactive lipopeptides derived from a known macrophage-activating lipoprotein to horse serum-derived apoA-1 (HS apoA-1). We propose that the apoA-1 component of HDL cholesterol binds to the bioactive lipopeptide region of a M. arthritidis surface lipoprotein, and, in so doing, acquiring bioactivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Organism and culture conditions

Toxigenic and arthritogenic M. arthritidis strain 158P10P9 was grown in modified Edward medium (6) consisting of Difco pleuropneumonia-like organisms broth base supplemented with 15% horse serum, 3% yeast extract, 0.25% L-arginine HCl, and 500 U of penicillin G. For growth in serum-free medium M. arthritidis, organisms were sequentially passed in medium containing decreasing amounts of horse serum, which was replaced with 3% BSA (Sigma-Aldrich) and 0.4% of a 250x cholesterol lipid concentrate (Invitrogen Life Technologies) similar to that described previously (1). Cultures harvested by centrifugation at 27,000 x g for 30 min were washed three times with normal saline (NS), and pellets were frozen at –70°C if not used immediately.

Mice

Female C3H/HeJ, C57BL/6, and SJL mice were purchased from The Jackson Laboratory. C57BL/6, TLR2 knockout (KO) mice originating from S. Akira (Osaka University, Suita, Japan) were provided by courtesy of T. Hawn (University of Washington School of Medicine, Seattle, WA) and were bred in our laboratory. All of the mice were bred in the Animal Resource Center of the University of Utah Health Science Center. All mice were maintained in specific pathogen-free conditions at the Animal Resource Center and were used at 8–12 wk of age. The Animal Resource Center guarantees strict compliance with regulations established by the Animal Welfare Act.

Chemicals, enzymes, and Abs

Sheep anti-human apoA-1 Ab and HRP-conjugated donkey anti-sheep IgG Ab were purchased from Novus Biologicals. Pam3-Cys-Ser-(Lys)4 3HCl (Pam3CSK4), macrophage-activating lipopeptide-2 (MALP-2), and LPS from Escherichia coli R515 were purchased from Alexis Biochemicals. LPS-free, homogenous native MAM was prepared, as described previously (21), and was stored in aliquots at –70°C. Endotoxin-free NS used for all of the experiments in this study was purchased from Baxter Healthcare, and n-octyl-beta-glucopyranoside (OG) was purchased from Sigma-Aldrich.

Protein assay and endotoxin assay

Protein concentration was determined by modified bicinchoninic acid assay for determination of lipoprotein concentration (22). Endotoxin concentration was determined by using Endospecy (Seikagaku America).

Detection of bioactivity and cytokine quantitation

Splenocytes, RAW 264.7 cells, and murine adherent peritoneal cells were cultured in RPMI 1640 medium, as described previously (4). Macrophage suspensions were prepared from pools of three to five mice. Each inducer or control was added to three cell suspensions, and individual supernatants were collected after 18 h of incubation. Serum was collected from two to three individual mice 90 min after i.v. injection of each agonist or control (6). Cytokines were determined by ELISA kits from eBioscience or BD Pharmingen. For all cytokine assays, the results are expressed as means ± SD.

Lipoprotein extraction and purification

Lipoproteins were extracted by OG, as described (23). Briefly, a 15-ml vol of cell membrane suspensions (7.5 mg of protein/ml) was treated twice with 15 ml of chloroform-methanol (2:1, v/v) at room temperature. The delipidated interphase was freed of the organic solvents in vacuo at 37°C and lyophilized to remove water. The lyophilized material was suspended in 50 mM OG in NS, treated for 6 min in boiling water, and centrifuged at 20,000 x g for 30 min. The supernatant was collected, filtrated through a 0.22-µm pore size filter, and used as OG extracts (OGex). OGex was passed over a 10% SDS-PAGE gel and stained. Another portion of the gel was blotted to a 0.45-µm cellulose membrane (Bio-Rad), cut into 2-mm strips, and each dissolved in 1 ml of DMSO, as described (24). Protein-coated particles were formed by adding sodium carbonate buffer, and then tested for bioactivity. For purification, the most active region of OGex was rerun on SDS-PAGE, the gel was stained with zinc, and the previously identified active lipoprotein band was cut out from the gel and eluted (Bio-Rad). A total of three different batches of organisms was processed similarly and assayed for activity before use in the various experiments conducted. OGex was also fractionated by reverse-phase chromatography using a 3-ml Resource 15 RPC column on a fast protein liquid chromatography system (Amersham Biosciences) with a 0–100% isopropanol gradient. Fractions were lyophilized and tested for bioactivity. The most active fraction was put on SDS-PAGE and again blotted to cellulose. Processed strips were assayed for activity, as before.

Partial amino acid sequence analysis

Purified lipoprotein was excised, and Edman sequencing was performed on an ABI Procise sequencer (Applied Biosystems) to investigate its N-terminal amino acid sequence. For mass spectrometry, lipoproteins were digested with trypsin from Promega (modified trypsin, mass spec grade). It was analyzed with an ion-trap mass spectrometer (LCQ-Decca; Thermo/Finnigan) to detect internal amino acid sequence of purified lipoprotein.

Infrared (IR) absorption analysis

The dried fractions in a KBr pellet were measured with an IR spectrometer (model IFS 88; Bruker). IR data were analyzed using established absorbance signatures of organic functional groups (25) and making comparisons of known spectra for similar or related molecules from previous publications (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Purification of the 25- to 30-kDa macrophage-activating lipoprotein from M. arthritidis

Washed M. arthritidis cells were extracted with OG, and extracts (OGex) were shown to induce TNF-{alpha} production in RAW 264.7 cells in a dose-dependent manner (Fig. 1A). SDS-PAGE of OGex was blotted to a nitrocellulose membrane, and strips were processed and assayed for TNF-{alpha} induction. Several active regions were seen the most active corresponding to a molecular mass of ~28 kDa (Fig. 1B). These results were similar to those obtained previously using TX-114 detergent extraction (6). OGex was again subjected to SDS-PAGE, and the gel was stained with zinc. The 28-kDa band was extracted and rerun on SDS-PAGE, revealing a homogeneous 28-kDa component (Fig. 1C). OGex were also run on a reverse-phase column, and eluted fractions were assayed for TNF-{alpha} induction and tested again on SDS-PAGE. Two active peaks were seen, the highest activity being in fraction 55 at very high hydrophobicity (Fig. 1D). SDS-PAGE, blotting, and assay of strips indicated that most of the activity of fraction 55 was also at the 28-kDa region (Fig. 1E), confirming the data in Fig. 1, B and C.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. Purification of a 28-kDa hydrophobic component. TNF-{alpha} production by RAW 264.7 cells was detected by ELISA (A, B, D, and E). A, Dose dependency of OGex TNF-{alpha} induction. B, Determination of bioactive regions in OGex. C, SDS-PAGE gel of molecular mass standards (lane A) and extracted 28-kDa component (lane B; 1 µg of protein) after silver staining. D, Protein profile obtained by reverse-phase chromatography of OGex. E, Determination of bioactive regions in fraction 55. The data presented in A–C are representative of three separate batches of purification for the 28-kDa component of M. arthritidis.

 
The active 28-kDa component of M. arthritidis is an apoA-1-like moiety

Edman degradation of the N-terminal amino acid sequence of the first 25 residues of the 28-kDa moiety closely matched that of dog apoA-1 precurser, but also had homology with apoA-1 molecules from other species, including human. In addition, the molecular mass of the 28-kDa moiety and apoA-1 was similar. As mycoplasmas require cholesterol for growth that is donated by LDL and HDL (1, 2), we surmised that the apoA-1 originated from the HDL complex present in the horse serum used in mycoplasma culture medium. Therefore, HS apoA-1 was extracted and purified from uninoculated mycoplasma medium in the same manner as was used for purification of the 28-kDa moiety (Materials and Methods). The N-terminal sequence of HS apoA-1 had identity with the 28-kDa mycoplasma moiety (Fig. 2A). To confirm this, tryptic digests of HS apoA-1 and the 28-kDa M. arthritidis component were subjected to mass spectrometry, and two fragments were found that were common to both (Fig. 2B). The first was identical with the N-terminal sequence, and the second was a distinct 18-residue fragment. Western blotting of the purified 28-kDa mycoplasma moiety and HS apoA-1 showed that they comigrated on the gel and both reacted with Ab to human apoA-1 (Fig. 2, C and D). Finally, M. arthritidis was grown in a semidefined medium replacing serum with protein-free cholesterol, and BSA and OGex were run on SDS-PAGE. The 28-kDa moiety was absent from serum-free grown organisms (Fig. 2E) and must therefore originate, at least in part, from serum. Importantly, the mycoplasma 28-kDa moiety, but not HS apoA-1, induced significant (p < 0.0005) TNF-{alpha} production (Fig. 3).


Figure 2
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 2. The mycoplasma 28-kDa component is related to apoA-1. A, N-terminal amino acid sequence of the 28-kDa moiety, dog apoA-1 precursor, and HS apoA-1. B, Sequences of fragments of tryptic digests of the 28-kDa moiety, dog apoA-1 precursor, and HS apoA-1. C and D, SDS-PAGE of the 28-kDa moiety or HS apoA-1 stained with Coomassie blue (C) or treated to Western blotting analysis with diaminobenzidine substrate (D). The amount of protein was 1.2 µg (lanes A and D), 0.24 µg (lanes B and E), and 0.048 µg (lanes C and F), respectively. E, SDS-PAGE of molecular mass standards (lane A), the 28-kDa moiety (lane B), OGex from M. arthritidis grown without serum (lane C) using silver stain.

 

Figure 3
View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 3. TNF-{alpha} production by RAW 264.7 cells stimulated by the 28-kDa moiety and HS apoA-1. The data shown are representative of three experiments.

 
Characterization of mycoplasma-modified apoA-1 (Mm apoA-1) and comparison with OGex lipopeptides

To gain insight as to how apoA-1 acquired bioactivity, experiments were conducted to more fully characterize the Mm apoA-1. First, we excluded the possibility that Mm apoA-1 was contaminated with other macrophage activators during preparation. LPS was not responsible, as levels were <0.0005% EU LPS/µg protein and the activity of Mm apoA-1 was not significantly (p > 0.2) inhibited with polymyxin B, in contrast to the inhibition (p < 0.0005) seen with LPS (Fig. 4A). The MAM SAg was excluded because Mm apoA-1 failed to induce IL-2 in splenocyte cultures from MAM-reactive mice (Fig. 4B), whereas MAM elicited high levels. Also, macrophages from the MAM-nonresponsive SJL mouse (3) produced high levels of TNF-{alpha} with Mm apoA-1 (Fig. 4C).


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Test for contamination with known active stimulants. RAW 264.7 cells (A), C3H/HeJ mouse splenocytes (B), and SJL peritoneal macrophages (C) were treated with stimulants or NS. Cytokines were tested by ELISA. A, Effect of polymyxin B on the activity of NS, Mm apoA-1 (50 ng/ml), or LPS (100 ng/ml). Cells were incubated with polymyxin B for 10 min at 37°C, and then they were stimulated. B, IL-2 production by splenocytes from C3H/HeJ mice stimulated with MAM, Mm apoA-1, or LPS. C, TNF-{alpha} production by Mm apoA-1 in SJL macrophages. The data shown are representative of two experiments.

 
Digestion of Mm apoA-1 with proteinase K (PK) resulted in loss of the 28-kDa band (Fig. 5A) and did not affect activity (Fig. 5B), suggesting that small lipopeptides bore the active moiety. Bioactivity was also resistant to 95°C for 10 min (data not shown). Both of these latter procedures deactivate MAM (data not shown). Exposure to H2O2, which deactivates thioester groups, inhibited the activity of Mm apoA-1 (Fig. 5C) as has been seen for MALP-2 and other microbial lipoproteins (11). Because thioesters are only present in microbial lipoproteins, this suggests that Mm apoA-1 contains a microbial moiety. Alkaline hydrolysis, which removes ester-linked lipids from the N terminus of lipoproteins (27), also reduced Mm apoA-1 activity (Fig. 5D).


Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 5. Characterization of Mm apoA-1 activity. The activity was tested by stimulating RAW 264.7 cells (B–D). A, Mm apoA-1 was treated with NS or PK (at 37°C for 2 h), heated to 95°C for 10 min, and subjected to SDS-PAGE. Molecular mass standards (lane A), Mm apoA-1 treated with NS (lane B) or PK (lane C) were stained by Coomassie blue. The faint band on lane C is PK. B, Effect of PK on the activity of Mm apoA-1 (50 ng/ml). C, Effect of H2O2 on the activity of Mm apoA-1 (50 ng/ml). D, Alkaline hydrolysis of Mm apoA-1 (25 ng/ml). Mm apoA-1 was treated with 0.2 M NaOH, neutralized with 0.1 M HCl, and then assayed for TNF-{alpha} induction. The data shown in B–D are representative of two experiments.

 
We next compared the IR spectra of HS apoA-1 and Mm apoA-1 with that of the bioactive synthetic microbial lipopeptide, Pam3CSK4, which exhibits distinct absorbances at 2900 cm–1 due to fatty acid alkyl chains, and at 1662 cm–1, indicating a microbial thioester bond (26) (Fig. 6A). Importantly, the IR spectra of Mm apoA-1 and HS apoA-1 were markedly different (Fig. 6, B and C), the latter showing a more complex pattern indicating that some significant changes had been made by culture with mycoplasmas. Also, careful alignment of apoA-1 and Pam3CSK4 profiles at 1662 cm–1 indicates that the absorbance with Mm apoA-1 was greater (OD of 0.22) than with HS apoA-1 (OD of 0.16), possibly indicating a thioester group in Mm apoA-1; this would be consistent with the loss of activity after H2O2 oxidation (Fig. 5C).


Figure 6
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 6. IR spectra of Pam3CSK4 (A), Mm apoA-1 (B), and HS apoA-1 (C). Dotted vertical lines indicate positions of potential bioactive groups.

 
In the next series of experiments, we compared the activity of PK-generated lipopeptides prepared from mycoplasmas grown in serum-free medium with the Mm apoA-1. Both induced TNF-{alpha} and IL-6 in RAW 264.7 cells in a dose-dependent manner (data not shown). In addition, lipopeptides, Mm apoA-1, and LPS all induced significant (p < 0.0005) levels of TNF-{alpha} and IL-6 in peritoneal macrophages from C57BL/6 mice, whereas only LPS induced TNF-{alpha} and IL-6 in macrophages from C57BL/6 TLR2 KO mice (Fig. 7A), indicating that the activity is dependent upon expression of TLR2, as is that for many other microbial lipoproteins (8). The TLR4-using LPS induced high levels of cytokines in wild-type and in TLR2 KO mice. We also showed that the activity of Mm apoA-1 and OGex lipopeptides as well as LPS were all significantly (p < 0.0005) inhibited by Ab to CD14, an agonist coreceptor for LPS and MAM, whereas that of the MALP-2 lipopeptide, which does not use CD14, was not significantly (p = 0.2) inhibited (Fig. 7B). Thus, Mm apoA-1 and OGex peptides activate cells by a similar pathway that is fundamentally distinct from that of MALP-2 derived from Mycoplasma fermentans. Finally, we demonstrated that Mm apoA-1 and mycoplasma peptides both induced significant levels of TNF-{alpha} (p < 0.002) and IL-12 p40 (p < 0.005), an important molecule in directing Th1 inflammatory responses, in the sera of i.v. injected C57BL/6 mice, but not in sera from TLR2 KO mice (p > 0.003) (Fig. 7C). To further confirm inflammatory cytokine production in vivo, Mm aopA-1 and lipopeptides were also injected into C3H/HeJ TLR2 KO mice. As with the C57BL/6 background mice, both agonists induced serum TNF-{alpha} and IL-12p40 in C3H/HeJ mice, but not in the TLR2 KO mice (data not shown). The combined results imply a potential inflammatory role for Mm apo-A-1 and perhaps for released mycoplasma-derived lipopeptides during the course of mycoplasma infection.


Figure 7
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 7. Involvement of TLR2 and CD14 on the activity of Mm apoA-1. A, TNF-{alpha} and IL-6 production in peritoneal macrophages from C57BL/6 and C57BL/6 TLR2 KO mice stimulated with various stimulants. All of their concentration was 50 ng/ml. B, Effect of anti-CD14 mAb on the bioactivity of Mm apoA-1 and lipopeptides on RAW 264.7 cells. The cells were preincubated with anti-CD14 mAb (10 µg/ml) for 1 h, and then stimulated with various stimulants. C, Cytokine production in sera of mice injected i.v. with various stimulants. All of their concentration was 1 µg/mouse. The data shown are representative of two experiments.

 
Bioactive lipopeptides from M. arthritidis bind to apoA-1

The observed differences between Mm apoA-1 and HS apoA-1 could be due to enzymatic changes induced by M. arthritidis due to its metabolism or uptake of cholesterol, or to acquisition of small bioactive lipopeptides of mycoplasmal origin. To test the latter, M. arthritidis was grown without serum, washed, OGex made, and digested with PK, and the peptides were tested for activity. After incubation of purified HS apoA-1 with peptides for 3 h, the mixture was run on SDS-PAGE, blotted, strips processed, and assayed for TNF-{alpha} induction. An HS apoA-1/NS mixture and a peptide/NS mixture similarly treated served as controls. As expected, HS apoA-1/NS exhibited no activity (Fig. 8A), and the activity of the peptide/NS mixture migrated with the dye front (Fig. 8B). The activity of the HS apoA-1/peptide mixture at the dye front was decreased, and significant activity (p = 0.001) was now seen in the 28-kDa region, where HS apoA-1 and Mm apoA-1 comigrate (Fig. 8C). Similar results were also obtained by using an incubation time of 6 h (data not shown). Increasing the amount of peptides in the HS apoA-1 mix by ~2-fold did not increase activity in the 28-kDa band, but did increase that at the dye front, suggesting that binding sites on HS apoA-1 were already saturated (data not shown).


Figure 8
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 8. Lipopeptides bind to HS apoA-1. A, SDS-PAGE and binding assay of HS apoA-1/NS. B, OGex lipopeptides/NS. C, HS apoA-1/OGex lipopeptides. The amounts of HS apoA-1 and OGex lipopeptides were 7 and 0.5 µg, respectively. Proteins were blotted to nitrocellulose, strips processed, and assayed for TNF-{alpha}. D–F were as for A–C, respectively, except that the peptides used were from PK digestion of purified M. arthritidis 41-kDa lipoprotein. The data shown in A–C are representative of three experiments, and in D–F of two experiments.

 
To confirm that the bioactive lipopeptides did in fact originate from M. arthritidis, the purified 41-kDa lipoprotein of M. arthritidis prepared from organisms grown without serum was PK treated to generate peptides (A. Hasebe, H. Mu, L. Washburn, F. Chan, N. Pennock, M. Taylor, and B. Cole, submitted for publication), which were then tested for their ability to bind to HS apoA-1, as performed for the OGex peptides (Fig. 8, A–C). The results show that the 41-kDa derived peptides, known to be of mycoplasmal origin, can significantly (p < 0.0005) bind to HS apoA-1, thereby imparting bioactivity (Fig. 8, D–F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown that a macrophage-activating, TLR2-dependent, 28-kDa lipoprotein purified from cells of M. arthritidis grown in standard mycoplasma serum-containing medium is a modified apoA-1-like molecule. The bioactive region of the molecule is borne on a small peptide derived by PK digestion that migrates close to the dye front on SDS-PAGE gels. A bioactive form of apoA-1 could also be produced by incubating purified HS apoA-1 with bioactive peptides derived from a known macrophage-activating lipoprotein of M. arthritidis described previously (A. Hasebe, H. Mu, L. Washburn, F. Chan, N. Pennock, M. Taylor, and B. Cole, submitted for publication). In the latter case, the lipoprotein, related to MlpD (7), was isolated from organisms that were grown in a serum- and apoA-1-free modified culture medium. We propose that the bioactive form of apoA-1 obtained from whole mycoplasmas was due to the binding of this molecule to the bioactive region of a bioactive mycoplasma surface lipoprotein.

Members of the genus Mycoplasma (Mollicutes) are the smallest self-replicating organisms known, and, as a result of a limited genome, lack a cell wall and are highly fastidious. In the absence of a rigid cell wall, mycoplasmas possess a triple-layered membrane, the structural integrity of which is maintained by incorporation of cholesterol derived from their animal hosts or from serum added to culture medium. We first considered that serum apoA-1 may have cosedimented with organisms and that the Mm apoA-1 comigrated with a biologically active component of M. arthritidis of similar molecular mass (3), i.e., the MAM SAg. This was unlikely, however, because an OGex mock preparation of apoA-1 prepared from uninoculated mycoplasma medium failed to activate macrophages (data not shown). Also, we excluded contamination with LPS and the MAM SAg as being responsible for activity. Thus, our results suggest that there is a physical association of mycoplasma organisms with HS apoA-1, although the mechanisms involved are not clear. Our subsequent observation, that peptides derived from M. arthritidis grown without serum and peptides from the purified 41-kDa lipoprotein of M. arthritidis avidly bind to purified HS apoA-1, provides a means whereby apoA-1 can in fact acquire inflammatory properties.

A comparison of Mm apoA-1 and M. arthritidis lipopeptides indicated that both induced TNF-{alpha} and IL-6 in RAW 264.7 cells as well as in peritoneal macrophages from C57BL/6 mice, and, in addition, TNF-{alpha} and IL-12p40 were induced in the sera of C57BL/6 mice. The activity of both was also resistant to PK treatment, but was destroyed by H2O2 oxidation and alkaline hydrolysis and was TLR2 dependent. The activity of Mm apoA-1 and the M. arthritidis lipopeptides was dependent upon CD14, a coreceptor for LPS (28) and for some other bioactive bacterial-associated lipopeptides (29), but was distinct from the MALP-2-derived lipopeptide from M. fermentans (6).

Bioactive peptides may also be naturally cleaved from growing mycoplasmas either in culture medium or possibly in vivo into plasma. Precedence for this is that a truncated MALP-404 lipoprotein is cleaved from M. fermentans, but in this case the bioactive MALP-2-like N-terminal lipopeptide remains cell associated (30). In Fig. 1B, there was evidence of a small band of activity that migrated with the dye front. Also, OGex of mycoplasma-free culture supernatants of M. arthritidis had some bioactivity that also migrated to the dye front. Thus, these active lipopeptides may exist in the free state, bound to HS apoA-1, or remain as lipoproteins on the mycoplasma membrane. Although it is not entirely clear how bioactive apoA-1 becomes associated with M. arthritidis cells, we propose that the bioactive lipid portion of a surface mycoplasma lipoprotein, such as the 41-kDa MlpD or related moiety (7), acts as the receptor for free apoA-1 or HDL-bound apoA-1. This process may in fact aid the mycoplasmas in acquisition of cholesterol, possibly by cleavage of HDL by mycoplasmal cholesterol esterases (31). In this regard, the presence of a protease-sensitive receptor for serum lipoproteins on the surface of the mycoplasmas described earlier might have relevance because this receptor was not present on the surface of the related, but sterol nonrequiring acholeplasmas (2). It remains to be determined whether our observations are specific for M. arthritidis, specific for cholesterol-requiring mycoplasmas, or whether shed bioactive lipopeptides from other organisms can also bind to apoA-1. Another implication of our observation concerns the potential role of bioactive apoA-1 as an inflammatory mediator in vivo.

Chronic inflammation due to microbial infection within the arterial vessel walls is receiving increasing attention as one etiological factor in atherosclerosis (16, 17). New epidemiological studies have implicated Helicobacter pylori (32), Chlamydia pneumoniae (33, 34), Mycoplasma pneumoniae (35, 36), and a number of oral bacteria such as Porphymonas gingivalis and Bacteroides forsythus (37, 38) as contributors to atherosclerosis. Experimental models using these organisms, such as P. gingivalis (39), show that they can indeed induce atherosclerotic inflammation, although the mechanism(s) by which they might promote this disease is not clear. It has been proposed recently that innate recognition by TLR2 in conjunction with either TLR1 or TLR6 of a wide range of microbial agonists is a potential inflammatory pathway whereby microbial moieties might be involved (17, 40). In fact, Mullick et al. (19) recently demonstrated that the systemic administration of the synthetic TLR2-using Pam3CSK4 lipopeptide could greatly enhance atherosclerosis in a mouse model using the LDL receptor KO–/– mouse, and that disease was ablated when TLR2 was eliminated through use of Ldl–/–, TLR2–/– mice.

An additional mechanism has been suggested, one that directly involves the HDL complex. ApoA-1, the major protein component of HDL, has traditionally been considered to be protective because its function as a reverse cholesterol transporter can scavenge unwanted lipids to the liver for removal (41). However, there is evidence that HDL structure and function change during the acute-phase response to irritants (42) or to infection (43), as seen by decreased levels of plasma HDL, a decreased ability of HDL to counteract LDL-induced inflammation, as well as the acquisition of inflammatory properties by HDL itself (42). This molecule has also been shown to become inflammatory during development of atherosclerosis, and the administration of an apoA-1 mimetic peptide to a murine model of atherosclerosis can inhibit disease, suggesting a key role for apoA-1 in inflammation (43). Our observation on the binding of bioactive mycoplasmal peptides to mammalian apoA-1, thus rendering it inflammatory, suggests a novel mechanism whereby infectious agents might contribute to the genesis of atherosclerotic disease. Work to assess the in vivo consequences of our observations on the binding of bioactive microbial peptides to apoA-1 using the well-defined M. arthritidis models of inflammatory disease is currently underway.


   Acknowledgments
 
Amino acid sequence analysis and IR spectrometry were done by the University of Utah Health Sciences Center Core Research Facilities. We thank Dr. Peter Muhlradt for insightful discussions and Dr. Shmuel Razin for critical review. We also thank Chenhong Lee for excellent technical assistance. C57BL/6 TLR2 KO mice were received, with the permission of Dr. Shizuo Akira, courtesy of Dr. Thomas R. Hawn.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Nora Eccles Treadwell Foundation and by Grant AR 02255 from the National Institute of Arthritis and Metabolic Diseases. B.C.C. is the holder of the Nora Eccles Harrison Presidential Endowed Chair in Rheumatology. Back

2 Address correspondence and reprint requests to Dr. Barry C. Cole, Division of Rheumatology, Department of Internal Medicine, University of Utah School of Medicine, 30 North 1900 East, Salt Lake City, UT 84132. E-mail address: Barry.Cole{at}hsc.utah.edu Back

3 Abbreviations used in this paper: HDL, high-density lipoprotein; apoA-1, apolipoprotein A-1; HS apoA-1, horse serum-derived apoA-1; IR, infrared; KO, knockout; LDL, low-density lipoprotein; MALP-2, macrophage-activating lipopeptide-2; MAM, M. arthritidis mitogen; Mm apoA-1, mycoplasma-modified apoA-1; NS, normal saline; OG, n-octyl-beta-glucopyranoside; OGex, OG extract; Pam3CSK4, Pam3-Cys-Ser-(Lys)4 3HCl; PK, proteinase K; SAg, superantigen. Back

Received for publication March 23, 2006. Accepted for publication July 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Washburn, L. R., N. L. Somerson. 1979. Lipoproteins as substitutes for serum in mycoplasma culture medium. J. Clin. Microbiol. 10: 586-589. [Abstract/Free Full Text]
  2. Razin, S., H. Efrati, S. Kutner, S. Rottem. 1982. Cholesterol and phospholipid uptake by mycoplasmas. Rev. Infect. Dis. 4: S85-S92. [Medline]
  3. Cole, B. C., A. Sawitzke, H. H. Mu. 2000. Mycoplasma arthritidis and its superantigen M. arthritidis mitogen as a model for inflammatory and autoimmune disease. M. W. Cunningham, and R. S. Fujinami, eds. Effects of Microbes on the Immune System 93-107. Lippincott, Williams and Wilkins, Philadelphia.
  4. Mu, H. H., N. D. Pennock, J. Humphreys, C. J. Kirschning, B. C. Cole. 2005. Engagement of Toll-like receptors by mycoplasmal superantigen: down-regulation of TLR2 by MAM/TLR4 interaction. Cell Microbiol. 7: 789-797. [Medline]
  5. Washburn, L. R., B. C. Cole. 2002. Mycoplasma arthritidis pathogenicity: membrane, MAM and MAV1. R. Herrmann, and S. Razin, eds. Molecular Biology and Pathogenicity of Mycoplamas 473-489. Kluver Acdemic/Plenum Publishers, New York.
  6. Cole, B. C., H. H. Mu, N. D. Pennock, A. Hasebe, F. V. Chan, L. R. Washburn, M. R. Peltier. 2005. Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2. Infect. Immun. 73: 6039-6047. [Abstract/Free Full Text]
  7. Washburn, L. R., E. J. Miller, S. Mukherjee, D. Dannenbring. 2004. Mycoplasma arthritidis bacteriophage MAV1 prophage integration, deletions, and strain-related polymorphisms. Plasmid 52: 31-47. [Medline]
  8. Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg, J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf, D. T. Golenbock. 1999. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J. Biol. Chem. 274: 33419-33425. [Abstract/Free Full Text]
  9. Medzhitov, R., C. Janeway, Jr. 2000. The Toll receptor family and microbial recognition. Trends Microbiol. 8: 452-456. [Medline]
  10. Takeuchi, O., A. Kaufmann, K. Grote, T. Kawai, K. Hoshino, M. Morr, P. F. Muhlradt, S. Akira. 2000. Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a Toll-like receptor 2- and MyD88-dependent signaling pathway. J. Immunol. 164: 554-557. [Abstract/Free Full Text]
  11. Morr, M., O. Takeuchi, S. Akira, M. M. Simon, P. F. Muhlradt. 2002. Differential recognition of structural details of bacterial lipopeptides by Toll-like receptors. Eur. J. Immunol. 32: 3337-3347. [Medline]
  12. Akira, S.. 2001. Toll-like receptors and innate immunity. Adv. Immunol. 78: 1-56. [Medline]
  13. Janeway, C. A., Jr, R. Medzhitov. 1998. Introduction: the role of innate immunity in the adaptive immune response. Semin. Immunol. 10: 349-350. [Medline]
  14. Pasare, C., R. Medzhitov. 2004. Toll-like receptors and acquired immunity. Semin. Immunol. 16: 23-26. [Medline]
  15. Iwasaki, A., R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987-995. [Medline]
  16. Mandal, K., M. Jahangiri, Q. Xu. 2004. Autoimmunity to heat shock proteins in atherosclerosis. Autoimmun. Rev. 3: 31-37. [Medline]
  17. Ludewig, B., P. Krebs, E. Scandella. 2004. Immunopathogenesis of atherosclerosis. J. Leukocyte Biol. 76: 300-306. [Abstract/Free Full Text]
  18. Redecke, V., H. Hacker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, E. Raz. 2004. Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J. Immunol. 172: 2739-2743. [Abstract/Free Full Text]
  19. Mullick, A. E., P. S. Tobias, L. K. Curtiss. 2005. Modulation of atherosclerosis in mice by Toll-like receptor 2. J. Clin. Invest. 115: 3149-3156. [Medline]
  20. Kol, A., P. Libby. 1998. The mechanisms by which infectious agents may contribute to atherosclerosis and its clinical manifestations. Trends Cardiovasc. Med. 8: 191-199. [Medline]
  21. Atkin, C. L., S. Wei, B. C. Cole. 1994. The Mycoplasma arthritidis superantigen MAM: purification and identification of an active peptide. Infect. Immun. 62: 5367-5375. [Abstract/Free Full Text]
  22. Morton, R. E., T. A. Evans. 1992. Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content. Anal. Biochem. 204: 332-334. [Medline]
  23. Shibata, K., A. Hasebe, T. Into, M. Yamada, T. Watanabe. 2000. The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts. J. Immunol. 165: 6538-6544. [Abstract/Free Full Text]
  24. Wallis, R. S., M. Amir-Tahmasseb, J. J. Ellner. 1990. Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte Western blot. Proc. Natl. Acad. Sci. USA 87: 3348-3352. [Abstract/Free Full Text]
  25. Solomons, T. W., C. B. Fryhle. 2000. Organic Chemistry 7th Ed. John Wiley & Sons, Indianapolis.
  26. Hasebe, A., A. Yoshimura, T. Into, H. Kataoka, S. Tanaka, S. Arakawa, H. Ishikura, D. T. Golenbock, T. Sugaya, N. Tsuchida, et al 2004. Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease. Infect. Immun. 72: 1318-1325. [Abstract/Free Full Text]
  27. Hantke, K., V. Braun. 1973. Covalent binding of lipid to protein: diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur. J. Biochem. 34: 284-296. [Medline]
  28. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. V. Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, et al 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088. [Abstract/Free Full Text]
  29. Sellati, T. J., D. A. Bouis, R. L. Kitchens, R. P. Darveau, J. Pugin, R. J. Ulevitch, S. C. Gangloff, S. M. Goyert, M. V. Norgard, J. D. Radolf. 1998. Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide. J. Immunol. 160: 5455-5564. [Abstract/Free Full Text]
  30. Davis, K. L., K. S. Wise. 2002. Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans. Infect. Immun. 70: 1129-1135. [Abstract/Free Full Text]
  31. Smith, P. F.. 1959. Cholesterol esterase activity of pleuropneumonialike organisms. J. Bacteriol. 77: 682-689. [Free Full Text]
  32. Oshima, T., R. Ozono, Y. Yano, Y. Oishi, H. Teragawa, Y. Higashi, M. Yoshizumi, M. Kambe. 2005. Association of Helicobacter pylori infection with systemic inflammation and endothelial dysfunction in healthy male subjects. J. Am. Coll. Cardiol. 45: 1219-1222. [Abstract/Free Full Text]
  33. Kuo, C. C., A. M. Gown, E. P. Benditt, J. T. Grayston. 1993. Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain. Arterioscler. Thromb. 13: 1501-1504. [Abstract]
  34. Arcari, C. M., C. A. Gaydos, F. J. Nieto, M. Krauss, K. E. Nelson. 2005. Association between Chlamydia pneumoniae and acute myocardial infarction in young men in the United States military: the importance of timing of exposure measurement. Clin. Infect. Dis. 40: 1123-1130. [Medline]
  35. Ngeh, J., S. Gupta, C. Goodbourn, G. McElligott. 2004. Mycoplasma pneumoniae in elderly patients with stroke: a case-control study on the seroprevalence of M. pneumoniae in elderly patients with acute cerebrovascular disease: the M-PEPS Study. Cerebrovasc. Dis. 17: 314-319. [Medline]
  36. Ngeh, J., C. Goodbourn. 2005. Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila in elderly patients with stroke (C-PEPS, M-PEPS, L-PEPS): a case-control study on the infectious burden of atypical respiratory pathogens in elderly patients with acute cerebrovascular disease. Stroke 36: 259-265. [Abstract/Free Full Text]
  37. Haraszthy, V. I., J. J. Zambon, M. Trevisan, M. Zeid, R. J. Genco. 2000. Identification of periodontal pathogens in atheromatous plaques. J. Periodontol. 71: 1554-1560. [Medline]
  38. Desvarieux, M., R. T. Demmer, T. Rundek, B. Boden-Albala, D. R. Jacobs, Jr, R. L. Sacco, P. N. Papapanou. 2005. Periodontal microbiota and carotid intima-media thickness: the Oral Infections and Vascular Disease Epidemiology Study (INVEST). Circulation 111: 576-582. [Abstract/Free Full Text]
  39. Gibson, F. C., III, H. Yumoto, Y. Takahashi, H. H. Chou, C. A. Genco. 2006. Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis. J. Dent. Res. 85: 106-121. [Abstract/Free Full Text]
  40. Laberge, M. A., K. J. Moore, M. W. Freeman. 2005. Atherosclerosis and innate immune signaling. Ann. Med. 37: 130-140. [Medline]
  41. Frank, P. G., Y. L. Marcel. 2000. Apolipoprotein A-I: structure-function relationships. J. Lipid Res. 41: 853-872. [Abstract/Free Full Text]
  42. Van Lenten, B. J., S. Y. Hama, F. C. de Beer, D. M. Stafforini, T. M. McIntyre, S. M. Prescott, B. N. La Du, A. M. Fogelman, M. Navab. 1995. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response: loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J. Clin. Invest. 96: 2758-2767. [Medline]
  43. Navab, M., G. M. Anantharamaiah, A. M. Fogelman. 2005. The role of high-density lipoprotein in inflammation. Trends Cardiovasc. Med. 15: 158-161. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Q. Zhou and S. Amar
Identification of Signaling Pathways in Macrophage Exposed to Porphyromonas gingivalis or to Its Purified Cell Wall Components
J. Immunol., December 1, 2007; 179(11): 7777 - 7790.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
L. M Agius
Complicated atheromatous plaque as integral atherogenesis
J. Clin. Pathol., June 1, 2007; 60(6): 589 - 592.
[Full Text] [PDF]

Home page
 Infect. Immun.Home page
A. Hasebe, H.-H. Mu, L. R. Washburn, F. V. Chan, N. D. Pennock, M. L. Taylor, and B. C. Cole
Inflammatory Lipoproteins Purified from a Toxigenic and Arthritogenic Strain of Mycoplasma arthritidis Are Dependent on Toll-Like Receptor 2 and CD14
Infect. Immun., April 1, 2007; 75(4): 1820 - 1826.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hasebe, A.
Right arrow Articles by Cole, B. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hasebe, A.
Right arrow Articles by Cole, B. C.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS