Published online before print December 3, 2007
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* Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany;
Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany;
Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany; and
EMC microcollections GmbH, Tübingen, Germany
1Correspondence: Cellular Immunology and Cell Biology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. E-mail: ajulmer{at}fz-borstel.de
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Key Words: gene expression • macrophages • microarray • signal transduction • receptor-ligand interaction
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and AP1, which in turn, leads to the production of proinflammatory cytokines such as TNF-
, IL-6, IL-1β, and IL-12 [7
, 8
]. For most of the TLR, except TLR10, TLR12, and TLR13, specific, natural ligands have been identified, including bacterial lipopeptides (LP), lipoteichoic acid, LPS, and oligonucleotides such as CpG-rich DNA, small RNA, as well as ssRNA [9 ]. The receptor for a functional recognition of bacterial LP is TLR2 [10 11 12 ]. Beside the expression on cells of the innate immune system, this receptor could also be detected on B and T cells [10 , 13 14 15 ]. Furthermore, TLR2 appears to be up-regulated on mononuclear cells during disorders such as chronic obstructive pulmonary disease [16 ], influenza virus infections [17 ], and sepsis [18 ]. Unlike other TLR, which are functionally active as homomers, TLR2 has evolutionarily developed its unique ability to form heteromers with TLR1 or TLR6 to attain specificity for the diverse LP repertoire [19 20 21 22 ]. Unlike other TLR, which are functionally active as homomers, TLR2 has evolutionary developed its unique ability to form heteromers with TLR1 or TLR6 to attain specificity for the diverse LP repertoire [19 20 21 22 ]. Comparison of the amino acid sequence reveals that TLR2, TLR1, and TLR6 form a TLR subfamily, which presumably diverged from one common ancestral gene. In humans, TLR10 is also a member of this TLR2 subfamily. Among all TLR, TLR1 and TLR6 have the highest identity of overall amino acid sequence, which is 66%, and a similar genomic structure. In addition, they are located in tandem on the same chromosome. Therefore, it is assumed that they are the evolutionary products of gene duplication [9 , 23 ]. However, the relevance for the innate immune response remains vague.
Bacterial LP are the most ubiquitous bacterial modulins synthesized by Gram-negative and Gram-positive bacteria as well as by cell wall-less bacteria such as mycoplasma. They are composed of di-O-acylated-S-(2,3-dihydroxypropyl)-cysteinyl residues, which are coupled to the N terminus of distinct polypeptides. The S-(2,3-dihydroxypropyl)-cysteine can be N-acylated with a third fatty acid, as it is the case in Gram-negative bacteria such as Escherichia coli, whose LP was the first to be sequenced and characterized by Braun [24 ]. The acylation pattern depends on the existence of an N-acyl-transferase, which is mostly absent in Gram-positive bacteria and mycoplasma to result in the production of only diacylated LP. Gram-negative bacteria, on the other hand, synthesize mainly triacylated LP [25 ].
Using a synthetic LP collection and cells from TLR1-, TLR2-, and TLR6-deficient mice, it was possible to decipher many structural requirements that are necessary to determine the TLR dependency of LP [26 , 27 ]. It has been shown that according to the length and arrangement of the fatty acids, as well as the assembly of the polypeptide tail, LP signal through TLR2 in a TLR1- or TLR6-dependent manner. However, we and others [26, 27] could also demonstrate that there are di- and triacylated LP, which signal even in TLR1-deficient murine cells and TLR6-deficient murine cells or in human epithelial cell lines transfected with TLR2/TLR1 or TLR2/TLR6 [28 ]. These findings indicate that there are LP that can signal in a TLR1- and TLR6-independent manner or alternatively, through TLR2/TLR1 and TLR2/TLR6 heteromers.
In this paper, we have now addressed the question of whether the existence of these different TLR2 dimers represents an evolutionary mechanism, which leads to a broad spectrum for the huge structure variety of LP or enables the innate immune system to respond to different invading pathogens by the activation of different signal transduction pathways. We made use of the synthetic LP, palmitic acid (Pam)octanoic acid (Oct)2C-(VPGVG)4VPGKG (TLR2/TLR1-dependent), fibroblast-stimulating LP-1 (FSL-1; Pam2C-GDPKHPKSF, TLR2/TLR6-dependent), and Pam2C-SK4 (active in TLR1-deficient murine cells and in TLR6-deficient murine cells), to specifically activate their signal transduction pathways (see Fig. 1 ). Therefore, we investigated the downstream events at different states of the signaling cascade. Dominant-negative (DN) plasmids of several adaptor molecules as well as analysis of MAPK revealed that all TLR2 dimers activate downstream effects using the same signaling molecules. Based on the assumption that activation of different signaling pathways should lead to different gene expression profiles, we used microarray analysis as a powerful tool to investigate the complex, transcriptional events in response to the various LP. With this approach, we furthermore demonstrated that the pattern of gene expression induced by the different LP is identical, resulting in the conclusion that the same signaling pathways are initiated. Therefore, it is reasonable to conclude that the heterodimerization of TLR2 with TLR1 and TLR6 is an evolutionary mechanism to broaden the ligand spectrum for this TLR.
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Figure 1. Denotation, TLR dependency, and structure of the three synthetic LP used in this study. All LP are strongly TLR2-dependent.
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Cell lines and cell culture
HEK293 cells (a HEK cell line, ECACC) were cultured in DMEM supplemented with 10% FCS, 1% PS, and 1% LG. Cells were seeded at 0.5 x 106 and 0.3 x 106 cells/10 ml in 75 cm3 flasks and cultured at 37°C, 5% CO2, for 3 or 4 days, respectively.
Transient transfection and stimulation of HEK293 cells
HEK293 cells were seeded at 0.2 x 105 cells/150 µl in 96-well culture dishes and transfected after 24 h using TransPEI transfectant reagent according to the provider’s instructions (Eurogentec, Seraing, Belgium). Cells were stimulated for 24 h with 100 nM LP, and supernatants were analyzed for IL-8 production using commercial ELISA (Biosource, Camarillo, CA, USA).
Preparation of murine bone marrow-derived macrophages (BMDM) and spleen cells
TLR1- and TLR6-deficient mice and the corresponding wild-type siblings were F2-interbred from the 129/C57BL/6 F1 strain and had been generated by gene targeting as described previously [20
, 21
]. Mice at the age of 8–12 weeks were killed after CO2 anesthesia, and cell suspensions were prepared from spleen. BM cells were isolated from the femur through rinsing with warm DMEM medium containing 10% FCS, 1% LG, 0.5% PS, 1% Hepes, and 1% sodium pyruvate. Collected cells were separated and subsequently differentiated into macrophages through incubation with 100 ng/ml human M-CSF for 7 days in DMEM. Spleen was isolated, and cells were cultured as described [29
].
All animal experiments were approved by the Ministerium für Umwelt, Naturschutz und Landwirtschaft, Schleswig-Holstein (Germany).
Stimulation of BMDM and spleen cells
BMDM were seeded at 2.5 x 105 cells/400 µl DMEM in 48-well culture dishes and stimulated after 2 h for the indicated time-points with 100 nM LP and 10 ng/ml LPS. Isolated spleen cells were stimulated at 4 x 105/150 µl DMEM with 1, 10, and 100 nM LP for 24 h and pulsed with (3H)thymidine [(3H)TdR], 0.9 µCi/ml, for another 24 h of culture. Cells were harvested and counted in a liquid scintillation spectrometer. The results are expressed in cpm per culture [29
].
RNA isolation and cDNA synthesis
RNA was obtained using the RNeasy Mini kit, as recommended by the manufacturer (Qiagen, Hilden, Germany). DNase digestion was performed on column (RNase-Free DNase set, Qiagen). Total RNA (1 µg) was taken for cDNA synthesis with the SuperScript III RT system for subsequent real-time PCR.
Real-time PCR analysis
Quantitative real-time PCR was performed with the Light Cycler 2.0 system (Roche Diagnostics, Switzerland) using LightCycler® Fast Start DNA MasterPLUS, according to the manufacturer’s instructions. Relative cDNA amounts of Adora2a, Arf3, Cnr, CXCR4, GPCR155, IL-6, Irf2, Lipocalin2, Ptgs2, Rab31, Sec61a1, and TNF were calculated compared with the expression of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT; for primer, see Supplemental data, Table S1).
Immunoblotting
Generation of cell lysates, SDS-PAGE, and immunoblotting were performed as described in our previous study [26
].
Affymetrix gene chip analysis and TreeView clustering
Murine BMDM were stimulated with 100 nM LP for 2 h and 6 h. Control samples were treated only with medium, and gene chip analysis was performed for each experiment. Total RNA (3 µg) was processed and hybridized to the murine expression array MOE430 2.0, according to the manufacturer’s protocols (Affymetrix, Santa Clara, CA, USA). Microarrays were scanned and initially analyzed using Affymetrix GCOS software. CEL files were processed for global normalization and generation of expression values using the robust multiarray analysis algorithm implemented in the R affy package (www.bioconductor.org [30
]). Changes in gene expression between conditions were analyzed on the probe data from 11 oligos per probe set using the s-score test. Scatterplot analysis of total gene expression of control cells versus stimulated cells was generated with SigmaPlot 9.0. For comparing the experimental groups (unstimulated vs. stimulated), the threshold level was set at threefold differences. Fold induction values were adjusted and filtered with the Cluster program. Average linkage clustering was performed and visualized using TreeView 2.0 [31
].
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Figure 2. FSL-1, Pam2C-SK4, and PamOct2C-(VPGVG)4VPGKG show specific TLR dependencies in murine spleen cells, and spleen cells from wild-type or TLR1- or TLR6-deficient mice were stimulated with different concentrations of LP for 24 h. (3H)TdR incorporation into B-lymphocytes was measured after another 24 h of incubation. The experiment shown is representative for five independent experiments.
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Figure 3. LP signaling was diminished by transfecting HEK293 cells with DN forms of signal transducing molecules. HEK293 cells were transiently transfected each with 100 ng murine TLR1, TLR2, and TLR6 construct, alone or together with 800 ng DN IRAK1, DN MyD88, DN TRAF6, and DN IRAK2. Cells were stimulated 24 h with 10 ng/ml TNF- and 100 nM FSL-1, Pam2C-SK4, or PamOct2C-(VPGVG)4VPGKG, and IL-8 release was measured by ELISA. One hundred percent is equivalent to IL-8 production of cells transfected only with TLR and control plasmid pcDNA3. The experiments shown are representative for three independent experiments. Pam2C-, Pam2C-SK4; PO2C-, PamOct2C-(VPGVG)4VPGKG.
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B was determined. Murine BMDM were stimulated with 100 nM of the different LP analogs for 5–90 min and subsequently, analyzed for phosphorylation of the MAPK, p38, Erk, and JNK and degradation of the regulatory protein IB.
In the first 5–15 min after stimulation, phosphorylation of all kinases could be detected. In this time-frame, p38, Erk, and JNK were phosphorylated, and IB could no longer be detected as a result of IB kinase-dependent degradation (Fig. 4
). After 30 min, IB recovered, and all MAPK converted into the unphosphorylated state upon stimulation with all three LP.
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Figure 4. Phosphorylation of MAPK and degradation of IB in murine BMDM upon stimulation with FSL-1, Pam2C-SK4, and PamOct2C-(VPGVG)4VPGKG. BMDM from wild-type mice were stimulated with 100 nM LP for the indicated times. Cell lysates were subjected to SDS-PAGE and immunoblotted for phosphorylated p38 (pp38; 43 kDa), Erk (pErk; 42 kDa), and JNK (pJNK; 47 kDa), as well as for IB (41 kDa). p38 (43 kDa) served as loading control. The experiment shown is representative for three independent experiments.
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Figure 5. Scatterplot analyses show the gene expression pattern induced in murine BMDM after stimulation with various LP. (A) Murine BMDM were stimulated with 100 nM FSL-1, Pam2C-SK4, and PamOct2C-(VPGVG)4VPGKG for 2 h (upper panel) and 6 h (lower panel). mRNA was isolated and processed for hybridization to Affymetrix MG 430 2.0 microarray. Gene expression values of untreated control cells are plotted against expression values of stimulated cells using Sigma Plot 9.0. Each dot represents the normalized signal intensity of a single probe set. (B) Gene expression values of stimulated cells are plotted against each other. Differently modulated genes spread from the diagonal, where gene values and expression behave similar.
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Figure 6. Verification of array results via real-time PCR. BMDM were stimulated with 100 nM LP, and mRNA was isolated after 2 h, 4 h, and 6 h. Expression levels of various up- and down-regulated genes were analyzed by normalization against HPRT. The experiment shown is representative for three independent experiments.
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500 probe sets (Table 1
). Roughly two-thirds of these modulated probe sets were up-regulated. The fold induction values indicate that PamOct2C-(VPGVG)4VPGKG down-regulates more probe sets, namely 209, compared with FSL-1 and Pam2C-SK4 with 91 and 94 regulated probe sets, respectively. After 6 h, 1550 probe sets were found to be regulated. At this time-point, Pam2C-SK4 repressed 1012, FSL-1 831, and the threefold acylated LP 763 probe sets, and expression of 700 probe sets was up-regulated. Differences in the amount of modulated probe sets induced by the three stimuli may arise from fluctuations of the fold-induction values around the threshold of threefold (e.g., 2.9-fold vs. 3.1-fold). For our extended examinations, we therefore selected only such probe sets that showed a substantial different regulation, namely probe sets that were found to be threefold or more differently up- or down-regulated between the different LP. |
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Table 1. Gene Regulation upon Stimulation with Various LPa
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subunit 1 of the Sec61 complex, which is an essential translocation component of the endoplasmatic reticulum, there are six different probe sets targeting different regions of the gene. Four probe sets show no regulation of Sec61a1 mRNA, whereas two sets show a more than threefold different regulation upon stimulation with PamOct2C-(VPGVG)4VPGKG compared with FSL-1 or Pam2C-SK4. Using specific primer against the probe sets that behave differently, real-time PCR showed that in these cases, the expression is indeed never modulated, and there are no differences in the regulation of these genes after stimulation with the three LP. Table S1 (Supplementary data) shows the real-time PCR results of 20 of such genes. For all of them, we could not validate the differences obtained for one probe set by microarray analysis. |
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Table 2. Different Probe Sets of One Single Gene Show Controversial Resultsa
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The dendrogram shows that all LP were clustered strongly together, showing a high similarity between the stimuli and their induced gene regulations (Fig. 7 ; Supplemental data, Fig. S1). However, array data of single probe sets indicate that 6 h of stimulation with FSL-1 or Pam2C-SK4 leads to a stronger down-regulation compared with PamOct2C-(VPGVG)4VPGKG. Nevertheless, these values next to never exceeded a two- or even threefold different regulation and were mostly found in cases of a strong decrease of mRNA to background levels. Fluctuations in these low background ranges produce differences that are biologically not relevant in comparison with the high control value. Hence, microarray data, cluster analysis, and real-time PCR show that we could find an equal regulation of various genes after stimulation with the three LP.
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Figure 7. Hierarchical clustering of genes that are potently regulated upon LP stimulation. TreeView illustrates fold-induction values of 20 of the strongest up (red)- and down (green)-regulated genes arranged for 2 h and 6 h of stimulation. Data represent relative fluorescence intensity of the given probe sets.
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The TLR2 coreceptors TLR1 and TLR6 have a high identity of overall amino acid sequence of 66%. Although they share even 90% identity within the TIR domain, the possibility for the induction of different signal transduction pathways cannot be excluded. In addition, the region that is responsible for the ligand recognition shows 67% divergence, which may be responsible for different conformational changes and discrete interactions between TIR domains of receptor and adaptor molecules after binding of different ligands to the TLR ectodomain. In this context, a previous study using mutant TIR domains of TLR2 indicates that the BB-loop is intrinsically flexible [33 ]. Therefore, one may assume a combination of the receptor with various known (MyD88, Mal) as well as unknown adaptor molecules [34 ].
To address this query, we used three different synthetic LP. PamOct2C-(VPGVG)4VPGKG is a triacylated LP, which we recently have found to be strictly TLR2/TLR1-dependent [26 ] (Figs. 1 and 2) . FSL-1 is a TLR2/TLR6-dependent synthetic analog derived from the LP of Mycoplasma salivarium [35 , 36 ] (Figs. 1 and 2) . Pam2C-SK4 has been described to signal through human TLR2/TLR1 and human TLR2/TLR6 in transfected HEK293 cells as well as in TLR1-deficient murine cells and in TLR6-deficient murine cells [26 27 28 ] (Figs. 1 and 2) . Pam2C-SK4 has been described to signal through TLR2/TLR1 and TLR2/TLR6 in transfected HEK293 cells as well as in TLR1-deficient murine cells and in TLR6-deficient murine cells [26 27 28 ] (Figs. 1 and 2) . Using human TLR/CD4 recombinant fusion proteins, it has been shown that all TLR but not TLR2 are able to signal as homomers, whereas TLR2 forms heteromers with TLR1 or TLR6 to attain specificity for a given stimulus [19 ]. Thus, it is likely that there is no functional human TLR2 homodimer, and LP, like Pam2C-SK4, are recognized by TLR2/TLR1 and TLR2/TLR6 heterodimers. However, so far, there are no TLR1/TLR6 double-knockout mice available to investigate whether LP, like Pam2C-SK4, are recognized by TLR2 homomers or by TLR2/1 and TLR2/6 heteromers in murine cells.
Beside TLR2, TLR1, and TLR6, there are other molecules described that are involved in the initial step of recognizing and binding bacterial LP. CD14 is a membrane-associated GPI-linked protein and functions together with myeloid differentiation protein 2 as an essential coreceptor in TLR4-dependent LPS binding and recognition [37 , 38 ]. Previous studies also predict an involvement of CD14 in LP signaling [39 ]. Nakata et al. [40] reported that CD14 binds directly to triacylated LP and facilitates the recognition of the ligand by TLR2/1 heterodimers, whereas this could not be observed with diacylated LP. Although this coreceptor promotes a difference in LP recognition, the absence of an intracellular signaling domain excludes the possibility of inducing different downstream events. CD36 has been postulated to be a sensor for diacylated LP, although only the R-enantiomeric form of macrophage activator lipoprotein peptide-2 was clearly shown to interact with CD36 [41 ]. Both molecules, CD14 and CD36, are expressed on murine macrophages, and putative different effects on the signal transduction during activation by the various LP would have been observed in our experiments but as a matter of fact, were not found.
Many molecules have been described to be involved in the signaling cascades that are activated upon TLR stimulation. TLR2 is absolutely dependent on the adaptor molecule MyD88, as there is no cell activation by LP in MyD88-deficient mice [42 ]. Upon stimulation, MyD88 is recruited to the receptor complex and leads in turn to the recruitment of IRAK1 and -4 as well as TRAF6. In overexpression models, IRAK2 is also able to interact directly with MyD88 and TRAF6 to mediate signal transduction [43 ].
Our experiments using DN forms of MyD88, TRAF6, IRAK1, and IRAK2 show that all signaling molecules indeed participate in the transduction cascade of the different TLR2 dimers. No matter whether PamOct2C-(VPGVG)4VPGKG, FSL-1, and Pam2C-SK4 were used to stimulate HEK293 cells transfected with DN plasmids in addition to murine TLR1, TLR2, and TLR6, the percentile inhibition was identical in all approaches (Fig. 3) . In all cases, DN TRAF6 and DN IRAK2 reduced the TLR2-dependent IL-8 production nearly to a background level; DN MyD88 and DN IRAK1 lowered the response to 25% and 40%, respectively. These results show that the signal cascades activated by PamOct2C-(VPGVG)4VPGKG, Pam2C-SK4, and FSL-1 use these adaptor molecules during cell activation, and knockdown of one of these molecules influences the signaling induced by all three LP in the same way.
Going one step forward in the transduction cascade, we then investigated the activation of MAPK used in TLR signaling in BMDM. After stimulation with pathogens, four intracellular protein kinase cascades are activated, namely, the NF-B cascade (prefaced through IB degradation), the ERK p42/44 (Erk), JNK, and p38 MAPK cascades, leading to the induction of many key cytokine genes that are essential for the innate immune response [9
, 44
]. The involvement of all three MAPK subtypes in LP-induced cytokine production and release has already been documented [45
]. Nevertheless, we were interested in comparing activation of MAPK more carefully under time-dependent aspects (Fig. 4)
. Kinetics of p38 phosphorylation as well as IB degradation are found to be nearly identical for all stimuli. Phosphorylation of Erk, JNK, and p38 could be detected as early as 5 min after stimulation with all LP and was again absent after 30 min. Parallel to this, IB was degraded already after 5 min and reappears after 15–30 min of incubation time. The data indicate that phosphorylation of MAPK and IB degradation appears to be quantitatively similar in their stimulation profile.
So far, activation of the investigated adaptor molecules and kinases induced by the different LP seems to be identical. Nevertheless, these findings give no complete evidence for identical signaling events. A conclusion on the different TLR2 dimer signaling can be made by investigating the induced gene expression patterns. Different expression profiles would prove the use of diverse signal transduction pathways, whereas the same expression profile would indicate that all LP and the corresponding TLR2 dimers signal via the same pathway.
In fact, Scatterplot analysis of Affymetrix microarrays again uncovered highly similar effects induced by the three LP. Gene expression patterns look equal after 2 h and 6 h of stimulation (Fig. 5) , supporting the previous results. Real-time PCR of various genes, which were found to be regulated equally, confirmed these findings and additionally showed similar kinetics and dose requirements in gene modulation by the different stimuli (Fig. 6) . Hence, although activating different TLR2 dimers, the three LP are likely to induce the same signaling. However, microarray data of several probe sets of the same gene show rather diverse results. Whereas one probe set shows strong modulation or a different regulation induced by the three LP, other sets of the same gene indicate no such regulation or differences. Real-time PCR using specific primers against these probe sets revealed that in these cases, there is no regulation or differences in the expression (Table 2 ; Supplemental data, Table S2).
TreeView analysis of regulated genes proves these findings by clustering the three LP together, although there seem to be slight differences in the expression of down-regulated genes (Fig. 7 ; Supplemental data, Fig. S1). The triacylated LP appears to decrease the expression of some genes to a lesser extend compared with the diacylated stimuli. This can be explained by the fact that mRNAs are reduced to background levels, and variations at this background range can therefore reflect differences when judging the fold induction. By comparing the absolute mRNA levels of the three different approaches with unstimulated control cells, the biological relevance of this difference can be ruled out, as these varieties are a result of a down-regulation to background levels (Fig. 7) . One example is the regulator of G-protein signaling 2 mRNA, where fluctuations in this low background range implicate differences. The fluorescence values indicate that these differences are not crucial, as the diversity of the background levels of 52, 7, and 12 is likely to be biologically irrelevant in comparison to the high level of 1049 in control cells.
Anyway, the hierarchical clustering of 20 of the strongest modulated probe sets reflects the high similarity of the gene expression patterns induced by the three LP in the dendrogram (Fig. 7) . The same result can be observed when more than threefold regulated probe sets are used for clustering (Supplementary data, Fig. S2). Again, the LP appear in the same cluster showing their high similarity.
Taken together, our data about MAPK phosphorylation, IB degradation, inhibition experiments with DN plasmids for signaling molecules, in addition to the microarray data, which were confirmed by real-time PCR, indicate that identical signaling pathways are induced by FSL-1, Pam2C-SK4, and PamOct2C-(VPGVG)4VPGKG. Our data indicate that the function of different TLR2 dimers is to expand the ligand spectrum of TLR2 rather than to expand the pattern of the response to the various LP. By collaboration of TLR2 heteromers with other PRR, such as Nacht-like receptors, C-type lectin-like molecules (e.g., Dectin-1), or even by acting in concert with TCRs, the immune system provided a mechanism to act effectively against specific pathogens. Simultaneous stimulation of TLR2 and Dectin-1 by the TLR6-dependent yeast cell-wall component zymosan promotes synergistic effects of TNF- production and IL-12 secretion of macrophages and DC [46
], and stimulation of monocytes with nucleotide-binding oligomerization domain-1 (NOD1) and NOD2 ligands together with LP synergistically enhanced IL-8 release [47
]. Thus, during infection with whole pathogens, the combination of various microbial ligands will be recognized by the cell, depending on the repertoire of expressed receptors and functional cooperation between the signals that are induced.
It is reasonable to conclude that the heterodimerization of TLR2 with TLR1 or TLR6 has been evolutionary developed to enable the innate immune system to recognize the variety of different LP from Gram-positive and Gram-negative bacteria as well as mycoplasma.
Received August 31, 2007; revised October 30, 2007; accepted November 3, 2007.
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