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Keywords:

  • Porphyromonas asaccharolytica;
  • Bacteroides fragilis;
  • OmpA;
  • cytokine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1α, tumor necrosis factor (TNF)-α, IFN-γ, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Porphyromonas asaccharolytica is a Gram-negative nonsporulating anaerobic rod, that was formerly part of the genus Bacteroides (Nitzan et al., 1999). Infections by this pathogen are associated with soft-tissue infections below the waist, foot ulcers, appendiceal abscesses, and empyema (Jousimies-Somer et al., 2002). Porphyromonas asaccharolytica was also implicated in cases of bacteremia (Melon et al., 1997) and in a left cardiac myxoma (Revankar & Clark, 1998). Bacteroides fragilis, a nonspore-forming, Gram-negative rod, is the most common anaerobic organism isolated from clinical infections. It is frequently associated with extraintestinal infections such as abscesses and soft-tissue infections, as well as diarrheal diseases in animals and humans. Bacteroides fragilis has the ability to invade the host immune response, which contributes to the virulence of the bacterium. Its capsule can mediate resistance to complement-mediated killing and to phagocytic uptake (Wexler, 2007).

The outer membrane of Gram-negative bacteria acts as a dynamic interface between the cell and its surroundings, and the importance of this interaction in both pathogenesis of the infection and immune response of the host has been investigated. The major components of the outer-membrane proteins in P. asaccharolytica and B. fragilis are proteins of the OmpA family, Omp-PA and Bf-OmpA, respectively. The importance of OmpA in the pathogenic process has been increasingly recognized. OmpA has been implicated in the invasion of brain microvascular endothelial cells (BMEC) (Prasadarao et al., 1996, 1999) and has been shown to contribute to the ability of Escherichia coli to cross the blood–brain barrier (Huang et al., 2000).

The outer-membrane proteins of the porin class, in general, possess a variety of immunomodulatory and procoagulant activities (Gupta, 1998; Iovane et al., 1998; Biswas, 2000; Brinkman et al., 2000). Omp-PA of P. asaccharolytica is the major porin protein of that organism (Magalashvili et al., 2007). In contrast, the Bf-OmpA protein does possess some pore-forming activity in liposomes but is not the major porin protein of B. fragilis (Wexler et al., 2002). Nontoxic concentrations of porins from a variety of organisms stimulate the synthesis and release of platelet-activating factor and promote proinflammatory and immunomodulatory cytokine release from immunocompetent cells or other cellular sources (Perfetto et al., 2003). Porins can induce the release of tumor necrosis factor (TNF)-α, IL-1α, and IL-6 by human monocytes and of IFN-γ and IL-4 by human lymphocytes. This was seen with porins from Salmonella typhimurium (Galdiero et al., 1993, 1995; Gupta, 1998), Pseudomonas aeruginosa (Brinkman et al., 2000; Perfetto et al., 2003), and Pasteurella multocida (Iovane et al., 1998). The OmpA-like porin from Acinetobacter spp. stimulates the secretion of gastrin and IL-8 (Ofori-Darko et al., 2000), and Shigella dysenteriae type 1 porin induces the release of nitric oxide and IL-1 (Biswas, 2000). The accumulated evidence clearly indicates that porins mediate release of cytokines and other proinflammatory factors, and that this activity may vary from one porin type to another.

The aim of this study was to investigate and compare the abilities of the OmpA proteins P. asaccharolitica and B. fragilis to induce the release of different cytokines by murine splenocytes in vitro. The significance of this study is due to the fact that Bacteroides and anaerobes in general are quite different from other aerobic microorganisms.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Animals

BALB/cByJ male mice, 8 weeks old were used. The animals were housed at a constant temperature (20±2 °C) under a fixed 12 h light–dark cycle with free access to food and water.

Preparation of Omp-PA and Bf-OmpA

Porphyromonas asaccharolytica ATCC 25260 and B. fragilis ATCC 25285 were grown anaerobically for 72 and 96 h, respectively, at 37 °C in Brucella broth medium supplemented with hemin and vitamin K (5 and 1 μg mL−1). Porphyromonas asaccharolytica Omp-PA and B. fragilis OmpA isolation and purification from the outer membranes were performed using lithium dodecyl sulfate (LDS) as described earlier (Nitzan et al., 1999; Wexler et al., 2002; Magalashvili et al., 2007). Lipopolysaccharide contamination in the final preparations was detected by the Limulus test (Yin et al., 1972) and was 10 pg per 10 μg porins. To eliminate any biological effect of lipopolysaccharide, proteins were incubated for 1 h with 5 μg mL−1 polymyxin B (Sigma) at room temperature (Blanchard et al., 1986). In all of the tests performed, the porin with polymyxin B gave the same results as the porin alone (data not shown).

The supernatants containing the OmpA proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970).

Elution from the gel

In order to obtain purified preparations containing only Omp-PA (37 kDa) and Bf-OmpA (37 kDa) in their native forms, elution from the gel was performed as follows:. bands corresponding to the proteins were cut out from the gel, placed in dialysis bags, and mashed manually. Buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS, and 3 mM sodium azide was then added and the preparations were dialyzed against the same buffer overnight at 4 °C. The gel/buffer mixtures were centrifuged to remove the gel traces, which were re-extracted with the same buffer and recentrifuged. In order to remove the detergent traces, the supernatants from both extractions were combined and redialyzed against buffer containing 20 mM Tris–HCL and 3 mM sodium azide for 4 days at room temperature, with frequent changes of the buffer, and then lyophilized.

Cloning and expression of B. fragilis ompA in E. coli and purification of recombinant Bf-OmpA from inclusion bodies

Primers, to amplify the Bf-ompA gene from genomic DNA, were constructed with appropriate restriction sites (NdeI and BamH1) for subsequent cloning of the PCR product into pET-27b(+). The primers used are indicated in Table 1. The cycles were 95 °C for 3 min, followed by 30 cycles of 95 °C for 45 s, 62 °C for 30 s, 72 °C for 4 min, and finally 72 °C for 5 min. One microgram each of plasmid and PCR product were digested with BamHI and NdeI as directed by the manufacturer. Both the digested pET-27b(+) and the digested Bf-ompA were purified from a 1% agarose gel. The gel slices containing the desired bands were melted at 65 °C for 5 min. Fifty nanograms of digested vector and 100 ng of digested insert were mixed with 400 U of T4 ligase in T4 ligase buffer (20 μL total volume) for 4.5 h. Five microliters of the ligation mixture was used to transform XLBlue MRF' and the reaction was plated on Luria–Bertani (LB) agar with kanamycin. Transformants were screened by PCR and verified by miniprep plasmid analysis.

Table 1.   Primers used for the pET cloning
 Primer sequence (5′–3′)
OmpAfwdTGTTCATATGCAGCAGACTACAATTACGGGAT
OmpArevAAGTGGATCCTTATTTAACAGACTCTACTAATA

Purified plasmid DNA was used to transform BL21 according to the manufacturer's instructions and plated on LB agar with kanamycin. Overnight culture (0.5 mL) was used to inoculate 1 L of LB with kanamycin and grown in a 37 °C shaker until the OD600 nm reached 0.4 U. The culture was induced at this point by adding IPTG to a final concentration of 1 mM. Protein induction proceeded for 3 h, and then the cells were harvested by centrifugation for 20 min at 7450 g at 4 °C. The cell pellet was washed with 20 mM Tris-HCl, pH 7.4, recentrifuged, and then frozen and stored at −20 °C.

The frozen cell pellet from 1 L of induced culture was resuspended in 100 mL of 20 mM Tris-HCl, pH 7.4, with 100 μg mL−1 lysozyme. The suspension was sonicated at a power level of 4 for 9 min until homogenized. The lysate was centrifuged for 27 000 g for 5 min at 4 °C to pellet the inclusion bodies containing the recombinant Bf-OmpA. The inclusion bodies were washed twice with 100 mL of 20 mM Tris-HCl, pH 7.4, supplemented with 10 mM EDTA, 1%Triton X-100. The washed inclusion bodies were frozen at −20 °C.

Refolding of overexpressed Bf-OmpA

The frozen inclusion bodies were resuspended to 20 mg mL−1 (0.48 mM) in 8 M urea, 10 mM borate, 2 mM EDTA, pH 10. Twelve milligrams of the resuspended protein was incubated with 16 mM 3–14 zwittergent for 4 days at 37 °C. A band indicating the refolded Bf-OmpA was cut out of the gel, passively eluted, and then diluted in the cell culture at the appropriate concentration (0.1±10 mg mL−1) for the cytokine assays.

Murine splenocyte preparation and stimulation

Murine splenocytes were prepared according to conventional procedures from aseptically removed mouse spleens (known to contain more than 97% lymphocytes of different types). Erythrocytes were lysed using 0.155 M NH4Cl, washed three times in RPMI 1640 medium (Labtek Laboratories, Eurobio, Paris, France), resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), glutamine (2 mM), penicillin (100 U mL−1), and streptomycin (100 U mL−1) at a concentration of 3 × 106 cells mL−1, and incubated at 37 °C in a humidified atmosphere containing 5% CO2.

Murine splenocytes resuspended at 3 × 106 cells mL−1 in complete medium were divided into aliquots to be treated or left untreated. The proteins were prepared in pyrogen-free distilled water and then diluted in the cell culture at the appropriate concentration (0.1±10 μg mL−1). The incubation time was 24 and 48 h for cytokine assays and 3 h for mRNA analysis.

After incubation times were completed a lactate dehydrogenase (LDH) assay was carried out according to the manufacturer's instructions using a Cytotoxicity Detection kit (Promega). LDH is a stable cytoplasmic enzyme present in all cells and is rapidly released into cell culture supernatant upon damage of the plasma membrane. LDH activity was determined by a coupled enzymatic reaction whereby the tetrazolium salt (INT) was reduced to formazan. An increase in the number of dead or damaged cells results in an increase in LDH activity in the culture supernatant.

Cytokine assays

All assays were carried out with 3 × 106 cells mL−1 stimulated with various concentrations of Omp-PA, native Bf-OmpA, and recombinant Bf-OmpA, and were incubated at 37 °C in 5% CO2 for 24 and 48 h. At specified time intervals, cell viability was checked by the Trypan blue exclusion test. Culture supernatants were harvested by centrifugation and stored at −20 °C until assayed for cytokines. All measurements were carried out using monoclonal antibodies. IL-1α, IL-6, TNF-α, IFN-γ, and IL-10 were measured using immunoenzymatic methods (ELISA kits of Invitrogen, Biosource, Worcester, MA).

RNA isolation and cDNA preparation

The concentrations of Omp-PA used in the assay were 0.1 μg mL−1 for TNF-α, 1 μg mL−1 for IFN-γ and IL-10, and 5 μg mL−1 for IL-1α and IL-6. The concentrations of native and recombinant Bf-OmpAs used in the assay were 0.1 μg mL−1 for IL-1α and IFN-γ, 1 μg mL−1 for TNF-α, and 5 μg mL−1 for IL-6. Nonstimulated cells were used as negative controls. The stimulated and the nonstimulated cells were collected after 3 h of incubation. Mouse β-actin was used as an internal standard. Total RNA was extracted according to the method of Chomczynski & Sacchi (1987). The RNA pellet was resuspended in 75% ethanol, sedimented, vacuum-dried, and dissolved in 50 μL of RNAse free water. One microliter of oligo (dT) (Promega Biotechnology, Madison, WI) was added to the suspension containing 2 μg of RNA and the mixture was heated at 70 °C for 5 min. After cooling on ice, the mixture was incubated for 2 h at 42 °C with 14 μL of the following mixture: 20 mM dithiothreitol (Sigma, St Louis, MO); 1 mM (each) dATP, dGTP, dCTP, and dTTP; 35 U of RNasin (Promega); and 525 U of Moloney murine leukemia virus reverse transcriptase (Promega) in reverse transcription buffer.

PCR procedure

The primer pair sequences were designed on the basis of published cytokine gene sequences as reported in Table 2. The primer sequences were complementary to sequences in the exons or spanned exon±exon junctions and thus were RNA-specific. One microliter of cDNA prepared as described above was amplified in the presence of 1 μL of 5′ and 3′ primers, 0.5 μL of dNTP (Promega), 2.5 μL of Taq DNA polymerase 10 × buffer (Promega), and 0.5 μL of Taq DNA polymerase (Promega) in a final volume of 25 μL. The PCR reactions were performed in a DNA thermal cycler (Perkin-Elmer-Cetus Instruments, Norwalk, CT). All PCRs started with a 3-min denaturation step that was followed by 35 cycles of 1 min of denaturation at 94 °C, 1 min of annealing temperature, and 1 min of extension at 72 °C. A final 10 min at 72 °C was used in all cases. The annealing temperature used for primers was as follows: IL-1α 60 °C, TNF-α 60 °C, IFN-γ 60 °C, IL-6 60 °C, IL-10 60 °C, and β-actin 60 °C. Twenty-five microliters of the reactions were subjected on 1.5% agarose gel and electrophoresis was performed at 100 V. One microgram of GeneRuler, DNA Ladder Mix (#SM0331, Fermentas), was run in parallel as a molecular weight (MW) marker (providing bands at 100–1000 bp).

Table 2.   Primer sequences used for RT-PCR
CytokineOligonucleotide sequence
IL-1α5′-AAGATGTCCAACTTCACCTTCAAGGAGAGCCG-3′
5′-AGGTCGGTCTCACTACCTGTGATGAGTTTTGG-3′
TNF-α5′-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3′
5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′
IFN-γ5′-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-3′
5′-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3′
IL-65′-ATGAAGTTCCTCTCTGCAAGAGACT-3′
5′-CACTAGGTTTGCCGAGTAGATCTC-3′
IL-105′-CTGGAAGACCAAGGTGTCTAC-3′
5′-GAGCTGCTGCAGGAATGATGA-3′
β-actin5′-GTGGGCCGCTCTAGGCACCAA-3′
5′-CTCTTTGATGTCACGCACGATTTC-3′

Statistics

The immunoenzymatic assays were carried out in triplicate and the results were expressed as the mean±SD. Comparisons between tests were made by Student's t-test, with statistical significance considered to be P<0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Purity of Omp-PA and Bf-OmpA preparations

SDS-PAGE analysis of the purified proteins is shown in Fig. 1. SDS-PAGE of Omp-PA showed one band with a MW of 37 kDa (Fig. 1, Lane 2), as reported previously (Magalashvili et al., 2007). SDS-PAGE analysis of Bf-OmpA demonstrated one band of 37 kDa (Fig. 1, Lane 3), as reported previously (Wexler et al., 2002).

image

Figure 1.  Electrophoretic pattern of the purified porins from Porphyromonas asaccharolytica ATCC 25260 and Bacteroides fragilis ATCC 25285. Lane 1, MW standards; lane 2, purified sample of P. asaccharolytica porin (37 kDa); lane 3, purified sample of B. fragilis porin (36.8 kDa).

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Cloning and expression of B. fragilis ompA in E. coli and purification of OmpA from inclusion bodies

IPTG-induced E. coli BL21 harboring plasmid pET-27b(+)-ompA produced inclusion bodies that contained mostly Bf-OmpA (Fig. 2).

image

Figure 2.  SDS-PAGE of recombinant Bf-OmpA refolded in zwittergent 3–14. Lane 1, standard proteins; lane 2, recombinant Bf-OmpA recovered from inclusion bodies; lane 3, refolded recombinant Bf-OmpA incubated with 16 mM (3–14) for 5 days at 37°C.

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Refolding of B. fragilis OmpA from inclusion bodies

Densitometric analysis refolded Bf-OmpA indicated that maximal results were achieved with 16 mM zwittergent 3–14 after incubation for 5 days at 37 °C. Approximately 51% of the overexpressed protein could be refolded under these conditions. The refolded Bf-OmpA migrated at the same apparent MW of 36.8 kDa as the native Bf-OmpA (Fig. 2). It was shown earlier that the refolded Bf-OmpA has activity similar to the native protein in the liposome assay (Wexler et al., 2002).

Release of IL-1α, TNF-α, IFN-γ, IL-6, and IL-10 from murine splenocytes induced by Omp-PA from P. asaccharolytica and native and recombinant Bf-OmpA from B. fragilis

The highest release of the cytokines from murine splenocytes induced by Omp-PA and both native and recombinant Bf-OmpAs was observed after 48 h of incubation. Cells stimulated with 5 μg mL−1 Con A were used as positive controls (data not shown), and the nonstimulated cells served as negative controls. LDH levels presented in the supernatants of stimulated cells were similar to those detected in the supernatants of nonstimulated cells, suggesting that cytokine release was not due to cell lysis (data not shown). Omp-PA induced high-level secretion of the proinflammatory cytokines IL-1α, TNF-α, IFN-γ, IL-6, and anti-inflammatory cytokine IL-10 in a dose-dependent fashion and at a concentration ranging from 0.1 to 10 μg mL−1. The amounts (pg mL−1) of each released cytokine are demonstrated in Fig. 3. The highest levels of the cytokine secretion were observed with the Omp-PA concentrations of 0.1 μg mL−1 for TNF-α, 1 μg mL−1 for IFN-γ and IL-10, and 5 μg mL−1 for IL-1α and IL-6. Concentrations higher than these caused a decrease in cytokine production, and a concentration lower than 0.1 μg mL−1 showed no significant effect. Both native and refolded recombinant Bf-OmpAs were able to regulate release of IL-1α, TNF-α, IFN-γ, and IL-6 but in much lower levels compared with those obtained using Omp-PA. Both native and recombinant Bf-OmpAs had no significant effect on the release of IL-10 under the same experimental conditions. The highest levels of the cytokine secretion were observed with the protein concentrations of 0.1 μg mL−1 for IL-1α and IFN-γ, 1 μg mL−1 for TNF-α, and 5 μg mL−1 for IL-6.

image

Figure 3.  Release of IL-1α, TNF-α, IFN-γ, IL-6, and IL-10 from murine splenocytes stimulated by Porphyromonas asaccharolytica Omp-PA, Bacteroides fragilis native Bf-OmpA, and B. fragilis recombinant Bf-OmpA at different concentrations after 48 h of incubation. Each point represents the mean of three experiments±SDs. Points designated by asterisks indicate statistically significant differences (P<0.05) vs. nonstimulated cells.

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Cytokine mRNA expression induced by Omp-PA from P. asaccharolytica and native and recombinant Bf-OmpA from B. fragilis

Expression levels of IL-1α, TNF-α, IFN-γ, IL-6, and IL-10 mRNAs were evaluated by treating murine splenocytes with Omp-PA and native and recombinant Bf-OmpAs in concentrations that showed maximum release of each cytokine as measured by ELISA kits (Fig. 3). The mRNA levels of all five cytokines, IL-1α, TNF-α, IFN-γ, IL-6, and IL-10, were increased upon stimulation of the cells by adding appropriate concentrations of Omp-PA (Fig. 4). The mRNA levels of the cytokines IL-1α, TNF-α, IFN-γ, and IL-6, expressed by stimulation of the cells with native and recombinant Bf-OmpAs, were similarly increased, but were found to be lower compared with those expressed by Omp-PA. Very low expression of IL-10 mRNA was detected under the same experimental conditions. The nonstimulated cells did not show increased mRNA expression of the tested cytokines (Fig. 4).

image

Figure 4.  Cytokine mRNA expression induced by Porphyromonas asaccharolytica Omp-PA and Bacteroides fragilis native and recombinant Bf-OmpAs in murine splenocytes. Control indicates the nonstimulated cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

The severity of sepsis (defined as a systemic inflammatory response syndrome associated with infection) is related to the severity of the host response. One measure of this response is the production of cytokines (particularly TNF-α, IL-1β, IL-6, and IL-8); although important in host defense functions, these cytokines can also result in widespread tissue injury. The early cytokines, TNF-α and IL-1β, are thought to mediate the production of the later or distal cytokines, including IL-6 and IL-8 (Blackwell & Christman, 1996). Triggering the release of the cytokines TNF-α, IL-1, IL-6, IL-8, and IL-12 and the subsequent inflammatory response is critical to containing bacterial infection in the tissues. If, however, infection disseminates in the blood, the widespread activation of phagocytes in the bloodstream is catastrophic.

Gram-negative bacteria, and specifically the lipopolysaccharide component, are most often implicated in inducing the cytokine cascade. Humans injected with purified lipopolysaccharide develop a cytokine cascade in the serum. The early cytokine response (TNF-α, IL-6, and IL-8) coincides with the onset of fever and the activation of blood neutrophils, monocytes, and lymphocytes.

In this study, we investigated the ability of the OmpA proteins from P. asaccharolytica and B. fragilis to trigger release and expression of proinflammatory and immunoregulatory cytokines IL-1α, TNF-α, IFN-γ, IL-6, and IL-10 by murine splenocytes in vitro. Both native and refolded recombinant B. fragilis Bf-OmpA that are proved to be functionally identical (Wexler et al., 2002) could elicit the cytokine release. We were not able to refold the Omp-PA, and therefore, could not test it in this assay. The main reason for using recombinant refolded Bf-OmpA was to avoid the possibility that any capsule contamination in the purified protein preparation may be responsible for the cytokine production (Wexler, 2007). The cytokine release proceeded in a dose-dependent fashion with different concentrations of porins needed for the maximum release of each cytokine. The release of IL-1α and IL-6 stimulated by Omp-PA was relatively low compared with the high-level production of TNF-α, IFN-γ, and IL-10. The results obtained cannot be attributed to the contaminating lipopolysaccharide (10 pg per 10 μg of porin) in the porin preparations, because this trace amount of lipopolysaccharide had no ability to induce any cytokine production (data not shown). Moreover, porins were incubated with polymyxin B to neutralize the biological activity of lipid A (Galdiero et al., 1993). It is proved that the porin–polymyxin complex has the same activity in the induction of the cytokine secretion as the porin preparations alone, while the lipopolysaccharide–polymyxin complex is inactive (Galdiero et al., 1995). While E. coli lipopolysaccharide is more active (per μg) than Bacteroides lipopolysaccharide in inducing cytokine production, Bacteroides strains often outnumber Enterobacteriaceae in the feces and in mixed infections, and their role in inducing and/or modulating the host response in septic shock should not be overlooked. Nagy et al. (1998) assessed the ability of several species of heat-inactivated Bacteroides as well as isolated Lipopolysaccharide to induce TNF-α release and IL-6 production in human mononuclear cells and whole blood. As expected, E. coli lipopolysaccharide was more active than B. fragilis lipopolysaccharide in inducing these factors; slightly more TNF-α (>2.5-fold) and more IL-6 (>10-fold) were produced. Both TNF-α and IL-6 were induced by 10 μg mL−1 Lipopolysaccharide or by 109 CFU mL−1 heat-inactivated cells; we cannot compare the level of induction because their assay used human mononuclear cells and whole blood. In another study, the cytokine induction of Bacteroides lipopolysaccharide depended on the extraction method used, and lipopolysaccharide extracted by the phenol–water method had the highest TNF-α-inducing activity (Delahooke et al., 1995a, b). Compared with E. coli lipopolysaccharide, the phenol–water extract of B. fragilis was only marginally less active (five- to sevenfold) in the bioassay for TNF induction from human mononuclear leukocytes (Delahooke et al., 1995b; Poxton & Edmond, 1995). In Bacteroides, only lipopolysaccharide has been studied in terms of ability to induce cytokines, but other cell structures, including porins or other outer-membrane proteins, have not been investigated (other than this study), although such structures in other organisms are known to act as immune ‘modulins’ (Henderson et al., 1996). Porins in other organisms such as Salmonella, Yersinia, and Pseudomonas are known to induce cytokines; the number of cytokines induced by Salmonella and Yersinia porins was in the same general ranges as that induced by PA Omp (Galdiero et al., 1993; Tufano et al., 1994). Direct comparison of inducing ability in Pseudomonas (which would be the best analogy because the major porin is an OmpA-like molecule) of the amount of cytokine induced was in Units based on lysis or cell proliferation assays (Cusumano et al., 1997).

Interestingly, Omp-PA was able to induce cytokine release at much higher levels compared with those obtained with native and recombinant Bf-OmpAs. We were initially surprised by this because P. asaccharolytica is considered to be less pathogenic than B. fragilis. However, mice were used as the experimental animals, and B. fragilis is part of mouse intestinal microbial communities. Thus, the mouse was potentially immunized to the Bf-OmpA (Pumbwe et al., 2006), which may explain why the stimulation of murine splenocytes by B. fragilis porin did not result in high-level secretion of cytokines.

Alternatively, perhaps other porin proteins present on the B. fragilis outer membrane may have more cytokine-stimulating activity. For example, the B. fragilis Omp-200 has more pore-forming activity than OmpA (Wexler, 1997). On the other hand, only one active monomeric porin (Omp-PA) was isolated from the outer membrane of P. asaccharolytica.

As expected, exposure to Omp-PA and native and recombinant Bf-Omp resulted in increased levels of IL-1α, TNF-α, IFN-γ, IL-6, and IL-10 mRNA expression. The results obtained in reverse transcriptase (RT)-PCR analysis corresponded to those measured by ELISA kits. For comparison purposes, we also checked the ability of another outer-membrane protein, Omp-EA, to induce the release and expression of IL-6 and IFN-γ. Omp-EA is an outer-membrane protein from Erwinia amylovora, a pathogen that infects only plants (Elazar et al., 2007). As expected, Omp-EA acted as a negative control and did not induce release of the tested cytokines (data not shown).

Recent reports suggest that the ability to induce cytokine production by the porins is dependent on the existence of the externally exposed loops that have been described extensively (Galdiero et al., 2006). We have recently described these loops in monomeric porins from Acinetobacter baumannii (Gribun et al., 2003), B. fragilis (Wexler et al., 2002), and P. asaccharolytica (Magalashvili et al., 2007). Future work will include studying the role of the external loops of P. asaccharolytica Omp-PA and B. fragilis Bf-OmpA in induction of cytokine release.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

The authors wish to thank Dr Y. Kalechman and the laboratory of Prof. B. Sredni for the great assistance and guidance in the immunology part of the research. This work was supported in part by a grant from the Rappaport Foundation for Medical Microbiology to Y.N. and in part by a grant from the United States Department of Defense PRMRP to H.M.W. H.M.W. is supported by Merit Review Funds from the Department of Veterans Affairs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
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