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Keywords:

  • Mycoplasmas;
  • Mycoplasma hyorhinis;
  • glycerophosphodiesterase;
  • phospholipase A;
  • cardiolipin synthetase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade and survive within nonphagocytic melanoma cells. The invasion process may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. In this study, we show that M. hyorhinis membranes possess a nonspecific phospholipase A (PLA) activity capable of hydrolyzing both position 1 and position 2 of 1-acyl-2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoyl) phosphatidylcholine. In silico analysis of the M. hyorhinis genome shows that the PLA of M. hyorhinis shares no homology to described phospholipases. The PLA activity of M. hyorhinis was neither stimulated by Ca2+ nor inhibited by EGTA and had a broad pH spectrum. Mycoplasma hyorhinis also possess a potent glycerophosphodiesterase (GPD), which apparently cleaves the glycerophosphodiester formed by PLA to yield glycerol-3-phosphate. Possible roles of PLA and GPD in invading host eukaryotic cells and in forming mediators upon the interaction of M. hyorhinis with eukaryotic cells are suggested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

Mycoplasmas (class Mollicutes) are the smallest self-replicating bacteria. These bacteria lack a rigid cell wall and are parasites, exhibiting strict host and tissue specificities (Baseman & Tully, 1997; Rosengarten et al., 2000). Many mycoplasmas are pathogenic to humans and animals and are frequent contaminants of cell cultures (Rottem, 2003). Mycoplasma hyorhinis was first isolated from the respiratory tract of young pigs (Kobisch & Friis, 1996). This organism has been implicated in a variety of diseases in swine (Morita et al., 1995); Kobisch & Friis, 1996) and was shown to be the major contaminant of tissue cultures (Kotani et al., 1990). Interest in M. hyorhinis has been recently further increased after the detection of this organism in human gastric cancer tissues, suggesting a possible association between M. hyorhinis and carcinogenesis (Huang et al., 2001; Yang et al., 2010). A practically noncultivable mycoplasma tentatively identified as M. hyorhinis (to be referred to as strain MCLD) has recently been identified in LB33mel A1, a melanoma cell line. This organism was adapted to grow in a modified mycoplasma medium (Hayflick & Stinebring, 1960; Kornspan et al., 2010). Although M. hyorhinis has been considered to remain attached to the surface of host cells, we have recently shown that MCLD invades nonphagocytic eukaryotic cells (Kornspan et al., 2010). There is no doubt that invasion is associated with the attachment of the organisms to the host cells; nevertheless, attachment is not sufficient to trigger events that lead to invasion (Rosengarten et al., 2000). The invasion of MCLD may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. As phospholipids represent the major chemical constituents of the lipid bilayer, phospholipases are likely to be involved in the membrane disruption process (Weltzien, 1979; Vernon & Bell, 1992). Furthermore, phospholipases may play a fundamental role serving to generate signals required for invasion as well as an array of metabolites with distinct biologic function (Nishizuka, 1992). Cleavage of phospholipids by a mycoplasmal phospholipase C (PLC) will release diacylglycerol that activates protein kinases (Nishizuka, 1992). The activity of phospholipase A (PLA) will release free fatty acids (FFA) as well as lysophospholipids that may perturb the host cell membrane and generate active metabolites (Weltzien, 1979; Vernon & Bell, 1992). Evidence for PLC activity in a variety of mollicutes has been presented before (De Silva & Quinn, 1987; Shibata et al., 1995), and a potent phospholipase A1 (PLA1) was described in Mycoplasma penetrans (Salman & Rottem, 1995). In the present study, we show that M. hyorhinis possess PLA and glycerophosphodiesterase (GPD) activities. The possible role of these enzymes in the virulence of M. hyorhinis and in triggering signal cascades in the host cells is discussed.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

Cultivation of Mycoplasma hyorhinis and preparation of membranes

Mycoplasma hyorhinis strain MCLD was used throughout this study. The organism was grown for 48 h at 37 °C in a modified Hayflick's medium (Hayflick & Stinebring, 1960) supplemented with 10% heat-inactivated fetal calf serum (Biological Industries, Beit Haemek, Israel). Membrane lipids were metabolically labeled by growing the cells in a medium containing 0.3 μCi of [9,10(N)-3H] palmitic acid (53.0 Ci mmol−1; New England Nuclear) or [9,10(N)-3H] oleic acid (53.0 Ci mmol−1; New England Nuclear) per mL. The organisms were harvested at the mid-exponential phase of growth (A 595 nm of 0.08–0.12; pH 6.8) by centrifugation for 20 min at 12 000 g, washed once, and resuspended in a buffer solution containing 0.25 M NaCl and 10 mM Tris–HCl adjusted to pH 7.5 (to be referred as TN buffer). Cell extracts were obtained from washed cells by ultrasonic treatment for 2 min at 4 °C in W-350 Heat Systems sonicator operated at 200 W and 50% duty cycles (Salman & Rottem, 1995). Membranes were collected from the cell extracts by centrifugation at 34 000 g for 30 min, washed once, and resuspended in TN buffer. Total protein content in cells, cell extracts, and membrane preparations was determined by the method of Bradford (1976) using bovine serum albumin as the standard.

Phospholipase activity

Phospholipase activity of M. hyorhinis cell extracts or membrane preparations was measured utilizing either fluorescent or radioactive substrates. The standard reaction mixture (in a total volume of 100 μL) contained 40 μg protein in a buffer solution (0.15 M NaCl, 1 mM CaCl2, and 10 mM Tris–HCl adjusted to pH 8). For the fluorescence analysis, 2 μL of the fluorescent substrate 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (Invitrogen, C12-NBD-PC, 0.5 mg mL−1 in 10% ethanol) and 10 μL of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (Sigma, 0.14 mg mL−1 in 10% ethanol) were added. The reaction mixture utilizing a radioactive substrate contained radioactive phosphatidylglycerol (PG) obtained by growing Mycoplasma gallisepticum cells in Hayflick's medium (Hayflick & Stinebring, 1960) containing 0.25 μCi of [9,10(n)-3H]oleic acid (New England Nuclear) per mL. The radiolabeled lipids thus obtained were extracted (Salman & Rottem, 1995) and separated by thin-layer chromatography (TLC), and the PG spot was scraped off the plate and eluted with chloroform-methanol (1 : 1 by vol.). The radioactive PG was dried under a stream of nitrogen, resuspended in a solution of 0.25 M NaCl in 10 mM Tris–HCl (pH 8) containing 1.5 mg mL−1 of a commercial PG preparation (Sigma), and dispersed by sonication as described above. In control experiments, the M. hyorhinis membrane preparations were replaced with 5 units of snake venom phospholipase A2 (PLA2), 2.5 units of Clostridium welchii PLC, or 1 unit of peanut phospholipase D (PLD), all products of Sigma. The reaction was carried out at 37 °C for up to 4 h and was terminated by the addition of methanol/chloroform (2 : 1 by vol.). The entire mixture was extracted by the Bligh and Dyer procedure (Bligh & Dyer, 1959) and analyzed by TLC developed in chloroform-methanol-water (65 : 25 : 4 by vol.). The fluorescence of C12-NBD-free fatty acids (C12-NBD-FFA, R F = 0.82), C12-NBD-PC (R F = 0.33), and C12-NBD-lysophosphatidylcholine (C12-NBD-LPC, R F = 0.11) was detected using the luminescent image analyzer LAS-3000 equipped with a blue-light-emitting diode (460 nm EPI) and a Y515-Di filter, and quantification of the C12-NBD fluorescence was performed using tina 2.0 software (Ray Tests). Radioactivity in PG, lyso-PG, FFA, or diglyceride spots was determined in a scintillation spectrometer (Packard Tri-Carb 2900 TR). PLC activity in membrane preparations was determined as previously described (Kurioka & Matsuda, 1976), by measuring the release of p-nitrophenol (pNP) from p-nitrophenyl phosphorylcholine (pNP-PC; Sigma). The reaction mixture (in a total volume of 100 μL) contained 40 μg membrane protein and 20 mM pNP-PC in a buffer containing 0.25 M NaCl and 10 mM Tris-maleate (pH 7.2). The reaction mixture was incubated for up to 42 h at 37 °C, and the release of pNP was monitored using BMG FLUOstar Galaxy multifunctional microplate reader at 410 nm.

Bioinformatics analysis

Functional annotation of M. hyorhinis GPD and phospholipases was obtained by blast searching using default parameters in the nonredundant database (http://blast.ncbi.nlm.nih.gov). Protein analysis of GPD was performed using psort (http://www.psort.org) and ScanProsite (http://prosite.expasy.org). Multiple sequence alignment of GPD was carried out using the ClustalW2 program (http://www.ebi.ac.uk). Amino acids shading was performed using BoxShade 3.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

Phospholipase C activity of M. hyorhinis membranes

As phospholipases play an important role as bacterial virulence factors (Weltzien, 1979; Nishizuka, 1992; Vernon & Bell, 1992), we examined various phospholipase activities in M. hyorhinis. A rapid screening assay for PLC activity using the chromogenic substrate pNP-PC as a water-soluble analog of phosphatidylcholine was first described by Kurioka & Matsuda (1976). The hydrolysis of this compound yields phosphorylcholine and a yellow pNP that can be measured spectroscopically (Kurioka & Matsuda, 1976; Shibata et al., 1995). This assay may serve as a rapid screening assay for PLC activity in mycoplasmas (De Silva & Quinn, 1987) and accordingly was used to show PLC activity in Ureaplasma urealyticum (De Silva & Quinn, 1986), Mycoplasma fermentans, and M. penetrans (Shibata et al., 1995). Indeed, when the release of pNP from pNP-PC was measured with M. hyorhinis cell extracts or membrane preparations, we detected a pronounced increase in absorbance owing to the yellow color formed by the hydrolysis of pNP-PC. The hydrolysis of pNP-PC was affected by divalent cations, mainly by Mn+2 (20 mM), resulting in a fourfold increase in activity (Fig. 1). As expected, the activity was inhibited by EDTA (20 mM, data not shown).

image

Figure 1. GPD activity of Mycoplasma hyorhinis. The GPD activity was determined in the presence or absence of a divalent cation (20 mM) as described in Materials and methods. Open bars, 17 h of incubation; Closed bars, 42 h of incubation. The data are means ± SD of three independent experiments.

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Attempts to support the assumption that the hydrolysis of pNP-PC represents PLC activity were made by following the formation of diglycerides in reaction mixtures containing M. hyorhinis lysates or membrane preparations with radiolabeled PG or with PC labeled by fluorescent NBD linked with position 2 (C12-NBD-PC). The reactions were carried out for extended periods of time (0–4 h) with or without divalent cations (10 mM) at 37 °C. The reaction mixtures were extracted and analyzed by TLC. The results did not show any accumulation of diglycerides (data not shown). Furthermore, as the genome of M. hyorhinis (strain MCLD) has been recently fully sequenced and annotated (Kornspan et al., 2011), the genome was analyzed in silico for PLC. We failed to identify PLC but revealed the presence of a PLC-like GPD (GenBank accession no. AEC45694.1). Little is known about the role of GPD in the biology and pathophysiology of mycoplasmas. In M. pneumoniae, the glycerol-3-phosphate formed by an active GPD (GlpQ, GenBank accession no. NP_110108.1, Schmidl et al., 2011) is oxidized by glycerol-3-phosphate oxidase, resulting in the formation of hydrogen peroxide, the major virulence factor responsible for the cytotoxicity of this organism (Schmidl et al., 2011). Furthermore, it was suggested that the GPD of M. pneumoniae acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression (Schmidl et al., 2011). Our analysis of the M. hyorhinis GPD sequence predicted a polypeptide of 241 aa with a calculated molecular mass of 28 367 Da with no signal sequence or hydrophobic motifs common in membrane proteins. Nonetheless, GPD activity was detected almost exclusively in the membrane fraction. Extensive washing of the membrane preparations with increasing concentrations of NaCl (up to 1 M) in 10 mM Tris buffer (pH 7.5) with or without 10 mM EDTA did not affect the levels of GPD activity in the membranes (data not shown), suggesting that the GPD is not a loosely bound membrane protein adsorbed onto the membrane surface. The identification of the M. hyorhinis GPD was further strengthened by showing its homology to the active GPD of M. pneumoniae and to the GPD of Thermoanaerobacter tengcongensis (GenBank accession no. 2PZ0_A) where strictly conserved residues involved in the activity were identified (Fig. 2, Shi et al., 2008). We suggest that GPD is an essential enzyme in the turnover of glycerophospholipids, the major building blocks of the lipid bilayer of M. hyorhinis membranes. First, the fatty acids are cleaved resulting in the formation of glycerophosphodiesters, which are then further cleaved by GPD to yield glycerol-3-phosphate (Schmidl et al., 2011).

image

Figure 2. Multiple sequence alignment of the GPD gene product of Mycoplasma hyorhinis (MhGPD), Mycoplasma pneumoniae (MpGPD), and Thermoanaerobacter tengcongensis (TtGPD). A black background represents identical amino acids, while a gray background represents highly similar amino acids. Triangles indicate the strictly conserved residues involved in activity.

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Phospholipase A activity of M. hyorhinis membranes

Upon incubation of M. hyorhinis extracts with radiolabeled PG, a decrease in the radioactivity of the PG band with a concomitant increase in the radioactivity of the lysophospholipid and FFA fractions were noticed (data not shown), suggesting a phospholipase activity. The activity was almost exclusively associated with isolated membrane preparations (data not shown). When reaction mixtures containing M. hyorhinis membranes and the fluorescent substrate C12-NBD-PC were incubated for up to 4 h at 37 °C, two fluorescently labeled breakdown products were detected on the TLC plates, the major being C12-NBD-LPC with nonfluorescent fatty acid in position 1 hydrolyzed and the minor C12-NBD-FFA (Fig. 3), suggesting the activity of a PLA in M. hyorhinis membranes. In control experiments, using snake venom PLA2, the breakdown product of C12-NBD-PC was exclusively C12-NBD-FFA. The PLA activity of M. hyorhinis was neither stimulated by Ca2+ (0.1–10 mM) nor inhibited by EGTA (5 mM) and had a broad pH spectrum (pH 7.0–8.5). Quantitative analysis of the fluorescence products obtained by the hydrolysis of C12-NBD-PC by M. hyorhinis membranes is shown in Table 1. The ratio of C12-NBD-LPC to C12-NBD-FFA after treatment of C12-NBD-PC with M. hyorhinis membranes was 2.5 after a short incubation period (up to 1 h) and 0.8 after a prolonged incubation period (4 h), suggesting that M. hyorhinis possess a nonspecific PLA activity capable of hydrolyzing both position 1 and position 2 of the C12-NBD-PC, but with a somewhat higher affinity to position 1. The possibility that M. hyorhinis possess a PLA1 (Istivan & Coloe, 2006) or PLA2 (Rigaud & Leblanc, 1980) as well as a lysophospholipase (Gatt et al., 1982) was excluded as we were unable to demonstrate lysophospholipase activity using C12-NBD-LPC (data not shown). The in silico analysis of M. hyorhinis genome failed to identify PLA, suggesting that the PLA of M. hyorhinis shares no homology to phospholipases described so far.

image

Figure 3. Hydrolysis of C12-NBD-phosphatidylcholine by Mycoplasma hyorhinis. Phospholipase activity was assayed in a reaction mixture containing 40 μg of M. hyorhinis membrane protein incubated with fluorescent phosphatidylcholine for various periods of time, and the reaction mixtures were extracted and analyzed by TLC as described in Materials and methods. Fluorescence was detected using a luminescent image analyzer. Snake venom PLA2 (5 units) was used as a control. PC, C12-NBD -phosphatidylcholine; LPC, C12-NBD-lysophosphatidylcholine; FFA, C12- NBD-free fatty acid.

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Table 1. Hydrolysis of fluorescently labeled phosphatidylcholine by Mycoplasma hyorhinis membranes
Time (h)PLA activity (nmoles per mg protein)Fluorescence (percentage) in:
C12-NBD-PCC12-NBD-LPCC12-NBD-FFA
  1. Phospholipase activity was assayed in a reaction mixture containing 40 μg of M. hyorhinis membrane protein incubated with fluorescent phosphatidylcholine (C12-NBD-PC) for various periods of times as described in Materials and methods. The results were an average of three independent experiments utilizing different batches of membranes. PLA activity was presented as nmoles PC hydrolyzed per mg membrane protein. PC, phosphatidylcholine; LPC, lysophosphatidylcholine; FFA, free fatty acid.

00100.0 ± 000
160.4 ± 2.588.0 ± 1.98.1 ± 2.73.5 ± 1.0
2110.2 ± 7.278.4 ± 3.810.0 ± 1.89.8 ± 1.0
4230.6 ± 11.253.8 ± 7.719.6 ± 2.824.5 ± 6.1

The breakdown of C12-NBD-PC by M. hyorhinis cell extract or isolated membrane preparations did not yield C12-NBD-phosphatidic acid even after prolonged incubation periods (up to 24 h), excluding the presence of PLD in M. hyorhinis. Nonetheless, in silico analysis of M. hyorhinis genome revealed the presence of the conserved ‘HKD’ motif of PLD (HxK(x)4D(x)6GSxN) (Lee et al.,2009 that appears in two domains that reside between residues 253–270 and residues 440–457 of the predicted cardiolipin synthetase (GenBank accession no. AEC45753.1). These motifs were observed only in cardiolipin synthetase containing mycoplasmas fully sequenced so far (data not shown). The presence of the signature PLD motif was previously described in bacterial cardiolipin synthetases and in eukaryotic and bacterial phosphatidylserine synthases, indicating that PLD and these enzymes form a family of homologous proteins (Ponting & Kerr, 1996).

In this study, we have demonstrated that M. hyorhinis membranes possess a PLA that may be involved in the plasma membrane disruption process that occurs upon the invasion of host cells (Istivan & Coloe, 2006) and a potent GPD. Nonetheless, further research is required to identify the role of PLA and GPD activities in the pathogenesis and survival of M. hyorhinis.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References

The help and advice of H. Rechnitzer is greatly appreciated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgement
  7. References