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J. Biol. Chem., Vol. 280, Issue 29, 26680-26689, July 22, 2005
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From the
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm S-17177, Sweden, Instituto Clodomiro Picado, Facultad de Microbiología, and ¶Departamento de Bioquímica, Facultad de Medicina, Universidad de Costa Rica, San José, 2060 Costa Rica, **Defence Science and Technology Laboratory, Porton Down, Salisbury, Wilts SP4 0JQ, United Kingdom, Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Parkville, Melbourne, Victoria 3050, Australia, and Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan
Received for publication, January 10, 2005 , and in revised form, May 26, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-toxin and has been associated with enteritis in domestic animals, Crohn disease, and gas gangrene in humans (6).
Gas gangrene is an acute and life-threatening infection most frequently caused by C. perfringens and characterized by massive local edema, severe myonecrosis, and the accumulation of gas at the site of infection (7). Gas gangrene without external injury is associated with diabetes mellitus, peripheral vascular disease, or an underlying gastrointestinal or hematologic malignancy (8). More often, gas gangrene is associated either to trauma or surgery, occurring after the introduction of bacterial spores in a deep lesion or in a surgical wound (7). Despite the use of antibiotics and intensive care regimes, in many cases of gas gangrene radical amputation is the treatment of choice to avoid shock, multiorgan failure, and death (7). The plc gene, encoded in the C. perfringens chromosome, shows minimal inter-strain sequence variations and is highly expressed in all strains associated with gas gangrene (6, 9). Homologous genes are found in other Clostridium species such as Clostridium sordellii, Clostridium absonum, and Clostridium novyi, which occasionally cause gas gangrene (10, 11).
Several lines of evidence indicate that Cp-PLC is the major virulence factor in C. perfringens-induced gas gangrene. First, when injected intramuscularly in mice, recombinant Cp-PLC causes myonecrosis and reproduces the histopathological features of gas gangrene (12). Second, immunization with the recombinant Cp-PLC C-terminal domain protects mice from the intramuscular challenge with a lethal dose of C. perfringens vegetative cells (13). Third, a C. perfringens mutant strain, in which the plc gene has been inactivated by homologous recombination, is unable to produce gas gangrene (14). Although considerable progress has been made in recent years in the knowledge of the structure of Cp-PLC, our understanding of the cellular and molecular basis of its toxic effects is still incomplete (15).
A mutant cell line hypersensitive to Cp-PLC, referred to here as Don Q, was isolated previously (16). This cell line has a permanent low UDP-glucose (UDP-Glc) level due to a recessive point mutation in the UDP-Glc pyrophosphorylase (UDPG:PP) gene (17). We showed previously (18) that upon one allele reversion of the mutation or transfection with a wild type UDPG:PP, the mutant cell compensates the UDP-Glc deficiency and regains the same relative resistance to Cp-PLC as the one displayed by the parental cell. However, the molecular basis relating the cellular UDP-Glc deficiency to the sensitization to Cp-PLC has not been established.
UDP-Glc is required for the synthesis of the carbohydrate moiety of cell surface glycoconjugates, such as proteoglycans, N-glycosylated glycoproteins, and glycosphingolipids (GSLs) (17). Proteoglycans consist of variable polysaccharide chains, the glycosaminoglycans, linked to a core polypeptide. The mature glycosaminoglycans contain glucosamine or galactosamine alternating in glycosidic linkages with glucuronic acid, iduronic acid, or galactose and are sulfated or acetylated to varying degrees (19). The glycosaminoglycans are assembled in the lumen of the Golgi apparatus by sequential addition of monosaccharides to the linker tetrasaccharide GlcUA
1,3Gal1,3Gal1,4Xyl-, which is bound to a Ser or a Thr residue of the core polypeptide (20). UDP-Glc is essential for the synthesis of UDP-glucuronic acid and UDP-galactose, two monosaccharide donors used in glycosaminoglycans synthesis.
N-Glycosylated glycoproteins have covalently bound to an Asn residue, oligosaccharides of variable composition, all of which arise from the common core precursor Glc3Man9GlcNAc2-PP-dolichol (21). The oligosaccharide moiety of this precursor is synthesized by the stepwise addition of monosaccharide units onto dolichol pyrophosphate on the endoplasmic reticulum (ER) membrane and is then transferred en bloc onto the nascent polypeptide chains (21). UDP-Glc is actively transported to the lumen of the ER, where it serves as donor of the three terminal glucoses of the core precursor (21). Within the ER, UDP-Glc is also used by the UDP-Glc:glycoprotein glucosyltransferase, which recognizes and glucosylates misfolded glycoproteins (21). This glucosylation prolongs the contact of N-linked glycoproteins with the lectin-type chaperones calnexin and calreticulin and thus leads to their retention within the ER until they are correctly folded (21).
GSLs are composed of a variable carbohydrate moiety linked to ceramide and derive either from glucosylceramide (GlcCer) or galactosylceramide (GalCer) (22). GalCer is synthesized from ceramide and UDP-galactose on the luminal face of the ER membrane, whereas GlcCer is synthesized from ceramide and UDP-Glc on a pre-Golgi compartment or on the cytosolic face of the Golgi apparatus (23). Ceramide, a common precursor of GSLs and SM, is either synthesized de novo from L-serine and palmitoyl-CoA (22) or by a salvage pathway from sphingosine (23) (Fig. 1). The de novo synthesis of ceramide involves serine palmitoyltransferase, 3-ketosphinganine reductase, sphinganine/sphingosine N-acyltransferase, and dihydroceramide desaturase (22), whereas the salvage pathway requires only the N-acyltransferase (23) (Fig. 1). In addition, GSLs from the extracellular space are incorporated in the plasma membrane and internalized along the lysosomal pathway (23). In mammalian cells, the most abundant derivatives of GlcCer are gangliosides, for which lactosylceramide serves as a precursor (Fig. 1). Gangliosides may have either one (a series), two (b series), or three (c series) sialic acid (SiaAc) residues linked to the 3-position of the inner galactose moiety or may lack SiaAc at this position (0 series) (23). Complex gangliosides have another SiaAc moiety linked to the 3-position of the terminal galactose (23).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cells and Cell Culture—Chinese hamster lung fibroblasts, referred to here as Don wt, mouse melanoma cells (B16), Chinese hamster ovary cells (CHO-K1), and human larynx carcinoma cells (HEp-2) were from ATCC. The UDP-Glc-deficient mutant of Don wt cells, Don Q, which has a defective UDPG:PP, the spontaneous revertant of this mutant, Don QR, and the UDPG:PP transfectant clone. B9 was described previously (18). A proteoglycan-deficient mutant of CHO cells, referred to here as pgsG-110, which has a defective UDP-GlcUA:Gal1,3Gal-R glucuronosyltransferase I (GlcUAT-I), and the GlcUAT-I transfectant clone pgs-T were described previously (20). A ganglioside-deficient mutant of B16 cells, referred to here as GM95, which has a defective UDP-Glc ceramide glucosyltransferase (UDPG:CGT, EC 2.4.1.80
[EC]
), and the UDPG:CGT transfectant clone CG-1 were described previously (29). Don wt, Q, QR, B9, B16, GM95, and CG-1 cells were cultivated at 37 °C in Eagle's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 5 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) in a humid atmosphere containing 5% CO2. CHO, pgs-110, and pgs-T cells were grown in Ham's F-12 medium (Invitrogen), supplemented as above.
Cytotoxicity Assays—Cells in 96-well plates grown to 80–90% of confluence were exposed to Cp-PLC or to wheat germ lectin in 200 µl of medium/well, and cell viability was assessed 24 h later using a neutral red assay (18). Briefly, the cells were incubated for 2 h with 200 µl/well of neutral red (50 µg/ml) dissolved in supplemented culture medium, and the incorporated dye was extracted with 100 µl of acetic acid/ethanol/water (1:50:49), before recording the absorbances at 540 nm. Cell survival was expressed as a percentage, considering as 100% the value of parallel cultures incubated with only medium or with medium and drugs at the indicated concentrations. Assays were performed with 3–6 replicate samples. When not shown, the standard deviation was <10%.
Pharmacological or Enzymatic Treatments and Evaluation of Their Effects—Cells were preincubated for 18–24 h with 1-deoxynojirimycin (1–90 µg/ml), castanospermine (1–90 µg/ml), swainsonine (1–90 µg/ml), brefeldin A (0.1–9 µg/ml), monensin (1–90 µg/ml), tunicamycin (0.1–9 µg/ml), -chloroalanine (1–90 µg/ml), L-cycloserine (3–300 µg/ml), myriocin (3–300 µg/ml), fumonisin B1 (0.3–30 µM), PPMP (0.3–30 µg/ml), C. perfringens large sialidase (0.3–100 milliunits/ml) or V. cholerae sialidase (0.3–100 milliunits/ml) or for 2 h with galactocerebrosides (90–900 µg/ml), gangliosides (90–900 µg/ml), in supplemented medium containing 10% FBS, or for 18–24 h in Opti-MEM (Invitrogen) with S. plicatus endoglycosidase H (1–9 milliunits/ml) or C. meningosepticum endoglycosidase F1 (1–9 milliunits/ml).
Proteins in lysates of untreated and treated cells were submitted to SDS-PAGE and electroblotted onto 0.45-µm nitrocellulose membranes as described (17). Carbohydrate moieties in glycoproteins were oxidized with periodate and detected using biotin-hydrazide and streptavidin-alkaline phosphatase with the GlycoTrack carbohydrate detection kit (Oxford Glycosystems, Abingdon, UK).
GSLs were isolated from trypsinized cells and pelleted by centrifugation at 4 °C at 300 x g for 10 min. Cell pellets were lyophilized and extracted twice with chloroform/methanol 1:1 (v/v). The combined extracts were reduced to 25% of the original volume and kept at 4 °C overnight, and then the insoluble material was removed by centrifugation at 4 °C at 100 x g for 10 min. The supernatant was dried under nitrogen and lyophilized, and the lipids were partitioned twice in 6 ml of diisopropyl ether/1-butanol, 60:40 (v/v), and 3 ml of 0.1% aqueous NaCl. The aqueous phase was lyophilized and redissolved in 300 µl of distilled water. Salts were removed by gel filtration in Sephadex G-50. HPTLC separation and immunostaining were performed as described (30) using mAb DH2 (26) or a rabbit anti-glucosylceramide antiserum (31). Densitometric scanning analysis was performed with a Shimadzu CS-930 HPTLC scanner.
To evaluate ganglioside incorporation into the membrane, cells were trypsinized, centrifuged, and washed once with PBS, and the cell pellet was resuspended and fixed with 4% formaldehyde for 30 min at 37 °C. After three washes with PBS, cells were incubated with 20 µg/ml of the ganglioside-specific mAbs for 1 h on ice. After three washes with PBS, cells were incubated with fluorescein-labeled anti-mouse immunoglobulins for 30 min on ice. Flow cytometry analysis was performed on a FACSort flow cytometer (BD Biosciences). Data were collected and analyzed using CellQuest software (BD Biosciences).
To evaluate the effects of the treatments in sensitivity to toxins, cells were grown to 60–80% of confluence in 96-well plates and exposed to the treatments for the mentioned periods of time before and during exposure to serial dilutions of the toxins. Cell viability was measured 24 h later using the neutral red assay as described. At the concentrations used, none of the substances by itself caused significant cytotoxicity (viability was higher than 95% in comparison with untreated cells). In control experiments after ganglioside addition, mild trypsinization was carried out with 0.1% trypsin in phosphate-buffered saline (PBS), pH 7.8, for 15 min at 37 °C.
Hydrolysis of Choline-containing Phospholipids from Prelabeled Cells—Cells were incubated with 5 µCi/ml [methyl-3H]choline (Amersham Biosciences) for 18 h, washed three times with Hanks' balanced salt solution, and exposed to Cp-PLC (20 ng/ml) in supplemented medium for 0.5, 1.5, 2, 4, and 6 h. The amount of radioactive choline incorporated into membranes and the choline remaining after Cp-PLC treatment were determined by counting the radioactivity in the lipid phase after extraction with chloroform/methanol (18). The released counts/min were measured and related either to the amount of cellular protein determined in parallel cultures using the Bio-Rad DC protein assay or to the total counts/min incorporated into the membranes.
Release of Lactate Dehydrogenase—The lactate dehydrogenase (LDH, EC 1.1.1.27 [EC] ) released to the medium after exposure of cells to toxins for 2 h was determined by using a kit from Roche Applied Science. LDH release was expressed as a percentage, considering as 100 and 0% the values of parallel cultures exposed to 0.1% Triton X-100 or medium only. Assays were performed with 3–4 replicate samples.
Myotoxicity Studies—Myotoxicity was estimated by measurement of the creatine kinase (CK) activity in the plasma after intramuscular injection of the toxin in the right gastrocnemius in groups of five CD-1 mice, as described (24). Animals were housed, fed, and handled according to the principles and practices approved by the Institutional Committee for Care and Handling of Experimental Animals of Universidad de Costa Rica. The CK activity in plasma was determined using a kinetic assay (Sigma, CK-10) 3 h after injection of 0.7 µg of Cp-PLC alone or together with different amounts of C. perfringens sialidase.
Membrane-disrupting Effect in Liposomes—Unilamellar liposomes composed of cholesterol and either PC or SM (in 1:1 molar ratio) encapsulating carboxyfluorescein were prepared as described (32). In some cases purified gangliosides were incorporated into the liposomes at concentrations of 100, 33, and 11 µg/ml (which would correspond to PC/ganglioside or SM/ganglioside molar ratios of about 30:1; 90:1, and 270:1). After a 20-min exposure to Cp-PLC (20 µg/ml), fluorescence at 520 nm (excitation at 485 nm) was measured using a Fluoroskan Ascent fluorimeter (PerkinElmer Life Sciences). Samples were assayed in triplicate. Carboxyfluorescein release was expressed as a percentage, considering as 100 and 0% the values of liposomes exposed for 1 h to 1% Triton X-100 at 37 °C or to borate saline buffer, pH 7.6, respectively.
Biosensor Studies—Analysis was performed using a BIAcore 2000 biosensor and the carboxymethyl-dextran-coated sensor chip, CM5, or the lipophilic pioneer L1 sensor chip (Pharmacia Biosensor, Uppsala, Sweden). GT1b ganglioside was immobilized onto a CM5 surface as described previously (27). Briefly, ganglioside GT1b was dissolved in 50% methanol/ethanol at a final concentration of 1 mg/ml, and 50 µl of this solution was diluted (1:3 v/v) in running buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2) and injected at a flow rate of 5 µl/min over the unmodified surface. The surface was then washed with 10 mM NaOH until a stable base line was obtained (27). Approximately 0.12 ng/mm2 (120 RU) was immobilized onto the sensor surface. For immobilization onto the L1 chip, GT1b was incorporated in PC vesicles (100 µg of GT1b/1 mg of PC), and the surface was then washed with 10 mM NaOH to remove unattached vesicles. Approximately 3.0 ng/mm2 (3028 RU) was immobilized onto the sensor surface, whereas PC vesicles (
3.2 ng/mm2, 3254 RU) were immobilized onto the control surface. mAbs, Cp-PLC, or C-PLC were buffer-exchanged into the biosensor running buffer using a Fast desalt column (PC 3.2/10) connected to a SMART system (Amersham Biosciences). Anti-GT1b ganglioside IgG mAbs 101 and 102 (28) and the anti-GD3 ganglioside IgG mAb KM871 (27) were injected (30 µl, 165 nM) at a flow rate of 10 µl/min over immobilized GT1b ganglioside to confirm surface specificity. Cp-PLC or C-PLC were also injected (30 µl and 2.35 and 3.5 mM, respectively) at a flow rate of 10 µl/min over immobilized GT1b ganglioside. The sensorgrams shown have been subtracted with the corresponding signal obtained when the sample was passed over a nonderivatized CM5 surface or over the PC control surface (L1 chip).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-mannosidase II and thus preventing further glycoprotein processing at the Golgi apparatus (33). Brefeldin A and monensin interfere with the organization of the Golgi apparatus and thereby interrupt the trafficking of glycoproteins through this compartment (34). As reported for other cell lines, growth of Don wt cells in the presence of deoxynojirimycin, castanospermine, swainsonine, brefeldin A, or monensin altered the glycoprotein pattern (not shown). However, none of these treatments increase the sensitivity of Don wt cells to Cp-PLC (Fig. 3A, bars 1–5), in comparison with untreated cells. Consistently, the sensitivity of Don wt cells to the cytotoxic effect of Cp-PLC was not affected when the cells were pretreated neither with endoglycosidase H, which cleaves high mannose and hybrid N-linked oligosaccharides from glycoproteins, nor with endoglycosidase F, which cleaves high mannose, hybrid, and biantennary complex N-linked oligosaccharides (Fig. 3A, bars 6 and 7). In contrast, the exposure to the same inhibitors of glycoprotein processing/transport or endoglycosidases changed significantly the cellular sensitivity to the cytotoxic effect of wheat germ lectin (Fig. 3B), which requires binding to cell surface glycoproteins in order to cause cytotoxicity (35). These results corroborate that the treatments with the inhibitors or the enzymes had the predicted effect and indicate that a reduction in cell surface N-linked glycoproteins does not increase the cellular sensitivity to Cp-PLC. However, pretreatment of Don wt with tunicamycin, an inhibitor of the N-acetylglucosamine phosphotransferase, which blocks the synthesis of all N-linked glycoproteins (21), was found to increase the cellular sensitivity to both Cp-PLC and wheat germ lectin, in comparison with control cells (Fig. 3, A and B, bars 8). Because tunicamycin also blocks the synthesis of GSLs (36–38), we were prompted to study the effects of specific inhibitors of GSLs synthesis in the cellular sensitivity to Cp-PLC.
A Specific Reduction in Cellular Gangliosides Increases the Sensitivity to the Cytotoxic Effect of Cp-PLC—We initially assessed the role of GSLs in the cellular sensitivity to Cp-PLC by treating the cells prior to and during the toxin exposure with various inhibitors of the enzymes involved in GSLs synthesis (see Fig. 1). Myriocin, L-cycloserine, and -chloroalanine inhibit the serine palmitoyltransferase, blocking the pathway of ceramide synthesis de novo (22). Fumonisin B1 inhibits the sphinganine/sphingosine fatty-acyl-CoA N-acyltransferase, blocking both ceramide synthesis de novo and the salvage pathway (22), whereas PPMP inhibits UDPG:CGT, the enzyme that catalyzes glucosylceramide synthesis (39). The treatment of Don cells with these substances reduces the synthesis of glucosylceramide and GM3 to 20–35% in comparison with untreated cells (not shown) and all increased significantly their sensitivity to Cp-PLC (Fig. 4A). In contrast, none of these GSLs synthesis inhibitors affected the cellular sensitivity to the cytotoxic effect of the B. cereus PLC (not shown). Similar results were obtained with the same treatments in the cell lines CHO and HEp-2 (not shown). These results suggest that the reduction of glucosylceramide-derived GSLs specifically sensitizes different cell lines to Cp-PLC. To substantiate this conclusion further, we compared the sensitivity to Cp-PLC of two cell lines having extreme differences in their GSLs content due to a genetic reason. We used the ganglioside-deficient mouse melanoma cell line GM95, which has an impaired activity of UDPG:CGT (29), and the clone CG-1, which is a stable transfectant having a wild type UDPG:CGT and has the same ganglioside content as the parental cell (29). Remarkably, the ganglioside-deficient cell GM95 was found to be 105 times more sensitive to Cp-PLC than the transfectant clone CG-1, which is as resistant to the cytotoxic effect of the Cp-PLC as the parental B16 cell (Fig. 4B). In contrast, GM95, CG-1, and B16 exhibited the same relative resistance to the B. cereus PLC (not shown). These results conclusively demonstrated that a cellular deficiency of glucosylceramide-derived GSLs specifically causes hypersensitivity to the cytotoxic effect of Cp-PLC. Indeed, the content of the ganglioside GM3, evaluated by HPTLC and immunostaining with mAb DH2, was found to be similar in Don Q and GM95 cells (2.4 ± 0.3 and 2.0 ± 0.4 nmol/108 cells respectively) and is significantly reduced (about 4 and 19 times, respectively) in comparison with that of the corresponding wild type cells Don wt and B16.
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To evaluate whether the sensitization to Cp-PLC caused by tunicamycin was preventable by gangliosides, cells were treated with tunicamycin in the presence of different concentrations of bovine brain gangliosides. This mixture fully protected Don wt cells (Fig. 5A) or CHO cells (not shown) from the sensitization caused by tunicamycin, thus demonstrating that the sensitization caused by tunicamycin was indeed due to the inhibition of ganglioside synthesis rather than to the inhibition of N-linked glycoprotein synthesis.
To determine whether protection from the cytotoxic effect of Cp-PLC is conferred by specific type(s) of gangliosides, experiments were performed adding different gangliosides to the cells. Pretreatment of Don Q cells with the ganglioside asialo-GM1a did not protect from the cytotoxic effect of Cp-PLC (Fig. 5B). However, pretreatment with gangliosides of the a series (GM3, GM1a, or GD1a) or the b series (GD1b or GT1b) (Fig. 1) increased cell resistance to Cp-PLC in a dose-dependent manner (Fig. 5B). The protective effect increased with the content of SiaAc of the ganglioside oligosaccharide chain, indicating that multiple SiaAc units of complex gangliosides play a protective role. Remarkably, the protective effect of GM3 and GT1b could be reduced or even abolished if they are added in the presence of the "C. perfringens large sialidase" (Fig. 5C), which catalyzes the hydrolysis of 2,3 SiaAc residues in gangliosides (41). These results further substantiate the conclusion that the SiaAc of these GSLs play a role in their cytoprotective effect from Cp-PLC.
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Cp-PLC Causes Disruption of the Cellular Membrane in Ganglioside-deficient Cells—We have shown previously that there are no significant differences in the incorporation of [3H]choline among cells with different UDP-Glc levels (18). We now compared the degradation of choline-containing phospholipids and the disruption of the cellular membrane caused by Cp-PLC in the cell lines Don wt, Q, and QR, which differ in their cellular UDP-Glc concentration (17). The degradation of choline-containing phospholipids induced by Cp-PLC, measured as the release of radioactive choline from prelabeled cells, occurred in a dose- and time-dependent manner (Fig. 6, A and B). Remarkably, Cp-PLC induced a higher release of labeled choline from the membrane of Don Q than from Don wt or Don QR cells, which have partially compensated the UDP-Glc deficiency (17) (Fig. 7, A and B). Furthermore, it also induced a higher release of LDH from Don Q than from the UDPG:PP transfectant clone B9 (Fig. 8A). Similarly, the release of LDH induced by Cp-PLC from GM95 cells was higher than that induced from the UDPG: CGT transfectant clone CG-1 (Fig. 8A). In contrast, B. asper myotoxin II, a membrane-disrupting toxin with broad cytolytic specificity (25), induced a similar LDH release from Don Q and GM95 as from their transfectant counterparts (Fig. 8B). Therefore, these data show that Cp-PLC causes extensive membrane damage in Don Q and GM95 cells and suggest that their hypersensitivity to this toxin depends, at least in part, on an increased disruption of the cellular membrane.
Gangliosides Protect PC- or SM-containing Liposomes from the Membrane-disrupting Effect of Cp-PLC—Although Cp-PLC has a broad spectrum of substrates (1), it only disrupts artificial membranes made of cholesterol and PC or SM, but not those made of cholesterol and phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol (42). The influence of the addition of gangliosides on the susceptibility of artificial membranes to disruption by Cp-PLC was studied. Incorporation of asialo-GM1a to PC- or SM-containing liposome assays did not protect them from the membrane-disrupting effect of Cp-PLC (Fig. 9, A and B). However, incorporation of gangliosides of the a series (GM1a or GD1a) or b series (GD1b or GT1b) readily prevented the membrane disruption caused by Cp-PLC in a dose-dependent manner (Fig. 9, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Our observations shed light on general issues of muscle pathology during clostridial myonecrosis. When injected intramuscularly, Cp-PLC causes rapid and extensive damage to muscle fibers, inducing a significant release of CK to the plasma (24). However, the reason for the high sensitivity of muscle cells to this toxin is not clear. Muscle is known to have the lowest concentration of complex gangliosides among all mammalian tissues studied so far (45, 46). Therefore, our findings offer a possible explanation for the high susceptibility of muscle fibers to the cytotoxic effect of Cp-PLC. A UDP-Glc deficiency has been reported to occur in the muscular tissue during diabetes (47), probably as a consequence of the defective glucose transport and/or phosphorylation (48). The effect of this metabolic deficiency on ganglioside synthesis could be a predisposing factor to the severity of the tissue damage caused by C. perfringens infections in these patients.
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The damage inflicted by Cp-PLC to the muscle tissue during C. perfringens infections could be also potentiated by its effects on diverse signal transduction pathways in endothelial cells, platelets, and neutrophils (7). In these cells, Cp-PLC leads to the uncontrolled production of several intercellular mediators and adhesion molecules, which alters the traffic of neutrophils to the infected tissue and promotes thrombotic events leading to further ischemia (7). Because UDPG:CGT expression decreases rapidly in cells exposed to low oxygen tension (53), the perfusion deficit caused by Cp-PLC in the infected tissue might lead to a reduction in cellular gangliosides, further increasing the susceptibility of muscle cells to its membrane damaging effect.
The capacity of Cp-PLC to disrupt artificial membranes depends on its ability to bind and enzymatically degrade their constituent phospholipids (54). We showed that the disruption of PC- or SM-containing liposomes by Cp-PLC is prevented by the incorporation of gangliosides into the liposomes membrane. It has been reported that the incorporation of gangliosides into artificial membranes does not affect the binding of Cp-PLC (55). Therefore, the inhibition of the membrane-disrupting effect of Cp-PLC by gangliosides could result from a direct interaction of the Cp-PLC with the SiaAc units, which would prevent it from reaching the acyl-ester bonds to be cleaved. However, the results of the biosensor studies excluded that possibility. Because gangliosides reduce membrane fluidity and shift the cut-off pressure to lower values (56), their inhibitory effect on the Cp-PLC membrane-disrupting activity could result from a tighter packing of the phospholipids in the target membrane. Alternatively, because the bulky head groups of gangliosides protrude from the interface and create a polar and hydrated microenvironment (56), the inhibition could be due either to an alteration of the availability of substrate due to a steric effect or to the electrostatic changes induced at the interface. The latter possibility is supported by the fact that GD1a and GT1b, which carry higher number of charges, exert the highest inhibition.
The three-dimensional structure of Cp-PLC shows two domains joined by a short flexible linker region (57). The N-terminal domain contains the active site, whereas the C-terminal domain, which is needed to interact with aggregated phospholipids, is analogous to C2 domains of intracellular eukaryotic proteins involved in signal transduction (57). Recent crystallographic studies have revealed that Cp-PLC exists in two conformations as follows: an open active form and a closed inactive form. In the open form three Zn2+ ions are bound, and the active site is accessible (58). In the closed form there are only two Zn2+ ions bound, and the active site is hindered by the loop encompassing residues 132–149 (58). It is suggested that the binding of the Cp-PLC C-terminal domain to the target membrane induces a conformational change in the N-terminal domain, which uncovers the active site, allowing hydrolysis of the aggregated phospholipid substrates (58). The changes induced by the reduction of the gangliosides content of the target membrane could favor that Cp-PLC acquires and/or maintains its active conformation. Regardless of the mechanism, our results demonstrate that the reduction of gangliosides in the target membrane increases the sensitivity to the Cp-PLC-induced disruption.
Most interestingly, gangliosides have been reported to modulate the activities of some other membrane-interacting enzymes in vitro: classical protein kinases C (PKCs), secreted class I phospholipase A2, and the B. cereus PLC (59–62). Gangliosides inhibit the responsiveness of classical PKCs to diacylglycerol (59). Although the mechanism of this inhibition has not been clarified, it is worth noting that classical PKCs possess a C2 domain (63), analogous to that of Cp-PLC (57). Gangliosides modulate the enzymatic activity of pancreatic phospholipase A2 without acting directly on the active center or the interfacial recognition region of the enzyme (55, 60). In addition, gangliosides also inhibit the fusion of large unilamellar liposomes induced by the B. cereus PLC (61, 62), which lack the C2-like domain present in Cp-PLC. This inhibition seems to occur at the level of the lipid-water interface, affecting both the maximum rate of catalysis of the enzyme and the availability of the substrate (61, 62). It is not known whether Cp-PLC could induce a similar effect in that type of liposomes, but it is clear that its cytotoxic effect is not associated with fusion of the cellular membranes of the target cells and syncytia formation (15). Considering the substantial structural differences between B. cereus PLC and Cp-PLC, it is difficult to speculate whether the mechanism by which gangliosides inhibit membrane fusion induced by the first enzyme is somehow related to the mechanism by which they inhibit the cytotoxic effect of the latter.
Previous studies with genetically engineered Cp-PLC variants have shown that its catalytic capacity is required for cytotoxicity (18), supporting a role for enzymatic phospholipids degradation and membrane damage in cell death. The capacity of cells to synthesize PC, required for an adaptive response of cells to Cp-PLC, is needed for cell resistance to this toxin, and therefore, cells with a lowered capacity to synthesize PC are hypersensitive to Cp-PLC (64). However, we have found previously that a cellular UDP-Glc deficiency is not associated with a defective synthesis of choline-containing phospholipids (18). The present data showed that Cp-PLC causes a higher degradation of choline-containing phospholipids in cells deficient in UDP-Glc than in cells with normalized levels of this metabolite. Therefore, the cytotoxicity of Cp-PLC in UDP-Glc deficient cells correlates with the extent of hydrolysis of choline-containing phospholipids of the plasma membrane and not with differences in the phospholipids-synthesizing metabolic pathways. Thus, it seems likely that upon Cp-PLC treatment in ganglioside-deficient cells, the synthesis of choline-containing phospholipids, albeit normal, cannot cope with the increased membrane degradation, leading to structural membrane damage.
In conclusion, this work provides new insights toward understanding the factors that determine the susceptibility to the toxic effect of Cp-PLC both in cultured cells and in the muscular tissue. The data provide compelling evidence that a decrease in the cellular content of gangliosides increases the sensitivity to the cytotoxic effect of Cp-PLC and showed that the disruption of the plasma membrane caused by this toxin is potentiated by the "large C. perfringens sialidase." This knowledge might provide a rationale for the development of novel therapeutic strategies to reduce tissue damage during clostridial myonecrosis.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, 2060 San José, Costa Rica. Tel.: 506-2290344 or 506-2293135; Fax: 506-2920485; E-mail: aalape{at}cariari.ucr.ac.cr.
1 The abbreviations used are: PLCs, phospholipases C; SM, sphingomyelin; PC, phosphatidylcholine; Cp-PLC, C. perfringens PLC; UDPG: PP, UDP-Glc pyrophosphorylase; ER, endoplasmic reticulum; GSL(s), glycosphingolipid(s); GlcCer, glucosylceramide; GalCer, galactosylceramide; SiaAc, sialic acid; C-PLC, C. perfringens PLC C-terminal domain; B. asper MT II, B. asper myotoxin II; mAb(s), monoclonal antibodies; L-cycloserine, L-4-amino-3-isoxazolidinone; PPMP, DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; GlcUAT-I, UDP-GlcUA:Gal1,3Gal-R glucuronosyltransferase I; UDPG:CGT, UDP-Glc: ceramide glucosyltransferase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HPTLC, high performance thin layer chromatography; LDH, lactate dehydrogenase; CK, creatine kinase; PKC(s), protein kinase(s) C; wt, wild type; CHO, Chinese hamster ovary; RU, resonance units.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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