Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flores-Díaz, M.
Right arrow Articles by Thelestam, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flores-Díaz, M.
Right arrow Articles by Thelestam, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 38, 24433-24438, September 18, 1998


UDP-glucose Deficiency Causes Hypersensitivity to the Cytotoxic Effect of Clostridium perfringens Phospholipase C*

Marietta Flores-DíazDagger §, Alberto Alape-GirónDagger §parallel , Richard W. Titball**, Michael MoosDagger Dagger §§, Isabelle Guillouard¶¶, Stewart Cole¶¶, Angela M. Howells**, Christoph von Eichel-StreiberDagger Dagger §§, Inger FlorinDagger , and Monica ThelestamDagger ||

From the Dagger  Microbiology and Tumorbiology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden, § Instituto Clodomiro Picado, Facultad de Microbiología and  Departamento de Bioquímica, Facultad de Medicina, Universidad de Costa Rica, San José, Costa Rica, ** Defense Evaluation and Research Agency, Chemical and Biological Defense Establishment Porton Down, Salisbury, Wiltshire Sp4 OJQ, United Kingdom, Dagger Dagger  Institut für Medizinische Mikrobiologie und Hygiene, Verfügungsgebäude 55101 Mainz, Germany, and ¶¶ Unité de Génetique Moléculaire Bactérienne, Institut Pasteur, 75724 Paris Cédex 15, France

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 105 times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Clostridium perfringens phospholipase C (PLC1), also called alpha -toxin, is the major virulence factor in the pathogenesis of gas gangrene (1-3). It is lethal, cytotoxic, hemolytic, necrotizing, and induces platelet aggregation (4, 5). This toxin displays lecithinase and sphingomyelinase activities, but the molecular mechanism by which it induces an extensive destruction of tissues remains unknown (3).

Structurally, the C. perfringens PLC is a single-chain zinc metalloenzyme comprising two domains joined by a hinge region: a catalytic N-terminal domain of 240 amino acid residues and a C-terminal domain of 120 residues (4, 5). The N-terminal domain has extensive amino acid sequence similarity with the nonlethal Bacillus cereus phosphatidylcholine (PC)-hydrolyzing phospholipase C (PC-PLC) (6). The crystal structure of the B. cereus PC-PLC was used as the basis for site-directed mutagenesis studies, which indeed showed that the critical amino acid residues for Zn2+ binding and catalysis are conserved between both enzymes (7-9). The C-terminal domain of the C. perfringens PLC, which has no counterpart in the B. cereus PC-PLC (10, 11), mediates interactions with membrane phospholipids in a calcium-dependent manner (12).

A Chinese hamster fibroblast mutant cell line (Don Q) that is 105 times more sensitive to C. perfringens PLC than the parental cell (Don wt) was previously isolated (13). Don Q is 104 times more resistant to Clostridium difficile glucosyltransferase toxin B (TcdB) (14) than Don wt, due to a UDP-Glc deficiency (15) caused by a recessive point mutation in the UDP-Glc pyrophosphorylase (UDPG:PP) gene (16). A spontaneous revertant cell (Don QR) was isolated from Don Q and showed to have the same sensitivity to TcdB as Don wt and a partially compensated UDP-Glc level (15, 16).

UDP-Glc deficiency occurs in cells cultured under hypoxia or in low glucose-containing media (17-20). Since these conditions are two of the hallmarks of ischemia, it is likely that a low UDP-Glc level occurs also in ischemic tissues (16). The aim of this work was to determine whether a cellular UDP-Glc deficiency affects the sensitivity to the cytotoxic effect of C. perfringens PLC as well as to map which parts of this toxin are needed for its cytotoxicity.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell Cultures-- Diploid Chinese hamster lung fibroblasts (Don cells, ATCC number CCL 16; Flow Laboratories, Irvine, Scotland; referred to here as Don wt), a mutant of this cell line (13) (referred to here as Don Q), and a spontaneous revertant of this mutant (16) (referred to here as Don QR) were cultivated in flasks (75 or 175 cm2; A/S Nunc, Roskilde, Denmark) or in polystyrene 96-well culture plates (Nunc) in Eagle's minimal essential medium (Flow) supplemented with 10% fetal bovine serum, 5 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) in a humid atmosphere containing 5% CO2 at 37 °C. Transfectant cells were cultured in the same medium in the presence of geneticin (0.4 µg/ml) (Life Technologies, Inc.).

Production and Purification of Toxins-- Wild type recombinant Clostridium bifermentans and C. perfringens PLCs, mutant variants (H11S, H68S, and D56N), and fragments of C. perfringens PLC were expressed in Escherichia coli and purified as described previously (8, 21, 22). The production of the N-terminal C. bifermentans-C-terminal C. perfringens hybrid PLC will be published separately.2 C. difficile TcdB and nonrecombinant C. perfringens PLC were purified as described (23-25). The B. cereus PC-PLC was from Calbiochem (San Diego, CA).

Protein Determination-- Protein concentrations were determined in 96-well plates using the Bio-Rad DC protein assay and bovine serum albumin as a standard.

Cytotoxicity Assays-- Cells were seeded in 96-well plates (500 to 1000 cells/well), cultivated to 90% confluency, and exposed at 37 °C to serial 10-fold dilutions of the purified toxins: C. difficile TcdB (500 µg/ml) or phospholipases C (recombinant C. bifermentans phospholipase C, B. cereus PC-PLC, wild type nonrecombinant or recombinant C. perfringens PLC) (214 µg/ml) in 200 µl of medium/well. Cell viability was measured 24 h later using a neutral red assay as described (16). All cytotoxicity assays were performed at least three times with 4-6 replicate samples in each experiment. The sensitivity to C. perfringens PLC was also determined in the Don wt cell line after a 24-h pretreatment with 2-deoxyglucose (15 mg/ml), 2-deoxygalactose (8 mg/ml), or D-galactosamine (6 mg/ml).

Choline Incorporation into Membranes-- Cell cultures were incubated with 0.4 µCi/ml [methyl-3H]choline (Amersham Pharmacia Biotech) for 90 min. The cells were then washed three times with Hanks' balanced salt solution and incubated in fresh medium at 37 °C. After 0.5, 2, 4, and 6 h of incubation, the cellular lipids were extracted with chloroform-methanol (26), and the choline incorporated into membranes was measured by counting the radioactivity in the lipid phase using a microtiter plate beta -counter. The cpm were related to the amount of cellular protein determined in parallel cultures.

Stable Transfection of Bovine UDPG:PP to Don Q and QR Cells-- The bovine UDPG:PP cDNA cloned in pUC18 (27), kindly provided by Professor K. Tanizawa (University of Osaka), was digested with EcoRI (Fermentas AB, Graiciuno, Lithuania). The 1.69-kilobase insert was separated in an agarose gel, cut out, eluted using the QIAEX kit (Qiagen, Hilden, Germany), and subcloned in pCDNA3 (Invitrogen Corp., Carlsbad, CA) at the EcoRI site. The ligation product was used to transform E. coli JM101 using a gene pulser electroporation instrument (Bio-Rad). After isolation of transformants, plasmids were digested with Acc651 and KpnI (Fermentas AB), and the fragments were separated in agarose gels to determine the orientation of the insert. The Chinese hamster Don Q and QR cell lines were transfected with the pCDNA3 vector containing the UDPG:PP insert in the sense and in the antisense orientation, respectively. Transfection was performed using Lipofectin (Life Technologies, Inc.) as described previously (28). Stable transfectant clones were obtained by minimal dilution and selected by cultivation in the presence of geneticin (500 µg/ml).

Neutralization of C. perfringens PLC Cytotoxicity-- The monoclonal antibodies (mAbs) 3A4D10 and 3A4F2 against the holotoxin were produced and purified as described previously (29). Polyclonal antibodies against the recombinant C-terminal domain of C. perfringens PLC were generated as described (30). The capacity of these antibodies to neutralize the cytotoxic effect of the recombinant C. perfringens PLC was determined by preincubation of this toxin (0.12 µg/ml) with serial 2-fold dilutions of the antibodies (initial concentration 1 mg/ml) for 1 h at 37 °C. Then 200 µl of the mixtures were added to cells and incubated for 24 h, the cells were washed twice with phosphate-buffered saline, and cell viability was determined using the neutral red assay as described (16).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Don Q Is Hypersensitive Specifically to the Cytotoxic Effect of C. perfringens PLC-- As previously shown, Don Q and wt cells exhibit the same sensitivity to phospholipases A2, B, and D, whereas Don Q is hypersensitive to the cytotoxic effect of C. perfringens PLC (13). To determine whether Don Q is hypersensitive to all PC-hydrolyzing phospholipases C, the sensitivity of Don wt, Q, and QR to two other phospholipases C was studied. The B. cereus PLC showed the same cytotoxicity to the three cell lines (Fig. 1A) whereas the C. bifermentans phospholipase C was not cytotoxic to any of the cells (data not shown). Thus, Don Q seems to show a specific hypersensitivity to C. perfringens PLC, a hypersensitivity that has been lost in Don QR (Fig. 1B).


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Dose response of Don wt (black-triangle), Q (bullet ), and QR (open circle ) cells to treatment with B. cereus PC-PLC or C. perfringens PLC. Cells were exposed for 24 h at 37 °C to serial 10-fold dilutions of B. cereus PC-PLC (A) or C. perfringens PLC (B). Cell viability was determined using the neutral red assay as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in control cells incubated with only medium.

The C. perfringens PLC Hypersensitivity of Don Q Is Not Due to a Decreased Synthesis of PC-- Hypersensitivity to C. perfringens PLC has previously been found in a mutant cell with a lowered capacity to synthesize PC (31-33). To determine whether the C. perfringens PLC hypersensitivity of Don Q is caused by a defective synthesis of PC, we measured the incorporation of [3H]choline into the membranes of Don wt, Q, and QR (Fig. 2). Since no significant differences in the [3H]choline incorporation were detected among the three cell lines, the hypersensitivity of Don Q to C. perfringens PLC does not seem to be due to a defective PC synthesis.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Incorporation of labeled choline in the membrane lipids of wt (black-triangle), Q (bullet ), and QR (open circle ) cells. Cells were incubated with [methyl-3H]choline (0.4 µCi/ml) for 90 min, the choline was removed, and normal medium was added. The lipids were extracted after 0.5, 2, 4, and 6 h with chloroform-methanol as described under "Experimental Procedures," and the radioactivity incorporated into membranes was measured in a beta -counter. The cpm were related to the amount of cellular protein determined in lysates from parallel cultures.

The C. perfringens PLC Hypersensitivity Is Due to Cellular UDP-Glc Deficiency-- Since Don QR has partially compensated the UDP-Glc deficiency (16), the results in Fig. 1B suggested that a hypersensitivity to C. perfringens PLC is linked to the low level of UDP-Glc. Accordingly, it was observed that Don wt cells had an increased sensitivity to C. perfringens PLC after treatment with sugar analogues (Fig. 3) known to cause a cellular UDP-Glc deficiency by trapping the uridine nucleotide pool (34, 35).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   Dose response of Don wt cells pretreated with sugar analogues to treatment with C. perfringens PLC. Cells were treated with 2-deoxyglucose (open circle ), 2-deoxygalactosamine (black-square), or D-galactosamine (triangle ) as described under "Experimental Procedures" and then exposed for 24 h at 37 °C to serial 10-fold dilutions of C. perfringens PLC. Cell viability was determined using the neutral red assay as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in controls incubated with sugar analogues but without toxin. bullet , control.

To conclusively clarify the link between UDP-Glc deficiency and C. perfringens PLC hypersensitivity, stable transfectant cells with bovine UDPG:PP cDNAs were prepared. Don Q cells were transfected with a construct having the UDPG:PP insert in the sense orientation and Don QR cells with a construct having the insert in the antisense orientation. Transfectant clones were designated Q Sense (QS) and QR Antisense (QRA), whereas clones transfected with only the vector were designated Q Control (QC) and QR Control (QRC), respectively. Since the resistance of Don Q to TcdB is due to its UDP-Glc deficiency (15) and this resistance has been lost in Don QR (16), we used the sensitivity to TcdB as an indirect measure of the cellular UDP-Glc level in the transfectant clones (Fig. 4). Don QRA clones were resistant to TcdB, showing that transfection with the antisense construct indeed had induced a UDP-Glc deficiency. Correspondingly, Don QS clones were more sensitive to TcdB than QC clones, demonstrating that the UDP-Glc deficiency indeed had been compensated by the transfection. This conclusion has been further substantiated by 1H NMR measurement of the UDP-Glc concentration in lysates of the transfectant cells.3


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   Dose response of transfectant clones to treatment with C. difficile TcdB. Different clones of Don QR transfected with the UDPG:PP antisense cDNA (designated QRA2C5 (open circle ), QRA2B4 (), and QRA2B2 (triangle )) and their control (QRC (bullet )) (A) and clones of Don Q transfected with the UDPG:PP sense cDNA (designated QSG3 (open circle ), QS20 (), and QSB9 (black-triangle)) and their control (QC (bullet )) (B) were exposed for 24 h at 37 °C to serial 10-fold dilutions of C. difficile TcdB. Cell viability was determined using the neutral red assay as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in controls incubated without toxin.

The sensitivity of the transfectant clones to C. perfringens PLC was then measured (Fig. 5). Don QRA clones were as hypersensitive to the cytotoxic effect of this toxin as Don Q. Correspondingly, Don QS clones had regained the same relative resistance to C. perfringens PLC as displayed by Don wt and QR. These results therefore demonstrate that a low level of UDP-Glc really causes hypersensitivity to the cytotoxic effect of C. perfringens PLC.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 5.   Dose response of transfectant clones to treatment with C. perfringens PLC. Clones of Don QR transfected with the UDPG:PP antisense cDNA (designated QRA2C5 (open circle ), QRA2B4 (), and QRA2B2 (triangle )) and their control (QRC (bullet )) (A), and clones of Don Q transfected with the UDPG:PP sense cDNA (designated QSG3 (open circle ), QS20 (), and QSB9 (black-triangle)) and their control (QC (bullet )) (B) were exposed for 24 h at 37 °C to serial 10-fold dilutions of C. perfringens PLC. Cell viability was determined using the neutral red assay as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in controls incubated without toxin.

The Sphingomyelinase Activity and the C-domain Are Required for the Cytotoxicity of C. perfringens PLC-- Using Don Q as a model system, we undertook a mapping of which parts of the C. perfringens PLC are needed for its cytotoxic effect. Previous studies with mAbs demonstrated that mAb 3A4D10 neutralized the enzymatic activities of C. perfringens, whereas mAb 3A4F2 did not (29). The cytotoxic effect of C. perfringens PLC in Don Q was abolished by mAb 3A4D10 but not affected by mAb 3A4F2 (Fig. 6A), suggesting that the catalytic capacity of C. perfringens PLC is required for its cytotoxic effect on UDP-Glc-deficient cells. This was further substantiated by studies with enzymatically inactive C. perfringens PLC variants. Replacement of His-11 or His-68 by Ser or Asp-56 by Asn resulted in a loss of the enzymatic activities (7-9). All these three C. perfringens PLC variants were 104-105 times less cytotoxic to Don Q than the recombinant wild type PLC (Fig. 6B). These results therefore demonstrated that the catalytic capacity of C. perfringens PLC is required for its cytotoxic effect on UDP-Glc-deficient cells.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   Neutralization of C. perfringens PLC cytotoxic effect with mAbs and comparison of the cytotoxicity of recombinant C. perfringens PLC and enzymatically inactive variants. A, Serial 2-fold dilutions of mAbs 3A4D10 (black-square) and 3A4F2 (box-dot ) against C. perfringens PLC were preincubated with recombinant C. perfringens PLC (0.12 µg/ml) for 1 h at 37 °C. The mixtures were then incubated with cells for 24 h. B, cells were exposed for 24 h at 37 °C to serial 10-fold dilutions of recombinant C. perfringens PLC (black-triangle) and the mutated variant PLCs H11S (), H68S (X), and D56N (open circle ). Cell viability was determined as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in controls incubated without toxin.

A truncated version of the C. perfringens PLC containing the 249 N-terminal residues retains the lecithinase activity but lacks the sphingomyelinase activity (10). This truncated toxin had a cytotoxic potency 106 times lower than the wild type enzyme (Fig. 7A). This result therefore demonstrated that the cytotoxic effect of the C. perfringens PLC requires the C-terminal domain and the sphingomyelinase activity. The data also showed that the lecithinase activity is not enough to confer cytotoxicity.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 7.   Comparison of the cytotoxicity of recombinant C. perfringens PLC and its fragments. Neutralization of C. perfringens PLC cytotoxic effect with polyclonal antibodies and cytotoxicity of the C. bifermentans PLC and a hybrid PLC are shown. A, cells were exposed for 24 h at 37 °C to serial 10-fold dilutions of recombinant C. perfringens PLC (black-triangle), the truncated C. perfringens PLC (black-square), the C. perfringens C-domain (triangle ), the combination of the two last (open circle ), or glutathione S-transferase (X). B, serial 2-fold dilutions of polyclonal antibodies against the C-terminal domain of C. perfringens PLC (box-dot ) were preincubated with recombinant C. perfringens PLC (0.12 µg/ml) for 1 h at 37 °C, and the mixtures were incubated with cells for 24 h. black-square, control without antibody. C, cells were exposed for 24 h at 37 °C to serial 10-fold dilutions of C. bifermentans phospholipase C (triangle ) and the N-terminal C. bifermentans-C-terminal C. perfringens PLC hybrid (bullet ). Cell viability was determined using the neutral red assay as described under "Experimental Procedures." The results are expressed as the percentage of neutral red uptake in controls incubated with only medium.

The C-terminal domain, containing residues 247-370, is devoid of both enzymatic activities of the holotoxin (30). However, polyclonal antibodies against the C-domain of C. perfringens PLC prevented the cytotoxic effect of the holotoxin in Don Q cells in a dose-dependent manner (Fig. 7B), demonstrating that this domain plays an important role in the cytotoxicity exerted by this PLC. In addition, the cytotoxic effect was potentiated about 102 times when the truncated PLC and the C-terminal domain where added simultaneously (Fig. 7A). However, the C. perfringens C-terminal domain did not potentiate the cytotoxic effect of the B. cereus PC-PLC (data not shown), excluding the possibility that a simple complementation of the lecithinase with the C-terminal domain would suffice to confer full cytotoxicity. Accordingly, when the C-terminal domain of C. perfringens PLC was fused with the N-terminal domain of C. bifermentans phospholipase C, this recombinant enzyme (designated hybrid D) showed a cytotoxic potency 103 times lower than that of C. perfringens PLC (Fig. 7C). These results indicate that a specific interaction between the two domains of the C. perfringens PLC is required for full cytotoxicity.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

C. perfringens PLC is hemolytic, necrotizing, and lethal, in contrast to most other bacterial phospholipases (3) and is considered the major virulence factor in trauma-induced gas gangrene (2, 36, 37). This disease is of increasing significance in elderly and diabetic populations, especially in those who have undergone lower limb surgery, where impaired blood supply to tissues can lead to hypoxic conditions suitable for multiplication of anaerobic bacteria (38-40). C. perfringens PLC induces localized intravascular neutrophil accumulation and platelet aggregation, thus reducing the blood supply to tissues and further promoting the anaerobic environment required for spread of the bacteria (36, 41).

A UDP-Glc deficiency has been reported in the skeletal muscle of diabetic animals (42-44). This deficiency could be a consequence of the defective transport and/or phosphorylation of glucose in cells from diabetic subjects (45-47). A UDP-Glc deficiency also occurs in cells cultured under low glucose concentration or hypoxia (17-20), two of the hallmarks of ischemia.

The isolation of the UDP-Glc-deficient mutant cell line Don Q and its intriguing hypersensitivity to C. perfringens PLC were previously reported (13, 15). The reason for the low UDP-Glc level in this cell is a mutation in the UDPG:PP gene (16). In the present work we show that Don Q is specifically hypersensitive to C. perfringens PLC and not to other phospholipases C that degrade phosphatidylcholine. Since the revertant cell line Don QR has partially compensated the UDP-Glc deficiency and lost the C. perfringens PLC hypersensitivity, a link between a low UDP-Glc level and an increased sensitivity to C. perfringens PLC was hypothesized. Such a link was further supported by the finding that cells treated with sugar analogues, which induce UDP-Glc deficiency (34, 35), were more susceptible to the cytotoxic effect of C. perfringens PLC. Transfection of mutant and revertant cells with the bovine UDPG:PP cDNA in sense and antisense orientations affected their UDP-Glc levels and indeed changed their susceptibility to C. perfringens PLC. Therefore, these results conclusively demonstrate that a cellular UDP-Glc deficiency causes hypersensitivity to C. perfringens PLC.

Although a detailed three-dimensional structure of the C. perfringens PLC is not yet available, two separate domains have been identified: the N-terminal domain, which catalyzes phospholipid hydrolysis, and the C-terminal domain, which mediates interactions with membrane phospholipids (4, 5, 12). Ca2+ is required for the interaction of the toxin with membranes, whereas Zn2+ plays a structural role. Crystallographic studies have revealed that the B. cereus PC-PLC has two tightly bound and one loosely bound Zn2+ ions, which are required for the enzymatic activity (48). These Zn2+ ions are coordinated to several residues conserved in the C. perfringens PLC, which suggests that these two enzymes have the same catalytic mechanism. It has been previously shown by site-directed mutagenesis that the substitution of His-11, His-68, His-126, or His-136 in the C. perfringens PLC dramatically reduces its lecithinase and sphingomyelinase activities (7, 8), whereas their significance for cytotoxicity has not been studied. These His residues are very likely involved in the coordination of the loosely bound Zn2+ ion, which is required for C. perfringens PLC binding to membranes (7, 8). On the other hand, Asp-56, which is exposed on the surface within the active site cleft of the B. cereus PC-PLC, is also conserved in the C. perfringens enzyme. Replacement of Asp-56 in the latter enzyme abolishes its lecithinase and sphingomyelinase activities without affecting its membrane-binding capacity (8, 9).

Neutralization experiments with mAbs and polyclonal antibodies suggested that both the catalytic capacity and the C domain are required for the cytotoxic effect of the C. perfringens PLC. The enzymatically inactive C. perfringens PLC variants H11S, H68S, and D56N and the truncated enzyme having only the first 249 residues had a markedly reduced cytotoxicity. The cytotoxic effect was partially restored when the truncated phospholipase and the C-terminal domain were added simultaneously. In contrast there was not any potentiation of the cytotoxic effect when the B. cereus PC-PLC and the C. perfringens C-terminal domain where added together. Moreover, the hybrid PLC, having the C. perfringens C-terminal domain and the C. bifermentans N-terminal domain, displayed a cytotoxic potency much lower than that of C. perfringens PLC. These results showed that the lecithinase activity is not enough to confer cytotoxic activity to C. perfringens PLC and suggested that a specific interaction between the two toxin domains is needed for full cytotoxicity. In addition, the data demonstrated that both the sphingomyelinase activity and the membrane-interacting C-terminal domain are crucial for the cytotoxicity of C. perfringens PLC to UDP-Glc-deficient cells.

Treatment of cultured cells with C. perfringens PLC has been shown to activate endogenous phospholipases A2 and C, hence generating messengers such as leukotrienes, diacylglycerol, and inositol 1,4,5-triphosphate (49-52). Therefore we have considered the possibility that the cytotoxicity could be mediated by the activation of those cellular enzymes. However, we have not been able to protect Don Q cells from the cytotoxic effect of C. perfringens PLC by pretreatment with (i) inhibitors of the arachidonic acid pathway (mepacrine, indomethacin, nordihydroguaiaretic acid), (ii) inhibitors of cellular phospholipases C or D (D-609, ethanol, neomycin), or (iii) the diacylglycerol lipase inhibitor U-57908 (1,6-bis(cyclohexyloximinocarbonylamino)hexane).4 Therefore, the cytotoxicity of C. perfringens PLC on UDP-Glc-deficient cells seems to be independent of the activation of endogenous phospholipases. The interaction between this toxin and the plasma membrane in UDP-Glc-deficient cells may be favored by a putative alteration in the composition of the membrane glycolipids or glycoproteins. The C. perfringens PLC induces activation of protein kinase C and stimulates Ca2+ influx into the cytosol, hence modulating a variety of cell functions (4, 50). The role of these effects in the cytotoxicity of the C. perfringens PLC are currently being investigated in our laboratories. Regardless of the molecular mechanism of cytotoxicity, our results demonstrate that a UDP-Glc deficiency sensitizes cells to the cytotoxic effect of C. perfringens PLC. In addition the data raise the possibility that the low UDP-Glc level occurring in diabetic tissues and cells under ischemic conditions predisposes to the extensive tissue damage induced by C. perfringens PLC during gas gangrene.

Concluding Remarks-- This work demonstrated that cells with a low UDP-Glc level are hypersensitive to the cytotoxic effect of C. perfringens PLC. Using genetically engineered variants of this phospholipase, we showed that the C-terminal domain and the sphingomyelinase activity of C. perfringens PLC are required for its cytotoxic effect. Since lowered levels of UDP-Glc have been reported to occur in tissues of diabetic animals and in cells grown under low glucose concentration or hypoxia, we hypothesize that a UDP-Glc deficiency in the target tissues plays a role in the pathogenesis of gas gangrene. Furthermore we suggest that the Don Q cell line can be used as a model to elucidate the molecular mechanism of cytotoxicity induced by C. perfringens PLC on ischemic tissues.

    ACKNOWLEDGEMENTS

We thank Professor K. Tanizawa (University of Osaka) for providing the bovine UDPG:PP clone. We are also grateful to Professors J. M. Gutiérrez and B. Lomonte (Universidad de Costa Rica) for the critical reading of the manuscript.

    FOOTNOTES

* Part of this work was presented at the 8th ETOX Workshop, June 1997. This work was supported by grants from the Swedish Medical Research Council (16X-05969), the Swedish Cancer Society (3826-B96-01XAB), Vicerrectoría de Investigación, Universidad de Costa Rica (741-98-287), the Magnus Bergvall Foundation, and the Karolinska Institute Research Funds.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Recipient of a fellowship from the Karolinska International Research Training program.

§§ Supported by a grant from the Deutsche Forschungsgemeinschaft (Ei206 8-1).

|| To whom correspondence should be addressed: Microbiology and Tumorbiology Center, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden. Tel: +46-8-728 71 62; Fax: +46-8-33 15 47; E-mail: monica.thelestam{at}mtc.ki.se.

The abbreviations used are: PLC, phospholipase C; PC, phosphatidylcholine; PC-PLC, phosphatidylcholine-hydrolyzing phospholipase C; TcdB, C. difficile toxin BUDP-Glc, UDP-glucoseUDPG:PP, UDP-glucose pyrophosphorylasemAb, monoclonal antibody.

2 A. Howells, M. Jepson, H. L. Bullifent, J. Holley, J. Miller, D. Moss, and R. Titball, manuscript in preparation.

3 M. Flores-Díaz, A. Alape-Girón, M. Moos, P. Pollesello, C. Cordula, T. Bergman, C. von Eichel-Streiber, I. Florin, and M. Thelestam, manuscript in preparation.

4 M. Flores-Díaz, A. Alape-Girón, I. Guillouard, S. Cole, R. Titball, A. Basak, C. Naylor, and M. Thelestam, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Ninomiya, M., Matsushita, O., Minami, J., Sakamoto, H., Nakano, M., and Okabe, A. (1994) Infect. Immun. 62, 5032-5039[Abstract/Free Full Text]
  2. Awad, M. M., Bryant, A. E., Stevens, D. L., and Rood, J. (1995) Mol. Microbiol. 15, 191-202[CrossRef][Medline] [Order article via Infotrieve]
  3. Songer, J. G. (1997) Trends Microbiol. 5, 156-161[CrossRef][Medline] [Order article via Infotrieve]
  4. Titball, R. W. (1993) Microbiol. Rev. 57, 347-366[Abstract/Free Full Text]
  5. Basak, A. K., Stuart, D. I., Nikura, T., Bishop, D. H. L., Kelly, D. C., Fearn, A., and Titball, R. W. (1994) J. Mol. Biol. 244, 648-650[CrossRef][Medline] [Order article via Infotrieve]
  6. Leslie, D., Fairweather, N., Pickard, D., Dougan, G., and Kehoe, M. (1989) Mol. Microbiol. 3, 383-392[CrossRef][Medline] [Order article via Infotrieve]
  7. Nagahama, M., Okagawa, Y., Nakayama, T., Nishioka, E., and Sakurai, J. (1995) J. Bacteriol. 177, 1179-1185[Abstract/Free Full Text]
  8. Guillouard, I., Garnier, T., and Cole, S. (1996) Infect. Immun. 64, 2440-2444[Abstract]
  9. Nagahama, M., Nakayama, T., Michiue, K., and Sakurai, J. (1997) Infect. Immun. 65, 3489-3492[Abstract]
  10. Titball, R. W., Leslie, D. L., Harvey, S., and Kelly, D. (1991) Infect. Immun. 59, 1872-1874[Abstract/Free Full Text]
  11. Nagahama, M., Lida, H., Nishioka, E., Okamoto, K., and Sakurai, J. (1994) FEMS Microbiol. Lett. 120, 297-302[CrossRef][Medline] [Order article via Infotrieve]
  12. Guillouard, I., Alzari, P. M., Saliou, B., and Cole, S. (1997) Mol. Microbiol. 26, 867-876[CrossRef][Medline] [Order article via Infotrieve]
  13. Florin, I. (1991) Microb. Pathog. 11, 337-346[CrossRef][Medline] [Order article via Infotrieve]
  14. Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503[CrossRef][Medline] [Order article via Infotrieve]
  15. Chaves-Olarte, E., Florin, I., Boquet, P., Popoff, M., von Eichel-Streiber, C., and Thelestam, M. (1996) J. Biol. Chem. 271, 6925-6932[Abstract/Free Full Text]
  16. Flores-Diaz, M., Alape-Girón, A., Persson, B., Pollesello, P., Moos, M., von Eichel-Streiber, C., Thelestam, M., and Florin, I. (1997) J. Biol. Chem. 272, 23784-23791[Abstract/Free Full Text]
  17. Aw, T. Y., and Jones, D. P. (1984) Biochem. J. 219, 707-712[Medline] [Order article via Infotrieve]
  18. Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146[Abstract/Free Full Text]
  19. Sheed, S. F., Lutz, N. W., and Hull, W. E. (1993) NMR Biomed. 6, 254-263[Medline] [Order article via Infotrieve]
  20. Franks, S. E., Kuesel, A. C., Lutzs, N. W., and Hull, W. (1996) Anticancer Res. 16, 1365-1374[Medline] [Order article via Infotrieve]
  21. Titball, R. W., Hunter, S. E. C., Martin, K. L., Morris, B. C., Shuttleworth, A. D., Rubidge, T., Anderson, D. W., and Kelly, D. C. (1989) Infect. Immun. 57, 367-376[Abstract/Free Full Text]
  22. Tso, J. Y., and Siebel, C. (1989) Infect. Immun. 57, 468-476[Abstract/Free Full Text]
  23. von Eichel-Streiber, C., Harperath, U., Bosse, D., and Hadding, U. (1987) Microb. Pathog. 2, 307-318[CrossRef][Medline] [Order article via Infotrieve]
  24. Shoshan, M. C., Åman, P., Skog, S., Florin, I., and Thelestam, M. (1990) Eur. J. Cell Biol. 53, 357-363[Medline] [Order article via Infotrieve]
  25. Möllby, R., Thelestam, M., and Wadström, T. (1974) J. Membr. Biol. 16, 313-330[CrossRef][Medline] [Order article via Infotrieve]
  26. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  27. Konishi, Y., Tanizawa, K., Muroya, S., and Fukui, T. (1993) J. Biochem (Tokyo) 114, 61-68[Abstract/Free Full Text]
  28. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielson, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7413-7417[Abstract/Free Full Text]
  29. Logan, A. J., Williamson, E. D., Titball, R. W., Percival, D. A., Shuttleworth, A. D., Conlan, J. W., and Kelly, D. C. (1991) Infect. Immun. 59, 4338-4342[Abstract/Free Full Text]
  30. Titball, R. W., Fearn, A. M., and Williamson, E. D. (1993) FEMS Microb. Lett. 110, 45-50[CrossRef][Medline] [Order article via Infotrieve]
  31. Esko, J. D., and Raetz, C. R. H. (1980) Proc. Natl. Acad. Sci. U. S. A. 9, 5192-5196
  32. Sleight, R., and Kent, C. (1983) J. Biol. Chem. 258, 824-830[Abstract/Free Full Text]
  33. Sweitzer, T. D., and Kent, C. (1994) Arch. Biochim. Biophys. 311, 107-116[CrossRef][Medline] [Order article via Infotrieve]
  34. Sung, S. J., and Silverstein, S. C. (1985) Biochim. Biophys. Acta 845, 204-215[Medline] [Order article via Infotrieve]
  35. Pels-Rijcken, W. R., Ferwerda, W., van den Eijnden, D. H., and Overdijk, B. (1995) Glycobiology 5, 495-502[Abstract/Free Full Text]
  36. Bunting, M., Lorant, D. E., Bryant, A. E., Zimmerman, G. A., Mclntyre, T. M., Stevens, D. L., and Prescott, S. M. (1997) J. Clin. Invest. 100, 565-574[Medline] [Order article via Infotrieve]
  37. Stevens, D. L., and Bryant, A. E. (1997) Clin. Infect. Dis. 25, Suppl. 2, 160-164
  38. Rogstad, B., Ritland, S., Lunde, S., and Hagen, A. G. (1993) Infection 21, 54-56[CrossRef][Medline] [Order article via Infotrieve]
  39. Williamson, E. D., and Titball, R. A (1993) Vaccine 11, 1253-1258[CrossRef][Medline] [Order article via Infotrieve]
  40. Chapnick, E. K., and Abter, E. I. (1996) Infect. Dis. Clin. North. Am. 10, 835-855[CrossRef][Medline] [Order article via Infotrieve]
  41. Stevens, D. L., Tweten, R. K., Awad, M. M., Rood, J. I., and Bryant, A. E. (1997) J. Infect. Dis. 176, 189-195[Medline] [Order article via Infotrieve]
  42. Spiro, M. J. (1984) Diabetologia 26, 70-75[CrossRef][Medline] [Order article via Infotrieve]
  43. Sochor, M., Kunjara, S., Zaheer-Baquer, N., and McLean, P. (1991) Diabetes 40, 1467-1471[Abstract]
  44. Robinson, K., Weinstein, M. L., Lindenmayer, G. E., and Buse, M. G. (1995) Diabetes 44, 1438-1446[Abstract]
  45. Bonadonna, R. C., Del Prato, S., Saccomani, M. P., Bonora, E., Gulli, G., Ferrannini, E., Bier, D., Cobelli, C., and Defronzo, R. A (1993) J. Clin. Invest. 92, 486-494
  46. Rothman, D. L., Magnusson, I., Cline, G., Gerard, D., Kahn, C. R., Shulman, R. G., and Shulman, G. I. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 983-987[Abstract/Free Full Text]
  47. Buse, M. G., Robinson, K. A., Marshall, B. A., and Mueckler, M. (1996) J. Biol. Chem. 271, 23197-23202[Abstract/Free Full Text]
  48. Hough, E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., and Derewenda, Z. (1989) Nature 338, 357-360[CrossRef][Medline] [Order article via Infotrieve]
  49. Kent, C., Evers, A., and Haun, S. S. L. (1986) Arch. Biochim Biophys. 250, 519-525[CrossRef][Medline] [Order article via Infotrieve]
  50. Schiess, K., Kaszkin, M., Jordan, P., Seidler, L., and Kinzel, V. (1992) Biochim. Biophys. Acta 1137, 82-94[Medline] [Order article via Infotrieve]
  51. Sakurai, J., Ochi, S., and Tanaka, H. (1993) Infect. Immun. 61, 3711-3718[Abstract/Free Full Text]
  52. Sakurai, J., Ochi, S., and Tanaka, H. (1994) Infect. Immun. 62, 717-721[Abstract/Free Full Text]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 MicrobiologyHome page
R. W. Titball
Gas gangrene: an open and closed case
Microbiology, September 1, 2005; 151(9): 2821 - 2828.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Flores-Diaz, A. Alape-Giron, G. Clark, B. Catimel, Y. Hirabayashi, E. Nice, J.-M. Gutierrez, R. Titball, and M. Thelestam
A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C
J. Biol. Chem., July 22, 2005; 280(29): 26680 - 26689.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Flores-Diaz, J.-C. Higuita, I. Florin, T. Okada, P. Pollesello, T. Bergman, M. Thelestam, K. Mori, and A. Alape-Giron
A Cellular UDP-glucose Deficiency Causes Overexpression of Glucose/Oxygen-regulated Proteins Independent of the Endoplasmic Reticulum Stress Elements
J. Biol. Chem., May 21, 2004; 279(21): 21724 - 21731.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
E. S. Trombetta
The contribution of N-glycans and their processing in the endoplasmic reticulum to glycoprotein biosynthesis
Glycobiology, September 1, 2003; 13(9): 77R - 91R.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. Lai, S.D. Langley, F.W. Khwaja, E.W. Schmitt, and L.J. Elsas
GALT deficiency causes UDP-hexose deficit in human galactosemic cells
Glycobiology, April 1, 2003; 13(4): 285 - 294.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
M. Jepson, A. Howells, H. L. Bullifent, B. Bolgiano, D. Crane, J. Miller, J. Holley, P. Jayasekera, and R. W. Titball
Differences in the Carboxy-Terminal (Putative Phospholipid Binding) Domains of Clostridium perfringens and Clostridium bifermentans Phospholipases C Influence the Hemolytic and Lethal Properties of These Enzymes
Infect. Immun., July 1, 1999; 67(7): 3297 - 3301.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Flores-Díaz, M.
Right arrow Articles by Thelestam, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Flores-Díaz, M.
Right arrow Articles by Thelestam, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement