Tyrosine 331 and phenylalanine 334 in <i>Clostridium perfringens</i> α‐toxin are essential for cytotoxic activity

Jepson, Marie; Bullifent, Helen L.; Crane, Dennis; Flores-Dı́az, Marietta; Alape-Girón, Alberto; Jayasekeera, Pramukh; Lingard, Bryan; Moss, David S.; Titball, Richard W.

doi:10.1016/s0014-5793(01)02385-7

Cited by 22 publications

(15 citation statements)

References 30 publications

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“…Despite the recent identification of the residues critical for toxicity in Cp-PLC (24,43,44), the molecular mechanism of action of this toxin is still not completely understood. Cp-PLC is hemolytic, necrotizing, lethal, and highly toxic to various cell types, particularly those that are deficient in UDPGlc (18).…”

Section: Discussionmentioning

confidence: 99%

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Flores-Dı́az

Alape-Girón

Clark

et al. 2005

Journal of Biological Chemistry

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Clostridium perfringens phospholipase C (Cp-PLC), also called ␣-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 glycoconjugates 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.

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Section: Discussionmentioning

confidence: 99%

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Flores-Dı́az

Alape-Girón

Clark

et al. 2005

Journal of Biological Chemistry

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“…However, the present study showed that residues which correspond to D293, D305, and K330 are conserved in Csp and/or Cbp, suggesting that other residues may be required for the strong biological activities of Cpa. AlapeGiron et al (1) reported that D269, Y275, Y307, Y331, and D336 of Cpa were critical for toxicity, and Jepson et al (11) reported that Y331 and F334 of Cpa were essential for it. Among the six residues, those that correspond to Y307 and D336 are conserved in both Csp and Cbp but those that correspond to D269, Y275, Y331, and F334 are substituted for Y, N, R, and I, respectively, in Csp and Y, N, Y, and I, respectively, in Cbp.…”

Section: Discussionmentioning

confidence: 99%

“…Among the six residues, those that correspond to Y307 and D336 are conserved in both Csp and Cbp but those that correspond to D269, Y275, Y331, and F334 are substituted for Y, N, R, and I, respectively, in Csp and Y, N, Y, and I, respectively, in Cbp. As discussed in the literature on the subject (1,11), amino acid residues which are specific to Cpa and not to other nontoxic phospholipases should be responsible for the toxicity. The C-terminal domain of Cpa possesses three calcium-binding sites, termed Ca1 (E32, D269, E271, D336, and A337), Ca2 (D293, N294, G296, and D298), and Ca3 (T272, D273, N297, and D298) (21).…”

Section: Discussionmentioning

confidence: 99%

Clostridium sordellii Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of Clostridium perfringens and Clostridium bifermentans Phospholipase C

Karasawa

Wang²,

Maegawa³

et al. 2003

Infect Immun

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The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidinetagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.Clostridium perfringens elaborates lecithinase, known as alpha-toxin (Cpa), which is the best characterized of all clostridial lecithinases (10,29,30). Cpa is a phospholipase C enzyme (30). It is toxic to mammals and is considered to be one of the major virulence factors produced by C. perfringens (25,29,30). However, there are still many lecithinases produced by other clostridia that are poorly characterized, and their roles in the pathogenesis of disease have not yet been determined (29,30). Clostridial lecithinases whose primary structures have been determined are limited to only Cpa (13,22,23,26,32), Clostridium bifermentans phospholipase C (Cbp) (32), and Clostridium novyi type A phospholipase C (Cnp) (33). Additionally, clostridial lecithinases that have been purified and characterized are limited to Cpa and Cbp.C. bifermentans and C. sordellii resemble each other in their cultural and biological properties, but they have been determined to be genetically different species (19). C. sordellii lecithinase is one of the clostridial lecithinases whose molecular properties are not yet understood (28). Here we report the cloning of the C. sordellii lecithinase (Csp) gene, expression of its product using purified histidine-tagged (His-tag) proteins, and comparison of the enzymatic and biological activities of Csp with those of Cpa and Cbp. MATERIALS AND METHODSBacterial strains, plasmids, and culture. C. sordellii NCIB10717 (ATCC 9714), C. perfringens KZ 221 (33), and C. bifermentans KZ 1012 (SJ2) were used to isolate the lecithinase genes. To investigate the occurrence of the csp gene, 23 C. sordellii strains kept at our laboratory were used. Esherichia coli TOP10F' (Invitrogen) was used for transformation. PCRII-TOPO (Invitrogen) and pKF3 (Takara Shuzo) plasmid vectors were used. Clostridia were grown by using home-made liver broth, brain heart infusion (BHI; Becton Dickinson Microbiology Systems), BHI agar plate, or 10% (vol/vol) egg yolk BHI agar plate under anaerobic conditions at 37°C. E. coli w...

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“…We also reported that the C-terminal region contributes to the activities of the alpha-toxin (9). Jepson et al (4) reported that the C-terminal domains of alpha-toxin and CbPLC are responsible for the differences in activity of these proteins. Guillouard et al (1) showed that Asp-269 and Asp-336 in alpha-toxin are involved in the binding to Ca 2 which is essential for hemolytic activity.…”

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confidence: 99%

Role of the C‐Domain in the Biological Activities of Clostridium perfringens Alpha‐Toxin

Nagahama

Mukai

Morimitsu

et al. 2002

Microbiology and Immunology

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Clostridium perfringens alpha‐toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C‐domain (251–370 residues; CP251–370) in biological activities of the toxin. The N‐domain (1–250 residues; CP1–250) of the alpha‐toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC‐CP251–370) consisting of BcPLC and CP251–370 bound to the red cells and lysed them. Incubation of CP1–250 with CP251–370 completely complemented hemolytic and PLC activities. CP251–370 also conferred hemolytic activity on BcPLC. CP251–340 (251–340 residues) significantly stimulated PLC activity of CP1–250, but did not confer hemolytic activity on CP1–250. Kinetic analysis suggested that CP251–370 increased affinity toward the substrate of CP1–250. The results suggested that CP251–370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1–250. Acrylodan‐labeled CP251–370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazolyl)ethylene diamine (NBD)‐labeled CP251–370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251–370 to a hydrophobic environment. These observations suggest that interaction of CP251–370 of alpha‐toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1–250.

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Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α‐toxin are essential for cytotoxic activity

Cited by 22 publications

References 30 publications

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium sordellii Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of Clostridium perfringens and Clostridium bifermentans Phospholipase C

Role of the C‐Domain in the Biological Activities of Clostridium perfringens Alpha‐Toxin

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