Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin 1 1Edited by A Klug

Naylor, C.E.; Jepson, Marie; Crane, Dennis; Titball, Richard W.; Miller, Julie Ann; Basak, A.K.; Bolgiano, Barbara

doi:10.1006/jmbi.1999.3279

Cited by 62 publications

(55 citation statements)

References 39 publications

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“…Numerous studies have identified Cpa as one of the principal toxins involved in the pathophysiology of gas gangrene (25), and recent genetic studies have provided additional evidence that Cpa is an essential virulence factor in gas gangrene (3,21,36). Moreover, the pathophysiology in the progression of tissue destruction and in the cardiovascular system from gas gangrene has been elucidated.…”

Section: Discussionmentioning

confidence: 99%

“…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). The present work has shown that the amino acid residues involved in the three calcium-binding sites and in the neighboring regions are basically conserved in Csp, with the exception of Ca1 (K32, Y268, and D337), as well as in Cbp Ca1 (Q32, Y269, and D338) and Cnp Ca1 (A269 and D337), suggesting that the differences in toxic activities of these proteins are not due to differences in their calciummediated recognition of phospholipid (9).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…Both observations suggest that there is communication between the domains, and all x-ray structures available have revealed extensive contact interfaces that would allow for interdomain communication. Significantly, calcium binding to the C2-like domain of gangrene ␣-toxin results in the ordering of a short segment of the phospholipase domain (4,24), although it is unclear whether Ca 2ϩ impacts catalytic activity per se or simply enzyme localization.…”

Section: Discussionmentioning

confidence: 99%

Insights from the X-ray Crystal Structure of Coral 8R-Lipoxygenase

Oldham

Brash

Newcomer

2005

Journal of Biological Chemistry

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Lipoxygenases (LOXs) catalyze the regio-and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 Å resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca 2؉ -ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca 2؉ -regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly 428 (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.

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“…The most important classes of phospholipases that have been shown to play a significant role in bacterial pathogenesis are phospholipase C and phospholipase D (31). Phospholipase C has been demonstrated to be an important virulence factor in an increasing number of bacteria, including Clostridium perfringens (22,23), Bacillus cereus (25), the intracellular pathogen Listeria monocytogenes (32,37), the extracellular pathogen Pseudomonas aeruginosa (24,34), and other bacteria (20,35).…”

Section: No Hemolytic Activity Was Detected In M Smegmatis M Gordomentioning

confidence: 99%

Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

et al. 2001

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Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.Phospholipases have a wide spectrum of in vivo and in vitro effects, ranging from minor alterations in cell membrane composition and function to lethality. The most important classes of phospholipases that have been shown to play a significant role in bacterial pathogenesis are phospholipase C and phospholipase D (31). Phospholipase C has been demonstrated to be an important virulence factor in an increasing number of bacteria, including Clostridium perfringens (22, 23), Bacillus cereus (25), the intracellular pathogen Listeria monocytogenes (32, 37), the extracellular pathogen Pseudomonas aeruginosa (24, 34), and other bacteria (20,35).Phospholipase genes homologous to the hemolytic plcH and nonhemolytic plcN genes from P. aeruginosa have been described in Mycobacterium tuberculosis, M. bovis, M. marinum, and recently in M. ulcerans (3,11,16). Fusion of mpcA or mpcB plc genes of M. tuberculosis encoding glutathione S-transferase produced beta-hemolytic activity when expressed in Escherichia coli (19), and phospholipase C sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring recombinant mpcA or mpcB genes (16).Nontuberculous mycobacteria are free-living saprophytes that have been detected and isolated from a variety of environmental sources, including water, soil, and dust (6), and some of these mycobacteria are opportunistic pathogens.Nontuberculous mycobacteria, including M. kansasii and M. avium-M....

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Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin 1 1Edited by A Klug

Cited by 62 publications

References 39 publications

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

Insights from the X-ray Crystal Structure of Coral 8R-Lipoxygenase

Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

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