Structure and function of clostridial phospholipases C

Review

Structure and function of clostridialphospholipases C

Marie Jepson*, Richard Titball

Defence Evaluation and Research Agency, CBD, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK

ABSTRACT – A range of clostridial species produce phospholipases C. The zinc metallophospholipases C have related sequences but different properties. All of these enzymes may bearranged, like α-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxicproperties may be explained by differences in amino acids at key positions. © 2000 Éditionsscientifiques et médicales Elsevier SAS

α-toxin / structure / phospholipase C / clostridia

1. IntroductionThe phospholipases C are an important group of pro-

teins which are produced by many species of bacteriaincluding Listeria monocytogenes, Bacillus cereus, Sta-phylococcus aureus, and many Clostridia spp. [1–4]. Phos-pholipases C are characterised by the site at which theycleave phospholipids (figure 1). The products of hydroly-sis are a water soluble polar head group which is linked tophosphate and a water insoluble hydrocarbon tail linkedto a glycerol backbone. Phospholipids are often classifiedon the basis of this head group, for example choline ispresent in phosphatidylcholine. The phospholipid tailgroups can vary with respect to their degree of length anddegree of saturation. These differences in phospholipidstructure have profound influences on the efficiency withwhich they are hydrolysed by individual phospholipasesC, and some bacterial species such as C. novyi, producemore than one phospholipase C [5, 6] which catalyse thehydrolysis of a range of phospholipids.

Over the past 50 years, we have gained an understand-ing of the enzymatic properties of these proteins. Morerecently, with the application of modern molecular bio-logical techniques, we have gained knowledge of thegenetic basis of toxin production and the molecular archi-tecture of some of these proteins. The purpose of thisreview is to draw together the information obtained overrecent years, with a particular focus on the structure-function relationships of the clostridia phospholipases C.

2. Clostridial phospholipases CThe phospholipases C produced by the clostridia have

attracted attention since the end of World War I, particu-

larly those produced by C. perfringens, C. bifermentansand C. novyi. Other clostridia, such as C. sordellii and C.paraperfringens (table I) also produce phospholipases C[7]. However these enzymes are less well characterised.

The production of these enzymes can be detected bythe formation of a zone of turbidity surrounding coloniesgrown on agar media containing human sera which isattributed to the hydrolysis of serum phospholipids. Thisreaction forms the basis of the diagnostic test (the Naglerreaction) for C. perfringens [8]. The preferred substrates forthe C. perfringens enzyme are phosphatidylcholine andsphingomyelin. Other clostridial enzymes preferentiallycleave other phospholipids. For example, C. novyi α-toxinpreferentially hydrolyses phosphatidylglycerol and phos-phatidylethanolamine [9]. The phosphatidylinositol-preferring phospholipase C (PI-PLC) enzyme produced byC. novyi catalyses the hydrolysis of phosphatidylinositolbut is structurally unrelated to the other clostridialenzymes. For the purpose of this review the C. novyi�-toxin and PI-PLC will not be considered unless other-wise stated.

Some phospholipases C, such as those produced by C.perfringens and C. novyi, are haemolytic but only forerythrocytes from certain species of animals [9–11]. Thesephospholipases C are also toxic, supporting the suggestionof MacFarlane [3] that toxicity and haemolytic activity arelinked.

The immature C. perfringens, C. bifermentans and C.novyi phospholipases C, all possess an amino-terminalpeptide of 26-28 amino acid residues (α-toxin, 28; Cbp,26; C. novyi α-toxin, 28) which corresponds to a post-translational signal sequence in α-toxin [12]. Alignment ofthe deduced amino acid sequences of the mature phos-pholipases C from C. perfringens, C. bifermentans and C.novyi reveals proteins of similar lengths (C. perfringens* Correspondence and reprints

Microbes and Infection, 2, 2000, 1277−1284© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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371; C. bifermentans 373, and C. novyi 370 amino acidresidues) (figure 2). The deduced amino acid sequences ofthe mature C. perfringens and C. bifermentans proteins are53% identical. C. novyi also shares a high degree of aminoacid identity with the C. perfringens (62%) and C. bifer-mentans (55%) proteins. The crystal structure of C. perfrin-gens α-toxin shows that the mature protein is organisedinto two domains [13]. The amino-terminal domain hasphospholipase C activity [14] whilst the carboxy-terminaldomain is a calcium-dependent putative phospholipidbinding domain [15]. The sequence homology and thesimilarity in length of the C. bifermentans and C. novyienzymes with the C. perfringens enzyme suggest that theyare also organised into two domain structures. The C.bifermentans and C. novyi proteins show 57–68% aminoacid identity within the putative amino-terminal domainsand 43–49% within the putative carboxy-terminaldomains, when compared with C. perfringens α-toxin(table II).

2.1. C. perfringens α-toxin

The phospholipase C produced by C. perfringens,known as α-toxin, is the best studied of all of the clostridialphospholipases C. C. perfringens is the species of clostridiamost frequently associated with the disease gas gangreneand the main virulence factor was for many years sus-

pected to be α-toxin [16]. The pathological features of gasgangrene are extensive local tissue damage whichprogresses to profound shock [16–19]. There have beenmany lines of investigation into the precise role of α-toxinin gas gangrene but early studies were complicated by theuse of preparations which probably contained other pro-teins secreted by C. perfringens. Recently the technique ofreverse genetics was applied to study the pathogenesis ofC. perfringens-mediated gas gangrene. The chromosomalplc gene was insertionally inactivated and the resultantstrain showed a dramatic reduction in virulence whentested in the murine model of gas gangrene [20]. Whereasonly 30% of mice challenged with the wild-type bacte-rium survived, there were no deaths in the group chal-lenged with the plc– mutant. In comparison with thewild-type limb swelling, inflammation, muscle necrosis,haematuria, and muscle destruction were all markedlyreduced when mice were challenged with the plc– mutant.Complementation of the mutant with a recombinant plas-mid carrying the plc gene restored virulence [20]. Thisstudy provided proof that α-toxin was an essential viru-lence factor in gas gangrene.

The α-toxin is able to hydrolyse phosphatidylcholineand sphingomyelin in the presence of calcium ions [21].Also in the presence of calcium ions the enzyme ishaemolytic but only towards erythrocytes from some spe-cies, e.g., human and mouse [10]. Erythrocytes from otherspecies such as rabbit and horse are not lysed by α-toxin.Sheep erythrocytes are only lysed under hot-cold condi-tions, when the erythrocytes are incubated with α-toxin at37 °C and then cooled to 4 °C [10, 22]. The interaction ofα-toxin with membrane phospholipids is thought to be akey event in the haemolysis of erythrocytes. An explana-tion for the difference in susceptibility of erythrocytes fromvarious animal species could be the different phospholipidcompositions of the red cell membranes. For examplesheep erythrocytes contain a high sphingomyelin and lowphosphatidylcholine ratio (63%: < 1%) compared withhuman erythrocytes which have a similar phosphatidyl-choline and sphingomyelin content (39%: 37%) [23, 24].

The hydrolysis of membrane phospholipids by α-toxinresults in the activation of cell pathways which contributeto the cytotoxic effect observed. For example, the hydroly-sis of membrane phospholipids resulting in the accumula-tion of diacylglycerol can subsequently activate the arachi-donic acid pathway [25, 26]. The activation of this pathwaycan lead to the production of thromboxane A2, a potentmediator of the inflammatory response [26]. Diacylglyc-erol is also known to activate protein kinase C and hencethe hydrolysis of membrane phospholipids by α-toxincould also result in the activation of this enzyme. Proteinkinase C may in turn activate eukaryotic cell phospholi-pases C and D which would increase membrane phospho-lipid turnover, resulting in extensive cell damage [26, 27].

Much less information is known regarding the proper-ties and mode of action of C. novyi γ-toxin. The purifica-tion of this phospholipase C was finally achieved in 1975and the enzyme was shown to hydrolyse phosphatidyl-choline, sphingomyelin and lyso-phosphatidylcholine [5].The enzyme was haemolytic for horse but not sheeperythrocytes [5]. Haemolysis of horse erythrocytes by C.

Figure 1. Types of phospholipids cleaved by phospholipases C.Arrows depict the site of cleavage.

Review Jepson and Titball

1278 Microbes and Infection2000, 1277-1284

novyi γ-toxin is reported to parallel phosphatidylcholine-hydrolysing activity [5]. The extracted phospholipids arehydrolysed more rapidly than in intact cells. This was alsothe case for C. perfringens α-toxin [5] suggesting thataccess of phospholipid to the enzymes was a factor inhydrolysis. Sheep erythrocytes are resistant to C. novyiγ-toxin and are only hydrolysed by α-toxin under hot-coldconditions [5]. However C. novyi α-toxin hydrolyses sph-ingomyelin extracted from erythrocytes to the same extentas α-toxin [5]. Therefore differences in the phospholipidcontent of different erythrocytes are not sufficient to pro-vide a full explanation for the differences in erythrocytesusceptibility to lysis by C. perfringens α-toxin or C. novyiγ-toxin. Therefore other factors must be involved; one ofwhich may be the accessibility of the phospholipids inmembranes to the enzymes.

3. Structure-function relationships3.1. Amino-terminal domain

The determination of a crystal structure of C. perfrin-gens α-toxin revealed that it was a two domain protein(figure 3) [13] and this has provided important insights intothe mechanisms involved in toxicity. The putative activesite is located in the amino-terminal domain and thepresence of three zinc ions were detected in the active sitecleft [13]. Within this cleft amino acid residues involved incoordinating these zinc ions have been identified (resi-dues Trp1; His11; Asp56; His68; His126; Asp130; His136;His148; Glu152) [13]. These zinc coordinating ligands arepresent in other clostridial phospholipases C, such as C.bifermentans and C. novyi. This suggests that the activesite architecture is similar in each of these enzymes. Thepresence of zinc ions has been identified in other phos-pholipase C enzymes such as the phosphatidylcholine-preferring phospholipase C produced by Bacillus cereusand L. monocytogenes PLC-B [2, 28, 29]. The zinc coor-dinating residues mentioned above are also conserved inthese proteins. This group of enzymes has been termed thezinc metalloenzymes.

Chemical modification of the histidine residues withdiethylpyrocarbonate, post EDTA treatment, abolishes allenzyme activity. Enzyme activity was not altered without

pretreatment with EDTA, suggesting that the zinc ionsprotected the normally susceptible histidine residues [28].Site-directed mutagenesis of the zinc-coordinating resi-dues in α-toxin results in the abolition of the phospholi-pase C, haemolytic and lethal activities (less than 3%retention of activity of the wild-type toxin) [30–32]. All ofthese studies confirm the essential role of zinc ions for theenzymatic activity of α-toxin and that the phospholipase Cactivity of the amino-terminal domain is essential for all ofthe biological activities of the toxin.

3.2. Structure-function of the carboxy-terminal domain

The carboxy-terminal domain of α-toxin has beenshown to be comprised of eight �-sheets organised into aGreek key fold [13] (figure 3). This structural motif is foundin phospholipid binding proteins such as synaptotagminand protein kinase C which bind phospholipids in acalcium-dependent manner [33, 34]. The carboxy-terminal domains of the clostridial phospholipases C havelittle amino acid sequence homology with the C2 domainsof eukaryotic phospholipases C [13].

3.2.1. Role of calcium ions with the carboxy-terminaldomain

The carboxy-terminal domains of the clostridial phos-pholipases C show a high degree of amino acid sequenceconservation . Moreau [21] showed that calcium ionswere required for binding of α-toxin to lipid films and oneof the features of the carboxy-terminal domain is thepossession of three calcium-binding sites, termed Ca1,Ca2 and Ca3 [35] (figure 4). The ligands identified withinthe Ca1 site are Asp269; Glu271; Asp273; Asp274; and Asp336

[35]. Guillouard et al [36] have shown that the Asp resi-dues at positions 269 and 336 are important for α-toxinactivity and that when substituted with Asn the mutatedproteins showed a 20% reduction in phospholipase Cactivity. The substitution of Asp274 with asparagine did notaffect lecithinase activity. Each of these mutated proteinsretained haemolytic activity. However, the mutated pro-teins did show differences in calcium dependency forphospholipase C activity. Wild-type α-toxin showed fullphospholipase C activity at 1.3mM and 5.2mM calcium,whereas the aspartic acid mutants Asp269 and Asp336

required the higher calcium concentration for full activity.These results suggested that the mutation of the aspartic

Table I. The phospholipases C produced by the clostridia family.

Clostridium species Phospholipase C Gene cloned Mol W Substrate specificity

C. perfringens α-toxin Yes [12, 39, 40, 41] 42,500a PC, SPH, PS, LPCC. bifermentans phospholipase C; Cbp Yes [40] 42,642b PC, SPHC. novyi γ-toxin No 30,000c PC, SPH, LPC, PE, PS, PG

PI-PLC Yes [6] 30,000 PI�-toxin No 32,000 PC

C. sordellii phospholipase C No NR NRC. paraperfringens phospholipase C No NR NRC. absonum phospholipase C No NR NRa calculated from the deduced amino acid sequence of strain NCTC8237 mature protein, b calculated from the deduced amino acid sequence of strainATCC638 mature protein [15], c determined using gel filtration chromatography [19]. PC = phosphatidylcholine; SPH = sphingomyelin; PS =

phosphatidylserine; PI = phosphatidylinositol; LPC = lysophosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol. NR =not reported.

Structure and function of clostridial phospholipases C Review


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acid residues Asp269 and Asp336 lead to a change incalcium dependency for phospholipase C activity.

The remaining calcium-binding sites Ca2 and Ca3 [13]are in close proximity to the Ca1 site (figure 4) and interactwith highly conserved Asp residues (Ca2, Asp293, Asp298

and Ca3, Asp273, Asp298) (figure 4). All three calcium-binding sites are located at the putative membrane-bindingsurface of the protein and may therefore be involved inbinding of α-toxin to the membrane, allowing the activesite to be optimally positioned to bind phospholipid [35,37].

Within the designated Ca1 site in C. perfringens α-toxin[35] the amino acid residues Glu271; Asp273; Asp274; andAsp336 are conserved in C. bifermentans Cbp and in C.novyi α-toxin. The remaining amino acid residues withinthe Ca1 site are not conserved; Asp269 is replaced with Tyrin C. bifermentans Cbp and Ala in C. novyi γ-toxin, whilstAla337 is replaced with Asp in both proteins (figure 2). Theamino acid residue Asp269 has been shown to play animportant role in calcium binding [36] and these changesmay therefore influence the activity of these enzymes byaltering their abilities to bind calcium at this site.

Figure 2. The deduced amino acid sequences of C. perfringens α-toxin, C. bifermentans Cbp and C. novyi γ-toxin (PLC). * represents the endof the amino-terminal domain of C. perfringens α-toxin (residues 1–246). ** represents the start of the carboxy-terminal domain of C.perfringens α-toxin (residues 256–370). ← → highlights the lysine-rich region in C. perfringens α-toxin (RKRK) and C. bifermentans Cbp(RKER).



The techniques of circular dichroism and intrinsic fluo-rescence have also shown that the isolated carboxy-terminal domain of α-toxin binds calcium ions [35]. Theintrinsic fluorescence of the carboxy-terminal domainaltered in the presence of calcium ions, inferring that atryptophan residue(s) became less exposed to the environ-ment following calcium binding. The crystal structure ofα-toxin reveals that the side chain of Trp360 lies 4–5Å fromthe designated Ca1 site and is exposed to the aqueousphase [13]. It is conceivable that this tryptophan residuewould be sensitive to changes in the environment andcould relocate to a less polar environment following cal-cium ion binding. In the presence of the chromophoricchelator, 5-NBAPTA (5' nitro 1,2-bis (o-aminophenoxy)

ethane-N, N,-N', N', -tetra acetic acid), competitive bind-ing of Ca2+ to the carboxy-terminal domain was observedand a calcium affinity of ∼ 200 µM was determined. Thedata from the CD, fluorescence and chelator experimentssuggested that the carboxy-terminal domain possessed asingle calcium ion binding site with an affinity for calciumof approximately 200 µM [35]. In contrast three calcium-binding sites were predicted from the crystal structure ofα-toxin. Naylor et al. [35] proposed that this discrepancymight reflect differences in the conformations of the iso-lated carboxy-terminal domain compared with thecarboxy-terminal domain in the holotoxin (crystal struc-ture). They proposed that the isolated carboxy-terminaldomain may be partially unfolded and that the change in

Table II. Amino acid sequence identity between the mature C. perfringens, C. bifermentans and C. novyi phospholipasesC.*

C. bifermentans vs C.perfringens

C. novyi vs C. perfringens C. novyi vs C. bifermentans

Amino-terminal domain 57.8 % (144/249) 68.3 % (170/249) 58.6 % (146/249)Carboxy-terminal domain 43.9 % (54/123) 49.2 % (59/120) 47.5 % (57/120)Overall 53 % (198/373) 62 % (229/370) 55 % (203/370)

* Number of amino acid residues identical are shown in brackets.

Figure 3. The crystal structure of C. perfringens α-toxin. The amino-terminal domain is represented as α-helicies and the carboxy-terminaldomain is depicted as �-sheets. The interdomain connecting loop between the amino-terminal and carboxy-terminal domains is illustrated.The inset shows the amino acid residues involved in coordinating the three zinc ions of the active site within the amino-terminal domain.



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structure seen with circular dichroism in the presence ofCa2+ is indicative of correct folding. This phenomenonhas been reported previously for the C2A domain of thephospholipid binding protein synaptotagmin [34]. There-fore the binding of a calcium ion into the Ca1 site [13]would induce the complete folding of this domain and inthis case the remaining two calcium-binding sites wouldbe undetectable using their methods.

There are some striking differences in the activity of C.perfringens α-toxin and C. bifermentans Cbp towards dif-ferent substrates (table III) [38]. For example, α-toxin andC. bifermentans Cbp hydrolyse phosphatidylcholine inegg yolk or unilamellar liposomes to a similar extent,whereas α-toxin was > 100-fold more haemolytic than C.bifermentans Cbp [15]. The α-toxin was tenfold moreactive than C. bifermentans Cbp towards liposomes com-posed of sphingomyelin. The carboxy-terminal domain isthought to play a major role in phospholipid interaction

and this interaction plays a key role in haemolysis. There-fore it was proposed that differences in the properties ofthe carboxy-terminal domain of α-toxin and Cbp mightexplain the different properties of these enzymes. To inves-tigate this possibility a hybrid protein was constructedcomprising the amino-terminal domain of C. bifermentansCbp and the carboxy-terminal domain of C. perfringensα-toxin (NbiCα) [15]. The introduction of the carboxy-terminal domain of α-toxin resulted in increasedhaemolytic activity, toxicity and activity towards sphingo-myelin liposomes when compared with activity of C.bifermentans Cbp (table III). This suggested that althoughthe carboxy-terminal domain played an important role inhaemolysis, this domain alone did not provoke toxic orhaemolytic activity to the same extent as α-toxin. There-fore the amino-terminal domain must also play a role intoxicity and haemolytic activity.

Figure 4. The interaction of C. perfrin-gens α-toxin with membrane phospho-lipids. The amino acid residues involvedin this interaction are highlighted. Thepotential Ca2+-binding sites within thecarboxy-terminal domain are illus-trated.

Table III. Comparison of activity of the phospholipases C produced by C. perfringens, C. bifermentans, and C. novyi, anda constructed hybrid NbiCα comprising the amino-terminal domain of C. bifermentans and the carboxy-terminal domainof C. perfringens.

Clostridium speciesPhosphatidylcholinehydrolysing activity

Sphingomyelinhydrolysing activity

Haemolytic activity Lethality (per mouse)

C. perfringens α-toxin +++ +++ +++ 1 µgC. bifermentans Cbp +++ ++ ±a > 10 µgC. novyi γ-toxin +++ +++ +++b + c

Hybrid NbiCα +++ +++ ++ 10 µg

+++ and ++ indicate activity similar to or less than that of a-toxin respectively; ± a weakly haemolytic for mouse erythrocytes; b haemolytic for horseerythrocytes; + c lethality reported for C. haemolyticum attributed to C. novyi γ-toxin



4. Interactions between the amino-terminal and carboxy-terminal domains

It has been suggested that interaction between theamino-terminal domain and carboxy-terminal domain ofα-toxin occurs via a hydrophobic loop (residues 84–88,WYLAY). This loop is situated between the putative activesite of the amino-terminal domain and the calcium-bindingsites of the carboxy-terminal domain (figure 3). This loopcould allow communication between the putative activesite and the calcium-binding sites during membrane bind-ing [13].

5. Membrane interactions

Interaction with membrane phospholipids is essentialfor the toxic activity of α-toxin and calcium-mediatedphospholipid binding is one mechanism of membranephospholipid interaction. However other amino acid sidechains may also play a role in membrane phospholipidrecognition and a variety of candidate residues have beenidentified in α-toxin [13, 15]. These residues have hydro-phobic side chains and are located on the membrane-protein interface. The residues Tyr331 and Phe334 arereplaced by leucine and phenylalanine in C. bifermentansCbp, and by isoleucine and valine in C. novyi γ-toxin. Ithas been suggested that Tyr331 interacts with the polarheadgroups of membrane phospholipids promoting bind-ing to membranes [13]. The replacement of the polarresidue tyrosine with the non-polar leucine or phenylala-nine in C. bifermentans Cbp or C. novyi γ-toxin respec-tively, could alter this interaction with membranes. Thereplacement of Phe334 with isoleucine or valine with theresultant loss of the aromatic ring may alter the interactionbetween enzyme and the phospholipid tail group. Thelimited number of amino acid sequence differences withinthe carboxy-terminal domain could provide an explana-tion for the differences in activity of the C. perfringens, C.bifermentans and C. novyi phospholipases C.

Residues in the amino-terminal domain might also beimportant for membrane interaction. The side chain ofresidue Trp214 which is located on the membrane interfaceregion of the protein, has been suggested to interact withthe hydrophobic tail groups of the membrane phospholip-ids [13]. The side chains of the Tyr331 and Phe334 residuesin the carboxy-terminal domain are also appropriatelypositioned to interact with phospholipid tail groups. Thisresidue is conserved in C. bifermentans Cbp and replacedwith asparagine in C. novyi γ-toxin, which may be signifi-cant.

6. Conclusions

This review on the phospholipases C produced by theclostridia collates the growing body of information onthese enzymes. One of the landmark events in this fieldwas the determination of the crystal structure of C. perfrin-gens α-toxin [13]. This structure has provided new insights

into the mode of action of this toxin. It has for example,resulted in the identification of calcium-binding siteswithin the carboxy-terminal domain, which supports pre-vious suggestions that calcium played a key role in α-toxinactivity. The close structural relationship of the carboxy-terminal domain of α-toxin with the C2-domains of eukary-otic phospholipid binding proteins, such as synaptotag-min and protein kinase C, could provide insights into themechanisms involved in cell signalling and calcium-regulated second messenger systems. There are many keyquestions remaining. What are the precise roles of theamino acids thought to be important for membrane inter-actions and what at a molecular level determines the toxicnature of some phospholipases C? These studies will notonly allow the design of novel anti-toxic drugs, but couldalso allow the exploitation of these enzymes as noveltherapeutics.

Acknowledgments

The authors would like to thank Claire Naylor, BirbeckCollege for providing the crystal structure figures.

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Structure and function of clostridial phospholipases C

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