Accepted Article
Received Date : 25-Jul-2014 Revised Date : 26-Sep-2014 Accepted Date : 29-Sep-2014 Article type
: MiniReview
Editor
: Ezio Ricca
Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells
Francesco Celandroni1, Sara Salvetti1, Sonia Senesi2, Emilia Ghelardi1
1
Department of Translational Research and New Technologies in Medicine and Surgery, and
2
Department of Biology, University of Pisa, Pisa, Italy
Correspondence: Emilia Ghelardi, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via San Zeno 37, Pisa 56127, Italy. Tel.: +39-050-2213686; fax: +39-050-2213711; e-mail: [email protected] Present address: Sara Salvetti, San Giuseppe Hospital-AUSL 11, Empoli 50053, Italy.
Keywords: Bacillus thuringiensis; toxin; mammalian infection; PC-PLC; PI-PLC; hemolysin BL.
Abstract Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered non-pathogenic to other This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12615 This article is protected by copyright. All rights reserved.
Accepted Article
animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning associated diarrheas, periodontitis, bacteremia, as well as ocular, burn and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian
cells,
such
as
phosphatidylcholine-
and
phosphatidylinositol-specific
phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intra-vitreous, intranasal, or intra-tracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC and HBL are responsible for most of the pathogenic properties B. thuringiensis may exert in non-gastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis and the structural features of these membrane-damaging toxins.
Introduction Bacillus thuringiensis is a Gram-positive, rarely capsulated (10% of the strains), sporebearing microorganism that is widely distributed in the environment. The placement of B. thuringiensis as a separate species within the genus Bacillus has been controversial since the publication of The Genus Bacillus in 1973 (Gordon et al., 1973) and Bergey's Manual of Determinative Bacteriology in 1974 (Buchanan & Gibbons, 1974). B. thuringiensis belongs to the “Bacillus cereus group”, also known as Bacillus cereus sensu lato, comprising the closely related species Bacillus cereus sensu stricto, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis (Drobniewski, 1993; Lechner et al., 1998; Nakamura, 1998). Lately, a novel member of the group, Bacillus cytotoxicus, has been described (Guinebretière et al., 2013). The species B. mycoides and B. pseudomycoides This article is protected by copyright. All rights reserved.
Accepted Article
are phenotypically different from other bacteria belonging to the B. cereus group for their rhizoidal colony shape and typical fatty acid composition of the cell membrane (Stenfors Arnesen et al., 2008). B. anthracis is the causative agent of the highly infectious disease anthrax, B. cereus is a soil bacterium behaving as a human opportunistic pathogen, and B. thuringiensis is an entomopathogenic bacterium used as a bio-pesticide worldwide. B. thuringiensis is able to produce parasporal crystalline protein inclusions (parasporal bodies or crystals) that are encoded by plasmids carrying cry genes (Schnepf et al., 1998). Loss of these genes makes B. thuringiensis indistinguishable from B. cereus by other physiological or morphological traits. As a whole, B. cereus sensu lato organisms have an extremely high degree of chromosomal synteny and whole genome comparisons between these organisms reveal a highly similar gene content (Rasko et al., 2005; Han et al., 2006). Sequence analyses of the 16S rRNA and 23S rRNA genes, showing similarity >99% among members of the B. cereus group, indicate that these organisms should more accurately be viewed as a single species (Helgason et al., 2000) despite their phenotypic diversity. In addition, the various species of the group are phylogenetically interspersed among one another in several phylogenies (Tourasse et al., 2008; Tourasse et al., 2011). Although the population has a clonal character, there do not appear to be clonal lineages that are species-specific, with the exception of the B. anthracis lineage (Hoffmaster et al., 2008). Similar to B. cereus, B. thuringiensis is able to produce an array of virulence factors potentially acting against mammalian cells. These include several phospholipases, two enterotoxic complexes (hemolysin BL, HBL and the non-hemolytic enterotoxin NHE), and various hemolysins, such as cytotoxinK (CytK) (Lovgren et al., 1998; Agaisse et al., 1999; Gaviria Rivera et al., 2000; Swiecicka et al., 2006).
This article is protected by copyright. All rights reserved.
Accepted Article
In B. thuringiensis strain 407, a transcriptional activator that positively regulates the expression of phospholipase C genes during the late vegetative growth was identified in 1996 and named PlcR (Lereclus et al., 1996). PlcR is a 34 kDa protein that positively regulates its own expression. It is a pleiotropic regulator controlling the expression of 45 genes, including virulence factors such as the phosphatidylcholine-preferring and the phosphatidylinositolspecific phospholipase C (PC-PLC and PI-PLC), as well as HBL (Agaisse et al., 1999). PlcR activates the transcription of its target genes by binding to a consensus sequence defined as wTATGnAwwwwTnCATAw (Agaisse et al., 1999; Gaviria Rivera et al., 2000; Lereclus et al., 1996; Gohar et al., 2008). The activity of PlcR is under the control of the signaling peptide PapR (Slamti et al., 2002). papR belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48 aa peptide that is secreted and then reimported through the oligopeptide permease (OppABCDF) system (Gominet et al., 2001). Once inside the cell, its processed truncated form binds PlcR and promotes its recognition of the PlcR box.
B. thuringiensis virulence in mammals Although long considered non-pathogenic to humans, B. thuringiensis has been sporadically identified as responsible of various infectious diseases in humans. These include foodpoisoning associated diarrheas (Jackson et al., 1995; McIntyre et al., 2008), ocular infections (Samples & Beuttner, 1995; Callegan et al., 2006; Peker et al., 2010), periodontitis (Helgason et al., 2000), burn (Damgaard et al., 1997) and wound (Hernandez et al., 1998) infections as well as bacteremia (Ghelardi et al., 2007; Kuroki et al., 2009). Noteworthy, the frequency of B. thuringiensis infections can also be underestimated, since discrimination between B. cereus and B. thuringiensis is not routinely performed in the clinical microbiology laboratory. This consideration is supported by data showing that, when discrimination is performed, an
This article is protected by copyright. All rights reserved.
Accepted Article
equal number of B. cereus and B. thuringiensis strains are isolated from clinical (blood, catheter tip, gauze) and environmental samples (Kuroki et al., 2009). Pathogenicity of B. thuringiensis in gastrointestinal infections has been attributed to the production of potent diarrheal toxins: NHE (Stenfors Arnesen et al., 2008), HBL (Beecher et al., 1995), and CytK (Swiecicka et al., 2006; Swiecicka et al., 2013). Several animal models have been used to study the contribution B. thuringiensis toxins in the pathogenesis of non-gastrointestinal infections in humans (Salamitou et al., 2000; Callegan et al., 2002; Callegan et al., 2003; Ghelardi et al., 2007). By using an intra-vitreous infection model in rabbits, Callegan and co-workers demonstrated that B. thuringiensis causes extensive inflammation and loss of retinal function and that damage in ocular tissues is similar to that obtained in the experimental B. cereus endophthalmitis (Callegan et al., 2002). B. thuringiensis virulence in endophthalmitis has been shown to be due to toxin production during intraocular bacterial growth (Callegan et al., 2005), although the role of individual toxins remains controversial. Most likely, these factors work in concert to achieve the level of virulence observed in the experimental model of infection. The finding that disruption of the plcR gene causes a dramatic reduction of B. thuringiensis pathogenicity during endophthalmitis (Callegan et al., 2003; Callegan et al., 2005) further supports this hypothesis. The effect of plcR disruption on B. thuringiensis virulence was also evaluated using two other animal models, the lepidopteran insect Galleria mellonella and BALB/c mice (Salamitou et al., 2000). Galleria mellonella larvae were either feed or intra-hemocoelically injected with a wild type or a ΔplcR strain. This study indicated that the virulence factors governed by PlcR are important if bacteria enter the larvae via the digestive tract, suggesting a role of the plcR regulon in the early stages of infection. When B. thuringiensis vegetative forms were administered to mice by nasal instillation, 100% mortality of the animals was
This article is protected by copyright. All rights reserved.
Accepted Article
recorded within 4-6 h, with hemorrhagic symptoms (nose bleeding and lungs clearly hemorrhagic on autopsy) (Salamitou et al., 2000). These results agreed with previous data showing that some B. thuringiensis strains are pathogenic to mice following nasal instillation (Hernandez et al., 1998; Hernandez et al., 1999). In a study aimed at evaluating the contribution of single B. thuringiensis toxins to the virulence of this organism, we set up an experimental model of pulmonary infection by intratracheally injecting bacteria in BALB/c mice (Ghelardi et al., 2007). Isogenic strains (the clinical isolate RM, B. thuringiensis 407 Cry- and its derivative mutants), which only differed in the production of membrane-damaging toxins, exhibited different ability to persist/replicate in the mouse lungs, with the strain completely defective in membranedamaging toxin showing the lower ability to survive intrapulmonary. The study also revealed that the lack of PI-PLC, PC-PLC, or HBL could be replaced by an increase in the level of each of the other toxins. In addition, the finding that a strain repeatedly isolated from the blood of a neutropenic patient suffering from severe pulmonary disease produced a very high level of PC-PLC, but lacked HBL and PI-PLC (Ghelardi et al., 2007), suggested that only one of the three toxins could be sufficient for B. thuringiensis to establish a severe infection in humans. This review will focus on PC-PLC, PI-PLC, and HBL, the B. thuringiensis toxins whose role in extra-intestinal infections has been better defined.
PC-PLC Phospholipases C (PLC) are enzymes that cleave phospholipids just before the phosphate group, to provide a lipidic alcohol and a phosphorylated polar head group. Phospholipase activity is required for a great variety of biological processes, such as cell membrane homoeostasis (Scott, 1984), digestion (Borgström, 1980; Watkins, 1985), inflammation
This article is protected by copyright. All rights reserved.
Accepted Article
(Oestvang & Johansen, 2006; Triggiani et al., 2006; Zhang et al., 2010), infection (AlbertiSegui et al., 2007; Ghelardi et al., 2007) and signal transduction (Becker & Hannun, 2005; Mateos et al., 2006; Wang & Kazanietz, 2006). Bacillus PC-PLC encoding genes do not show significant sequence homology to eukaryotic genes, but cross-reaction of antibodies raised towards these proteins occurs with proteins of mammalian cells (Clark et al., 1986). Therefore, PC-PLCs from Bacillus spp. are considered as a model for studying the structural features, function and substrate preference of these enzymes (Benfield et al., 2007). Bacillus PC-PLC is a small (28 kDa; Table 1), monomeric enzyme, with three Zn2+ atoms in the active site likely to be involved in the binding to the substrate and essential for the enzymatic activity and protein conformational stability (González-Bulnes et al., 2010). The residues constituting the active site are Glu4, Tyr56, and Phe66, which comprise the substrate head group-binding pocket (Benfield et al., 2007). The carboxyl group on the side chain of Glu4 interacts with the nitrogen atom on the choline head group by a polar or ionic bound, while the Phe66 via a cation-π interaction (Dougherty, 1996; Burley & Petsko, 1988). Tyr56 is supposed to help in stabilizing the positive charge on the inhibitor or substrate and appears to play the role of determinant of specificity. Bacillus PC-PLC hydrolyzes phospholipids to give diacylglycerol and an alkyl phosphate (Fig. 1A). The enzyme recognizes three different phospholipid substrates, which only differ in the structure of the head group, with the following order of preference: phosphatidylcholine (PC) > phosphatidylethanolamine (PE) > phosphatidylserine (PS) (Martin et al., 2000), with specificity constants in the approximate ratio of 10:7:1, respectively (Antikainen et al., 2003). PC with six carbon atoms in each of the acyl side chains is processed with slightly greater catalytic efficiency than PC with two–four carbon atoms (El-Sayed et al., 1985; Martin et al., 2000). The spatial orientation of the glycerol side
This article is protected by copyright. All rights reserved.
Accepted Article
chains on the phosphatidylcholine moiety appears to be an important factor contributing to binding and catalysis (Snyder, 1987). B. thuringiensis PC-PLC is identical to the B. cereus protein. Using erythrocytes from different species as models for membranes with varied phospholipid contents, previous studies have demonstrated that cooperativity between PC-PLC and sphingomyelinase is required to lyse human (Gilmore et al., 1989) and swine red blood cells (RBCs) (Beecher & Wong, 2000), the last having phospholipid compositions similar to human RBCs. PC-PLC enhances hemolysis due to HBL in cells containing significant amounts of PC (swine, 22% PC; human, 31% PC), but inhibits HBL-mediated lysis of sheep erythrocytes (
Received Date : 25-Jul-2014 Revised Date : 26-Sep-2014 Accepted Date : 29-Sep-2014 Article type
: MiniReview
Editor
: Ezio Ricca
Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells
Francesco Celandroni1, Sara Salvetti1, Sonia Senesi2, Emilia Ghelardi1
1
Department of Translational Research and New Technologies in Medicine and Surgery, and
2
Department of Biology, University of Pisa, Pisa, Italy
Correspondence: Emilia Ghelardi, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via San Zeno 37, Pisa 56127, Italy. Tel.: +39-050-2213686; fax: +39-050-2213711; e-mail: [email protected] Present address: Sara Salvetti, San Giuseppe Hospital-AUSL 11, Empoli 50053, Italy.
Keywords: Bacillus thuringiensis; toxin; mammalian infection; PC-PLC; PI-PLC; hemolysin BL.
Abstract Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered non-pathogenic to other This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12615 This article is protected by copyright. All rights reserved.
Accepted Article
animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning associated diarrheas, periodontitis, bacteremia, as well as ocular, burn and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian
cells,
such
as
phosphatidylcholine-
and
phosphatidylinositol-specific
phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intra-vitreous, intranasal, or intra-tracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC and HBL are responsible for most of the pathogenic properties B. thuringiensis may exert in non-gastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis and the structural features of these membrane-damaging toxins.
Introduction Bacillus thuringiensis is a Gram-positive, rarely capsulated (10% of the strains), sporebearing microorganism that is widely distributed in the environment. The placement of B. thuringiensis as a separate species within the genus Bacillus has been controversial since the publication of The Genus Bacillus in 1973 (Gordon et al., 1973) and Bergey's Manual of Determinative Bacteriology in 1974 (Buchanan & Gibbons, 1974). B. thuringiensis belongs to the “Bacillus cereus group”, also known as Bacillus cereus sensu lato, comprising the closely related species Bacillus cereus sensu stricto, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis (Drobniewski, 1993; Lechner et al., 1998; Nakamura, 1998). Lately, a novel member of the group, Bacillus cytotoxicus, has been described (Guinebretière et al., 2013). The species B. mycoides and B. pseudomycoides This article is protected by copyright. All rights reserved.
Accepted Article
are phenotypically different from other bacteria belonging to the B. cereus group for their rhizoidal colony shape and typical fatty acid composition of the cell membrane (Stenfors Arnesen et al., 2008). B. anthracis is the causative agent of the highly infectious disease anthrax, B. cereus is a soil bacterium behaving as a human opportunistic pathogen, and B. thuringiensis is an entomopathogenic bacterium used as a bio-pesticide worldwide. B. thuringiensis is able to produce parasporal crystalline protein inclusions (parasporal bodies or crystals) that are encoded by plasmids carrying cry genes (Schnepf et al., 1998). Loss of these genes makes B. thuringiensis indistinguishable from B. cereus by other physiological or morphological traits. As a whole, B. cereus sensu lato organisms have an extremely high degree of chromosomal synteny and whole genome comparisons between these organisms reveal a highly similar gene content (Rasko et al., 2005; Han et al., 2006). Sequence analyses of the 16S rRNA and 23S rRNA genes, showing similarity >99% among members of the B. cereus group, indicate that these organisms should more accurately be viewed as a single species (Helgason et al., 2000) despite their phenotypic diversity. In addition, the various species of the group are phylogenetically interspersed among one another in several phylogenies (Tourasse et al., 2008; Tourasse et al., 2011). Although the population has a clonal character, there do not appear to be clonal lineages that are species-specific, with the exception of the B. anthracis lineage (Hoffmaster et al., 2008). Similar to B. cereus, B. thuringiensis is able to produce an array of virulence factors potentially acting against mammalian cells. These include several phospholipases, two enterotoxic complexes (hemolysin BL, HBL and the non-hemolytic enterotoxin NHE), and various hemolysins, such as cytotoxinK (CytK) (Lovgren et al., 1998; Agaisse et al., 1999; Gaviria Rivera et al., 2000; Swiecicka et al., 2006).
This article is protected by copyright. All rights reserved.
Accepted Article
In B. thuringiensis strain 407, a transcriptional activator that positively regulates the expression of phospholipase C genes during the late vegetative growth was identified in 1996 and named PlcR (Lereclus et al., 1996). PlcR is a 34 kDa protein that positively regulates its own expression. It is a pleiotropic regulator controlling the expression of 45 genes, including virulence factors such as the phosphatidylcholine-preferring and the phosphatidylinositolspecific phospholipase C (PC-PLC and PI-PLC), as well as HBL (Agaisse et al., 1999). PlcR activates the transcription of its target genes by binding to a consensus sequence defined as wTATGnAwwwwTnCATAw (Agaisse et al., 1999; Gaviria Rivera et al., 2000; Lereclus et al., 1996; Gohar et al., 2008). The activity of PlcR is under the control of the signaling peptide PapR (Slamti et al., 2002). papR belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48 aa peptide that is secreted and then reimported through the oligopeptide permease (OppABCDF) system (Gominet et al., 2001). Once inside the cell, its processed truncated form binds PlcR and promotes its recognition of the PlcR box.
B. thuringiensis virulence in mammals Although long considered non-pathogenic to humans, B. thuringiensis has been sporadically identified as responsible of various infectious diseases in humans. These include foodpoisoning associated diarrheas (Jackson et al., 1995; McIntyre et al., 2008), ocular infections (Samples & Beuttner, 1995; Callegan et al., 2006; Peker et al., 2010), periodontitis (Helgason et al., 2000), burn (Damgaard et al., 1997) and wound (Hernandez et al., 1998) infections as well as bacteremia (Ghelardi et al., 2007; Kuroki et al., 2009). Noteworthy, the frequency of B. thuringiensis infections can also be underestimated, since discrimination between B. cereus and B. thuringiensis is not routinely performed in the clinical microbiology laboratory. This consideration is supported by data showing that, when discrimination is performed, an
This article is protected by copyright. All rights reserved.
Accepted Article
equal number of B. cereus and B. thuringiensis strains are isolated from clinical (blood, catheter tip, gauze) and environmental samples (Kuroki et al., 2009). Pathogenicity of B. thuringiensis in gastrointestinal infections has been attributed to the production of potent diarrheal toxins: NHE (Stenfors Arnesen et al., 2008), HBL (Beecher et al., 1995), and CytK (Swiecicka et al., 2006; Swiecicka et al., 2013). Several animal models have been used to study the contribution B. thuringiensis toxins in the pathogenesis of non-gastrointestinal infections in humans (Salamitou et al., 2000; Callegan et al., 2002; Callegan et al., 2003; Ghelardi et al., 2007). By using an intra-vitreous infection model in rabbits, Callegan and co-workers demonstrated that B. thuringiensis causes extensive inflammation and loss of retinal function and that damage in ocular tissues is similar to that obtained in the experimental B. cereus endophthalmitis (Callegan et al., 2002). B. thuringiensis virulence in endophthalmitis has been shown to be due to toxin production during intraocular bacterial growth (Callegan et al., 2005), although the role of individual toxins remains controversial. Most likely, these factors work in concert to achieve the level of virulence observed in the experimental model of infection. The finding that disruption of the plcR gene causes a dramatic reduction of B. thuringiensis pathogenicity during endophthalmitis (Callegan et al., 2003; Callegan et al., 2005) further supports this hypothesis. The effect of plcR disruption on B. thuringiensis virulence was also evaluated using two other animal models, the lepidopteran insect Galleria mellonella and BALB/c mice (Salamitou et al., 2000). Galleria mellonella larvae were either feed or intra-hemocoelically injected with a wild type or a ΔplcR strain. This study indicated that the virulence factors governed by PlcR are important if bacteria enter the larvae via the digestive tract, suggesting a role of the plcR regulon in the early stages of infection. When B. thuringiensis vegetative forms were administered to mice by nasal instillation, 100% mortality of the animals was
This article is protected by copyright. All rights reserved.
Accepted Article
recorded within 4-6 h, with hemorrhagic symptoms (nose bleeding and lungs clearly hemorrhagic on autopsy) (Salamitou et al., 2000). These results agreed with previous data showing that some B. thuringiensis strains are pathogenic to mice following nasal instillation (Hernandez et al., 1998; Hernandez et al., 1999). In a study aimed at evaluating the contribution of single B. thuringiensis toxins to the virulence of this organism, we set up an experimental model of pulmonary infection by intratracheally injecting bacteria in BALB/c mice (Ghelardi et al., 2007). Isogenic strains (the clinical isolate RM, B. thuringiensis 407 Cry- and its derivative mutants), which only differed in the production of membrane-damaging toxins, exhibited different ability to persist/replicate in the mouse lungs, with the strain completely defective in membranedamaging toxin showing the lower ability to survive intrapulmonary. The study also revealed that the lack of PI-PLC, PC-PLC, or HBL could be replaced by an increase in the level of each of the other toxins. In addition, the finding that a strain repeatedly isolated from the blood of a neutropenic patient suffering from severe pulmonary disease produced a very high level of PC-PLC, but lacked HBL and PI-PLC (Ghelardi et al., 2007), suggested that only one of the three toxins could be sufficient for B. thuringiensis to establish a severe infection in humans. This review will focus on PC-PLC, PI-PLC, and HBL, the B. thuringiensis toxins whose role in extra-intestinal infections has been better defined.
PC-PLC Phospholipases C (PLC) are enzymes that cleave phospholipids just before the phosphate group, to provide a lipidic alcohol and a phosphorylated polar head group. Phospholipase activity is required for a great variety of biological processes, such as cell membrane homoeostasis (Scott, 1984), digestion (Borgström, 1980; Watkins, 1985), inflammation
This article is protected by copyright. All rights reserved.
Accepted Article
(Oestvang & Johansen, 2006; Triggiani et al., 2006; Zhang et al., 2010), infection (AlbertiSegui et al., 2007; Ghelardi et al., 2007) and signal transduction (Becker & Hannun, 2005; Mateos et al., 2006; Wang & Kazanietz, 2006). Bacillus PC-PLC encoding genes do not show significant sequence homology to eukaryotic genes, but cross-reaction of antibodies raised towards these proteins occurs with proteins of mammalian cells (Clark et al., 1986). Therefore, PC-PLCs from Bacillus spp. are considered as a model for studying the structural features, function and substrate preference of these enzymes (Benfield et al., 2007). Bacillus PC-PLC is a small (28 kDa; Table 1), monomeric enzyme, with three Zn2+ atoms in the active site likely to be involved in the binding to the substrate and essential for the enzymatic activity and protein conformational stability (González-Bulnes et al., 2010). The residues constituting the active site are Glu4, Tyr56, and Phe66, which comprise the substrate head group-binding pocket (Benfield et al., 2007). The carboxyl group on the side chain of Glu4 interacts with the nitrogen atom on the choline head group by a polar or ionic bound, while the Phe66 via a cation-π interaction (Dougherty, 1996; Burley & Petsko, 1988). Tyr56 is supposed to help in stabilizing the positive charge on the inhibitor or substrate and appears to play the role of determinant of specificity. Bacillus PC-PLC hydrolyzes phospholipids to give diacylglycerol and an alkyl phosphate (Fig. 1A). The enzyme recognizes three different phospholipid substrates, which only differ in the structure of the head group, with the following order of preference: phosphatidylcholine (PC) > phosphatidylethanolamine (PE) > phosphatidylserine (PS) (Martin et al., 2000), with specificity constants in the approximate ratio of 10:7:1, respectively (Antikainen et al., 2003). PC with six carbon atoms in each of the acyl side chains is processed with slightly greater catalytic efficiency than PC with two–four carbon atoms (El-Sayed et al., 1985; Martin et al., 2000). The spatial orientation of the glycerol side
This article is protected by copyright. All rights reserved.
Accepted Article
chains on the phosphatidylcholine moiety appears to be an important factor contributing to binding and catalysis (Snyder, 1987). B. thuringiensis PC-PLC is identical to the B. cereus protein. Using erythrocytes from different species as models for membranes with varied phospholipid contents, previous studies have demonstrated that cooperativity between PC-PLC and sphingomyelinase is required to lyse human (Gilmore et al., 1989) and swine red blood cells (RBCs) (Beecher & Wong, 2000), the last having phospholipid compositions similar to human RBCs. PC-PLC enhances hemolysis due to HBL in cells containing significant amounts of PC (swine, 22% PC; human, 31% PC), but inhibits HBL-mediated lysis of sheep erythrocytes (
