1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this report we describe the 1500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17 117.2 Da (PlcR2) and 78 417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the con-clusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In recent years, phosphatidylcholine preferring phospholipases C (PC-PLC) and sphingomyelinases (SMase) have been associated with a growing number of critical signal transduction mechanisms in eukaryotic cells (Cheng et al., 1997; Nofer et al., 1997; Li et al., 1998; Mathias et al., 1998; Tschaikowsky et al., 1998; Monick et al., 1999; Hannun and Luberto, 2000; Nofer et al., 2000; Zhang et al., 2001). The PC-PLCs hydrolyse phosphatidylcholine (PC), yielding diacylglycerol (DAG) and phospholcholine, whereas SMases hydrolyse sphingomyelin (SM) yielding ceramide and phosphocholine. In mammalian systems, the products ceramide and DAG are involved in powerful signal transduction cascades, many times acting as antagonists to each other. Ceramide has been shown to be involved in the eukaryotic stress response including regulation of growth, differentiation, and apoptosis (Mathias et al., 1998; Hannun and Luberto, 2000) whereas DAG has been implicated in transformation, proliferation, and inflammation (Exton, 1990; Nishizuka, 1992; Rhee and Bae, 1997). Whereas the evidence for mammalian SMases is clear (Liu et al., 1998; Bernardo et al., 2000), the existence of mammalian PC-PLCs is controversial (Luberto and Hannun, 1998). Most of the studies demonstrating PC-PLC activity in mammalian cells have relied on detection of elevated DAG levels and inhibition by the putative, specific PC-PLC competitive inhibitor, tricyclodecan-9-ylxanthate (D609) (Cheng et al., 1997; Li et al., 1998; Tschaikowsky et al., 1998; Monick et al., 1999; Nofer et al., 2000; Zhang et al., 2001). The compound D609 was originally demonstrated to com-petitively inhibit the Bacillus cereus PC-PLC (Amtmann, 1996). This lack of structural and mechanistic information for mammalian PC-PLC has accorded prokaryotic PC-PLC added significance. Consequently, bacterial PC-PLCs have been used to probe signalling events in mammalian cells. For example Moscat and colleagues were the first to show that a bacterial PC-PLC (B. cereus) externally applied to eukaryotic cells caused mimicking of both a significant portion of a mitogenic response and constitutive activation of protein kinase C (PKC) (Diaz-Laviada et al., 1990). Subsequently, this research group was able to demonstrate that the stable expression of the B. cereus PC-PLC gene in NIH 3T3 fibroblasts led to oncogenic transformation of these cells (Johansen et al., 1994).

The major class of well-characterized PC-PLCs is found in Gram-positive bacteria. These bacterial PC-PLCs play remarkably diverse roles in the biology and pathogenesis of Listeria monocytogenes, B. cereus, and Clostridium perfringens (Titball, 1993; Songer, 1997). It has been demonstrated that the PC-PLC of L. monocytogenes contributes to the cell-to-cell spread of this opportunistic pathogen (Vazquez-Boland et al., 1992; Marquis et al., 1997; Songer, 1997) whereas the alpha toxin of C. perfringens is the major agent of gas gangrene (Awad et al., 1995). Moreover, the crystal structures have been elucidated for both the B. cereus (Hough et al., 1989) and C. perfringens (Naylor et al., 1998) PLCs.

The Gram-negative, opportunistic plant and human pathogen, Pseudomonas aeruginosa, produces a haemolytic heat-labile PC-PLC (PlcH) and a non-haemolytic PC-PLC (PlcN) (Ostroff et al., 1990). PlcH and PlcN are highly homologous with greater than 40% identity and 60% similarity. PlcH has been demonstrated to be a virulence determinant in a wide variety of organisms including mammals (Hollsing et al., 1987; Chin and Watts, 1988; Ostroff et al., 1989; Meyers and Berk, 1990; Meyers et al., 1992; Wiener-Kronish et al., 1993), plants (Rahme et al., 1995; Rahme et al., 2000), yeast (Hogan and Kolter, 2002), and insects (Jander et al., 2000). In addition, mutants of M. tuberculosis deficient in phospholipase C homologues to PlcH were shown to have reduced virulence in a mouse pulmonary infection model (Raynaud et al., 2002).

In this report, we present an extensive biochemical characterization of the P. aeruginosa haemolytic PLC, demonstrating that this enzyme is a multimeric protein composed of PlcH, an enzyme specific for choline containing phospholipids (SM, PC, and plasmaglogens) and either one of two co-secreted chaperones, PlcR1,2. Furthermore, we demonstrate that the haemolytic phospholipase of P. aeruginosa is a paradigm of a novel class of phospholipases and phosphatases with a novel catalytic mechanism.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

General characteristics of PlcH, PlcR1,2 and the PlcH homologues

The haemolytic PC-PLC of P. aeruginosa was the first member of a now large family of enzymes with PC-PLC, SMase, and phosphatase activity identified in a number of microbial pathogens including Mycobacterium tuberculosis, Bordetella pertussis, Francisella tularensis, Burkholderia pseudomallei, and Xanthomonas campestris. Figure 1 shows a phylogenetic tree depicting relationships between the PC-PLC and APase homologues identified via a blast search, with PlcH (http:www.ncbi.nlm.nih.govblast/). The members of this class do not share any amino acid homology with the well-characterized B. cereus PC-PLC or similar enzymes in C. perfringens and L. monocytogenes. In comparison, the enzymes of this novel class are larger, ranging from 50 to 80 kDa, whereas the PC-PLC in the B. cereus class range from 25 to 43 kDa. Interestingly, the F. tularensis AcpA, which shows no homology to known phosphatases, shows 21% identity (50% similarity) to the amino-terminal 450 amino acids of PlcH. In addition to acid phospha-tase activity, the F. tularensis AcpA was reported to be active on the PC-PLC synthetic substrate ρ-nitrophenyl-phosphorylcholine (NPPC) suggesting that this enzyme also possesses PLC activity (Reilly et al., 1996). It is also worthy to note that some of the organisms that contain a PlcH homologue also contain an AcpA homologue (e.g. B. mallei, B. pseudomallei, B. thailandensis) (Burtnick et al., 2001). The similarities between these PC-PLCs and acid phosphatases support the hypothesis that these enzymes comprise a family of related proteins that hydrolyse phosphodiester and phosphomonoester bonds analogous to what has been described for the phospholipase D (PLD) superfamily (Gottlin et al., 1998; Waite, 1999; Liscovitch et al., 2000).


Figure 1. The PLC/APase superfamily. Neighbour joining phylogenetic tree depicting the relationships between the PlcH homologues, including the related APases. Accession numbers are included when available. Homologues listed with no accession number were identified via the unfinished genome databases. Units of distance are uncorrected ‘p’ values estimating the proportional differences between sequences. The alignment was performed using ClustalW (ver. 1.4).

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Figure 2A depicts a number of characteristics of PlcH and the other homologues in this class of PC-PLC. Although, these PC-PLCs are highly homologous to each other, it is likely that they have each evolved certain characteristics that impart additional roles and functions that suit the individual biology of each organism. Unique to the M. tuberculosis PC-PLCs is that they are all truncated with an additional 7th cysteine at the ultimate or penultimate (Cys-Ser) amino acid in comparison with the other homologues. Whereas this feature has not been examined in prokaryotes it is interesting to note that in eukaryotic cells C-terminal Cys or Cys-Ser are frequently lipid modified. Two independent groups have demonstrated that the M. tuberculosis PC-PLC activity is secreted to the cell surface, but not released into the extracellular environment like PlcH or PlcN are in P. aeruginosa (Johansen et al., 1996; Raynaud et al., 2002). The cell-associated nature of the mycobacterial PC-PLCs may contribute to the intracellular lifestyle of this organism in contrast to the extracellular nature of P. aeruginosa in opportunistic infections. Unique to PlcH and one of four homologue from B. pseudomallei (B. pseudomallei D, Fig. 1), but not found in any other homologue of this superfamily, is the presence of an Arg-Gly-Asp (RGD) motif. This tripeptide motif is commonly recognized by integrins and has been shown to be important in the pathogenesis of organisms including Borrelia burgdorferi, Yersinia spp., B. pertussis, adenovirus, foot-and-mouth disease virus, and Hantaviruses. In addition, PlcH possesses a 24 amino acid sequence, which is gapped when aligned with the other homologues. It is possible that the RGD motif and the 24 amino acid unique region of PlcH may be involved in the interaction with the chaperone proteins PlcR1,2 (see below).


Figure 2. Characteristics of the haemolytic PLC of P. aeruginosa.

A. Schematic representation of PlcH showing the shared and unique characteristics of PlcH and its homologues.

B. Schematic representation of PlcR1,2 illustrating important characteristics and differences between PlcR1 and PlcR2.

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The plcR1,2 genes are located 3′ to plcH and are expressed from in-phase overlapping genes (Shen et al., 1987). Previous reports have shown that these proteins are required for the extracellular secretion of PlcH (Cota-Gomez et al., 1997). Figure 2B illustrates some of the characteristics of the PlcR1,2 chaperone proteins. PlcR1 is synthesized with a typical sec-dependent signal sequence whereas PlcR2, which starts at an initiation codon internal to plcR1, lacks a N-terminal signal sequence. The predicted mature PlcR1 is larger than PlcR2 by 36 amino acids, of which 10 are prolines and 15 are hydrophobic amino acids (Cota-Gomez et al., 1997). PlcR2 is identical to PlcR1 except for the signal sequence and the above mentioned 36 amino acid region. The 151 amino acids in common to PlcR1 and PlcR2 contain four putative EF-hands, which are calcium-binding motifs (Cota-Gomez et al., 1997). Proteins with significant homology to PlcR1,2 have not been identified in any of the other organisms that possess a PlcH homologue. Nevertheless, because PlcR1,2 are mainly composed of EF-hand motifs, and such motifs do not necessarily demonstrate linear amino acid homology, it is possible that there are proteins in the other organisms which possess PlcH homologues that have a similar function as PlcR1,2.

Purification of the haemolytic PLC from P. aeruginosa

The purification of the haemolytic phospholipase C from P. aeruginosa is summarized in Table 1. Supernatants from P. aeruginosa ADD1976 over-expressing plcHR1,2 were batch bound and eluted from DE52. The decision to use batch purification instead of column purification was based on the observation that in the time it took to load, wash, and elute PlcHR2 from a column, PlcR2 was proteolytically degraded. The inclusion of the serine protease inhibitor phenylmethylsulfonyl floride (PMSF) in all solutions did not abrogate the observed degradation seen when using a column procedure. Accordingly, the protocol described in the Experimental procedures was adopted for the purification of the phospholipase C described in this report. Additionally, calcium was omitted from all buffers used in the purification because pilot experiments revealed that the inclusion of calcium resulted in degradation of PlcR2.

Table 1. . Purification of the haemolytic phospholipase C from P. aeruginosa.
Purification stepVolume(ml)Total proteina (mg)Total activityb (nmol min−1)Specific activity(nmol min−1 mg−1)Yield(%)Purification factor(-fold)
  • a

    . Total protein was measured using bicinchoninic acid method (Pierce).

  • b

    . Total activity was quantified using the synthetic substrate NPPC (Sigma).

  • c. Total protein was measured by SDS-PAGE using SYPRO® Orange molecular weight standards (Molecular Probes).

Supernatant15009825972 427    98.97100  1
DEAE-52 eluate 250 820391 298  477 40   4.8
Ammonium sulphate precipitation   6.5   8.4266 452 31 777 27 321
Native preparative gel  10.5   0.60c 89 640149 774  9.21513

After elution with 500 mM NaCl the DE52 matrix still reacted with the synthetic substrate NPPC indicating that a substantial amount of active enzyme remains bound to the matix. As shown in Table 1, 40% of the total activity was recovered from the DE52. Hence 60% of the initial activity is still bound to the matrix. In addition, PlcR1 is not detected in the DE52 eluate (Fig. 3A). Multiple attempts have been made to strip the remaining activity and PlcR1 from the DE52, including increasing ionic strength to 1 M NaCl, but none were successful. Following concentration of the DE52 eluate via 75% ammonium sulphate precipitation the sample was dialysed to remove excess salt to facilitate loading onto the native preparative gel. Twenty-seven per cent of the total activity remained following concentration and dialysis (Table 1). The concentrated, dialysed sample was loaded onto a native preparative gel and fractions were collected. Figure 3B shows the PLC activity found in each fraction with the peak activity in fractions 30–33. Corresponding Western blots show that the fractions with the peak activity contain both PlcH and PlcR2 (Fig. 3E). Mass spectrometry data also confirmed the presence of peptides from PlcH and PlcR2 in the purified sample (Fig. 4A and B). The fractions with peak activity were pooled, aliquoted, and stored at − 80°C. The purification resulted in a 1500-fold purification of the haemolytic PLC with a recovery of approximately 10% of the initial activity (Table 1). A SYPRO® Orange (Molecular Probes) stained SDS-PAGE of the purified enzyme shows two bands, one at 79 kDa (PlcH), and another at 17 kDa (PlcR2) (Fig. 3C). No other contaminating proteins were visualized in the purified sample. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the purified sample detected two proteins, with masses of 78409.5 ± 200 Da and 17 117.2 ± 1 Da corresponding to PlcH and PlcR2 respectively (data not shown). The deduced masses for PlcH and PlcR2 are 78 356.22 and 17 246.2 Da respectively. Removal of the N-terminal methionine from PlcR2 results in a deduced mass of 17 115 Da. The data that these two proteins of differing sizes and properties purify together suggests that the haemolytic PC-PLC from P. aeruginosa is an enzyme complex consisting of PlcH and PlcR2. Furthermore, Western blot analysis of the purified sample resolved under non-denaturing conditions demonstrates that both PlcR2 and PlcH migrate together as a heterodimeric complex, PlcHR2 (Fig. 3D). Additional data indicate that PlcH and PlcR2 are at a stoichiometry of 1:1 in the purified sample (Fig 3C and Fig 4C and D).


Figure 3. Purification of the haemolytic PLC from P. aeruginosa.

A. Samples from the purification steps were separated via SDS–PAGE, transferred to nitrocellulose, and probed with PlcR monoclonal antibodies.

B. Each 2 ml fraction collected from the native preparative gel was assayed for PC-PLC activity with the synthetic substrate NPPC.

C. Purified PlcHR2 was separated by SDS-PAGE and the proteins stained with SYPRO® Orange (Molecular Probes). The band intensities for PlcH and PlcR2 were determined with Quantity One software version 4.0.3 (Bio-Rad). A 1 : 1 stoichiometry of PlcH to PlcR2 was determined by comparing the band intensity ratio (4.57x) to the mass ratio (4.54x) for the two proteins. SYPRO® Orange has been demonstrated to stain proteins non-preferentially (Steinberg et al., 1996a, b). Molecular weight markers are shown to the left of the gel. The gel shown is a representative of multiple gels.

D. Purified PlcHR2 was separated via native-PAGE, transferred to nitrocellulose, and probed with either PlcH or PlcR monoclonal antibodies.

E. Native preparative gel fractions were separated via SDS-PAGE, transferred to nitrocellulose and probed with PlcH and PlcR monoclonal antibodies.

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Figure 4. Representative triple quadruple mass spectrometry data used to identify the PlcHR2 complex.

A. Reconstructed ion current trace from the semi-automated capillary LC/MS/MS experiment that identified PlcH and PlcR2 as co-purified proteins. Because the majority of ions were blocked by the first mass filter in CAD mode (see Experimental procedures), the total ion current of the resulting CAD scan was less than that for the main beam scan. Therefore, the RIC (reconstructed ion current) shows a series of alternating dips and rises in signal intensity as the observed ion current increases during main beam mode and decreases during CAD mode. Each dip in the trace represents several CAD scans of a particular peptide fragment parent ion mass. A total of 111 peptide fragments were examined over the entire chromatographic run in this particular experiment.

B. Representative CAD (MS2) mass spectrum showing the key ions used to sequence the peptide and identify the protein. The ions are labelled according to the nomenclature of Biemann (Biemann, 1992). An asterisk denotes a neutral loss of either water or ammonia (18 or 17 mass units respectively). In this case it was preferable to sequence the shorter tryptic subdigest peptide, QIFGEENPR, to confirm the Lys-C result shown in A, due to the complexity of the CAD spectrum generated by the strongly fragmentation directing internal P and R residues contained in the sequence QIFGEENPRLLQR. The sequest search results (see Experimental procedures) showed very high cross correlation scores for three peptide fragments from PlcH and five from PlcR. The sequest scores were subsequently verified by manual interpretation of the data.

C. A representative main beam (MS1) mass chromatogram (summed m/z for 784 [M + 4H]4+, 627 [M + 5H]5+, and 523 [M + 6H]6+) of the larger Asp-N fragments from PlcH that were used to confirm the Lys-C results and to determine the relative proportions of PlcH and PlcR2.

D. Representative Asp-N peptide main beam mass chromatogram (m/z 1050, [M + 2H]2+) data for PlcR2.

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Functional characterization of the haemolytic PLC

The purified PlcHR2 complex is enzymatically activity on PC and SM, but no activity was detected for PS, or PE assaying with the C6 short chain substrates (Fig. 5A). The substrate specificity has also been confirmed using mixed micelle assays either with BODIPY® substrates (PC, SM and PE) or with radiolabelled substrates (PC, SM and PS) (data not shown). In comparison, the B. cereus PLC (Sigma) is active on PC, PE and PS but no activity was detected for SM (Fig. 5A, inset).


Figure 5. PlcHR2 substrate specificity.

A. Purified PlcHR2 (0.3 µg ml−1) was incubated with varying concentration (1, 0.5, 0.25, 0.125, 0.063, 0.031 and 0.016 mM) of PC, SM, PE, or PS short chain C6 phospholipids (Avanti Lipids). The inset in A shows the substrate specificity of the B. cereus PLC (0.2 units) under the same conditions that PlcHR2 was assayed. The data is a representative of multiple determinations.

B. PC compounds (150 µM, including 33% plasmalogen content) extracted from bovine heart (Avanti Lipids) were incubated with PlcHR2 (0.1 µg ml−1) in a vesicle assay (50 mM Hepes, pH 7.2, 80 mM KCl) for the indicated times and then analysed by electrospray ionization mass spectrometry in full scan positive ion mode. The masses of the compounds determined were matched up to the masses of previously referenced compounds (including plasmalogens of this PC class) (Tables 7,1 RCM book).

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The similarities between PlcH and the acid phosphatases shown in Fig. 1 along with the report that the F. tularensis AcpA possesses activity on the PC-PLC synthetic substrate NPPC (Reilly et al., 1996) raised the question of weather PlcHR2 also possesses phosphatase activity. Phosphatase activity was assayed with the synthetic substrate NPP at both pH 6 and pH 7.2. No activity was detected for PlcHR2 on this substrate (data not shown).

Substrate specificity was examined further by investigating the activity of PlcHR2 on a subtype of the glycerophospholipids termed plasmalogens. Plasmalogens are defined as any glycerophospholipid displaying a vinyl ether at the sn-1 position. Plasmalogens make up approximately 18% of the phospholipid mass in humans, however, the plasmalogen content of individual tissues or cell types varies widely (Nagan and Zoeller, 2001). The compounds present in the mixture of bovine heart PC included several distinct plasmalogen containing molecules (m/z 742.5, 768.5) as well as diacyl phosphocholine molecules (m/z 758.5, 786.5). When exposed to PlcHR2 in a time course of 5, 10, 20 and 40 min it was apparent that the phosphocholine group was cleaved non-preferentially from all phosphocholine molecules (Fig. 5B). Plasmenyl compounds showed no preference, or diminished response to reaction with PlcHR2. Thus masses of the positively charged phosphocholine components present at the start of the experiment, m/z 786.5 (18 : 0, 18 : 2 PC), m/z 768.5 (36 : 3p), m/z 758.5 (16 : 0, 18 : 2 PC) and m/z 742.5 (34 : 2p) diminished as a function of increasing time with a corresponding increase in positive ion species 148 mass units less (m/z 638.5, 620.5, 610.4, and 594.4 respectively) (Fig. 5B). The mass difference of the transformed phosphocholine species represents a consistent loss of a neutral phosphocholine head group with subsequent charging of the residual diacyl lipid by co-ordination with ammonium ion present in the makeup solvent. Diacyl lipid standards (dipalmatoyl diacylglycerol, and steroyl, linoyl diacylglycerol) showed similar adduction of ammonium cation to the neutral lipids under these conditions to yield a positively charged ion at 18 mass units higher than the molecular weight of the compound (m/z 586.4 and m/z 638.5, respectively; data not shown). Taken together the data presented indicate that the moiety attached to the phosphocholine head group has no observed effect on the substrate specificity of PlcHR2. Consequently, the terminology phosphocholine specific phospholipase C has been adopted to describe PlcHR2 rather than phosphatidylcholine specific phospholipase C.

The kinetic parameters for PlcHR2 on the synthetic substrate NPPC, PC and SM in a mixed micelle assay, and PC in a vesicle assay were determined and are shown in Table 2. PlcHR2 had peak activity on the synthetic substrate NPPC but the relatively high Km (14 100 µM) indicates low affinity for this substrate. This weak affinity is most likely due to the nitro-phenyl group replacing the DAG or ceramide present in the natural substrates PC and SM respectively. Mixed micelle kinetics were performed using the surface dilution kinetic model in which the enzymes activity is determined under initial rate condition over a wide range of substrate concentrations (Deems, 2000). Under these conditions PlcHR2 had equivalent activity on both PC (kcat, 150 s−1) and SM (kcat, 154 s−1). The Km for PC and SM were 3.89 ± 1.16 and 7.84 ± 2.21 mol% respectively. The slower rate of hydrolysis of PC (kcat, 34 s−1) in a vesicle assay was expected because the lipids in a vesicle are packed into a bilayer making them less available then they are in a mixed micelle. Theoretically a phospholipase has to insert into the lipid bilayer before hydrolysing the phospholipids while in a mixed micelle assay the lipids are more accessible do to the inclusion of a neutral dilutor (Triton X-100). These kinetic data indicate that PlcHR2 is a highly active phospholipase and is comparable to the B. cereus PLC and C. perfringens in its ability to hydrolyse phospholipids.

Table 2. . PlcHR2 kinetic analysis.
Enzymatic assayVmax (µmol min−1 mg−1)Km (µM)kcat (s−1)kcat/Km (M−1 s−1)
ρ-NPPC611.9 ± 5.514 100 ± 5009796.9 × 104
PC vesicle 21.3 ± 0.8  347 ± 26 3498
Enzymatic assayVmax (µmol min−1 mg−1)Km (mol%)kcat (s−1)
  1. The initial rates of hydrolysis (µmol min−1mg−1) for the NPPC and vesicle assays were fitted to the Michaelis–Menten equation using the program Sigma Plot (SPSS ). The kinetic parameters Vmax and Km were obtained from the non-linear least squares fit of the data; kcat values were calculated using a Mr of 96 000 Da for PlcHR2. Each set of data is from three independent determinations. The mixed micelle kinetic values were determined using the surface dilution kinetic model (Deems, 2000).

PC mixed micelle93.66 ± 12.033.89 ± 1.16150
SM mixed micelle96.08 ± 11.127.84 ± 2.21154

Tricyclodecan-9-ylxanthate (D609) has been reported to be a potent and competitive inhibitor of PC-PLC because it inhibits both the B. cereus PC-PLC (Amtmann, 1996) and the C. perfringensα-toxin (Preuss et al., 2001). Although PlcH and its homologues have no primary amino acid homology to the B. cereus zinc-metalloprotein class of PC-PLCs, a similar catalytic mechanism for phospholipid hydrolysis may exist. Therefore, D609 was tested on purified PlcHR2 in a unilamellar vesicle assay. As shown in Fig. 6, D609 at concentrations up to 2 mM (400-fold excess inhibitor to substrate) did not inhibit PlcHR2. At 4 mM D609 (800-fold excess inhibitor to substrate) there is partial inhibition of PlcHR2, 65% of the control activity remained. The inhibition seen at 800-fold excess inhibitor to substrate is most likely due to non-specific interaction. In comparison an IC50 of 109 µM was determined for D609 on PLCBC. Both PlcN and the M. tuberculosis PLCs (PlcA, PlcB and PlcC) were also similarly not inhibited by D609 (data not shown). Furthermore, D609 did not inhibit PlcHR2 in both a NPPC assay and a short chain C6 assay (data not shown). These data suggest that the members of this class of PC-PLC possess a different mechanism for the hydrolysis of phospholipids than the well-characterized zinc-metallophospholipases C in B. cereus, L. monocytogenes and C. perfringens.


Figure 6. D609 effect on PlcHR2. Purified PlcHR2 (0.1 µg ml−1 ) was incubated with different concentration of D609 (500, 1000 2000 and 4000 µM) in a unilamellar vesicle assay consisting of 5 µM total PC (0.2 µM PC [choline-methyl-3H]), 25 mM Hepes (pH 7.2), 100 mM NaCl. For comparison PLCBC (0.125 µ ml−1) was similarly tested with different concentration of D609 (62.5, 125, 250, 500 and 1000 µM) in a unilamellar vesicle assay consisting of 5 µM total PC (0.2 µM PC [choline-methyl-3H]), 25 mM Hepes (pH 7.2), 100 mM NaCl, 1 mM CaCl2, 0.1 mM ZnCl2. The data are the means ± SE for four determinations.

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Cation effects on PlcHR2

Many SMases and PLCs including the bacterial PLCs from B. cereus, L. monocytogenes, C. perfringens and a newly characterized PLC from Pseudomonas fluorescens have a cation requirement (Titball, 1993; Liu et al., 1998; Bernardo et al., 2000; Preuss et al., 2001). The cation requirements for PlcHR2 were determined by incubating PlcHR2 with cations in both a NPPC enzymatic assay and a haemolysis assay. The cations tested include magnesium, calcium, copper, manganese, cobalt, zinc and nickel along with the metal chelators EDTA and EGTA. As shown in Fig. 7A, manganese, copper, cobalt, nickel and zinc all inhibit PlcHR2 activity, whereas calcium, magnesium, EDTA and EGTA did not significantly affect PlcHR2 enzymatic activity even at the highest concentrations assayed. This is in stark contrast to the haemolysis assay where calcium inhibited and the metal chelators, EGTA and EDTA, increased the haemolytic activity of PlcHR2 (Fig. 7B). Similarly, nickel inhibited haemolysis (Fig. 7B) as it did the enzymatic activity. Calcium also inhibited the haemolytic activity of free PlcH (Fig. 9B, filled circles) and free PlcH with PlcR2 added back (Fig. 9B, filled squares). These data suggest a difference between the enzymatic and biological properties of PlcHR2. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. These data also indicate that PlcHR2 does not have a detectable cation requirement for enzymatic activity on the substrates tested thus far.


Figure 7. Cation effect on PlcHR2.

A. PlcHR2 (0.025 µg ml−1) was incubated with 15 mM NPPC at 37°C in the presence of different cations or metal chelators. The initial rates of NPPC hydrolysis (nmol min−1mg−1) are plotted against cation concentration (mM). The data are the means ± SE for three determinations.

B. PlcHR2 (0.01 µg ml−1) was incubated with 1% human erythrocytes at 37°C alone or in the presence of cations or metal chelators for the indicated times. The asterisk (*) indicates hot/cold treatment of sample. The data are the means ± SE for three determinations.

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Figure 9. The effect of PlcR2 on the enzymatic and haemolytic properties of PlcH.

A. PlcH (5.1 nM) was incubated with increasing concentrations of PlcR2 in a NPPC enzymatic assay at 37°C.

B. PlcH (7.49 nM) alone or in combination with PlcR2 (10 nM), calcium, EDTA and EGTA were incubated with 1% human erythrocytes at 37°C for the indicated times.

C. PlcHR2 (150 ng), PlcH (125 ng), PlcR2 (250 ng) and PlcR2 (250 ng) added back with PlcH (125 ng) were separated on a non-denaturing polyacrylamide gel, transferred to nitrocellulose and probed with either PlcH or PlcR monoclonal antibodies. PlcH and PlcR2 were mixed and incubated at 37°C for 10 min before loading on the gel.

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The SMases from Staphylococcus aureus and B. cereus induce hot-cold lysis of sphingomyelin rich erythrocytes (Rogolsky, 1979; Ikezawa et al., 1986) therefore it was evaluated whether human erythrocytes treated with PlcHR2 were susceptible to hot-cold lysis. Figure 7B shows that PlcHR2 induces hot-cold haemolysis of human erythrocytes resulting in approximately a two to threefold increase in lysis.

PlcHR2 binds calcium

The experimental data that calcium affects the haemolytic activities of PlcHR2 and the observation that PlcR1,2 share significant features with calcium binding proteins of eukaryotes (Cota-Gomez et al., 1997) (Fig. 2B) suggests that PlcR1,2 and possibly PlcH bind calcium. Calcium binding was addressed with the inorganic dye ruthenium red (Sigma). Ruthenium red provides a simple, rapid method for detecting various types of Ca2+-binding proteins following electrophoresis (Charuk et al., 1990). In many circumstances it is more sensitive than calcium-45 autor-adiography. As shown in Fig. 8 both PlcH and PlcR2 stain with ruthenium red. However, the staining varies based on the electrophoresis conditions. Under denaturing conditions (Fig. 8A) only PlcH stains with ruthenium red. When the proteins are separated under non-denaturing conditions both the PlcHR2 complex and the free PlcR2 stain (Fig. 8B). These data suggest that both PlcH and PlcR2 along with the PlcHR2 complex bind calcium.


Figure 8. Calcium binding. PlcHR2 (150 ng), PlcH (125 ng), and PlcR2 (250 ng) were separated on a 10% SDS-PAGE (A) and a 10% native-PAGE (B), transferred to nitrocellulose and the proteins stained with ruthenium red.

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PlcR2 effect on the biological properties of PlcH

It has been previously demonstrated that PlcH requires the PlcR1,2 chaperones for secretion (Cota-Gomez et al., 1997) but their effect on the enzymatic and biological properties of PlcH had not been examined. In the process of purifying the PlcHR2 complex the individual proteins PlcH and PlcR2 were also purified (Fig. 3E). PlcR2 is seen in fractions 2–6 and there is a second minor peak of activity that corresponds to free PlcH at fraction 50–60 (Fig. 3B). The purification of the free proteins has allowed us to address whether PlcR2 plays any additional roles in the biology of this enzyme. As shown in Fig. 9A, addition of increasing amounts of PlcR2 to a constant concentration of PlcH results in a dose-dependent increase in enzymatic activity. The increased activity was approximately twofold that of free PlcH. In contrast, addition of PlcR2 to PlcH inhibited haemolysis (Fig. 9B). PlcR2 alone does not possess any detectable haemolytic, PC-PLC, or SMase activity (data not shown). Other non-specific proteins, BSA and HasA had no effect on the activities of PlcH (data not shown). In addition, as shown in Fig. 9C, the mixing of the individual proteins, PlcR2 and PlcH, results in the reformation of the PlcHR2 complex


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this report we describe an extensive characterization of an extracellular bacterial PC-PLC/SMase from the opportunistic pathogen P. aeruginosa. This enzyme is the paradigm member of a novel PC-PLC/phosphatase superfamily. Members of this family extend from pathogenic bacteria (e.g. M. tuberculosis, Burkholderia spp., F. tularensis, Xanthomonas spp., and Bodetella spp.) to fungi (e.g. Aspergillus fumigatus) and plants (e.g. Arabidopsis thaliana).

This PC-PLC/SMase was produced via a T7 overexpression system in P. aeruginosa and was purified 1500-fold by ion exchange chromatography and preparative native-PAGE. SDS-PAGE and MALDI-TOF spectrometry analysis revealed that there were two proteins present in the purified sample, a 78 417 Da protein (PlcH) and a 17 117.2 Da protein (PlcR2). Western blot analysis of native gels revealed that PlcH, and the chaperone protein PlcR2 form a heterodimeric complex, PlcHR2.

Data presented herein clearly illustrate that the catalytic mechanism of this new class of enzymes is remarkably distinct from other well-studied PC-PLCs, including those produced by the Gram-positive organisms B. cereus, L. monocytogenes and C. perfringens. Whereas, it has been demonstrated that these PC-PLCs require zinc for activity (Titball, 1993; Songer, 1997), PlcHR2 is strongly inhibited by zinc. Additionally, the cation chelators, EDTA and EGTA, had no affect on the enzymatic activity of PlcHR2. Furthermore, all the cations tested (Zn2+, Ni2+, Co2+, Cu2+ and Mn2+) except Ca2+ and Mg2+ inhibited the enzymatic activity of PlcHR2. These characteristics of PlcHR2 are highly distinct from other phospholipases, which in almost all cases have cation requirements for activity (Titball, 1993; Songer, 1997; Liu et al., 1998; Bernardo et al., 2000). Additionally, it is interesting to note that the F. tularensis AcpA, the APase representative of this enzyme superfamily, is likewise inhibited by zinc, and is unaffected by EDTA or EGTA (Reilly et al., 1996).

A further distinguishing characteristic of PlcHR2, PlcN and the M. tuberculosis PLCs is that their enzymatic activities are unaltered by the putative PC-PLC competitive inhibitor D609. This compound has been employed extensively to pharmacologically imply the existence of PC-PLCs in mammalian cells (Nofer et al., 1997; 2000; Li et al., 1998; Tschaikowsky et al., 1998; Monick et al., 1999; Ricard et al., 1999; Zhang et al., 2001). However, the presence of PC-PLCs enzymes in mammalian cells is controversial, and D609 has been shown to inhibit sphingomyelin synthase (Luberto and Hannun, 1998). Recently, Gonzalez-Roura et al. (2002) reported that the competitive inhibition of the B. cereus PC-PLC by D609 is associated with altering the co-ordination of the critical Zn2+ cations in the active sites of these metalloenzyme via a Zn-chelating dithiocarbonate head present in D609 and other xanthate derivatives. Therefore, it is possible that the reason D609 has no detectable effect on PlcHR2, PlcN and the M. tuberculosis PLCs is that zinc is not required for their enzymatic activity. Accordingly, our data indicate that caution should be used when applying D609 as a specific PC-PLC inhibitor. Implicit in our collective data is the conclusion that this superfamily of enzymes possesses a distinct catalytic mechanism for the hydrolysis of phosphodiester bonds (PC-PLCs, SMases) or phosphomonoester bonds (APases).

All PC-PLC members of this enzyme family examined to date hydrolyse choline phospholipids including PC and SM, the exception being PlcN, which hydrolyses PC and PS (Ostroff et al., 1990; Johansen et al., 1996). It is worthwhile to note that there can be significant diversity in the moiety that is linked to the phosphocholine headgroup. For example, both PlcHR2 and PlcN hydrolyse NPPC, which contains a nitrophenyl group instead of DAG. Furthermore, PlcHR2 along with the M. tuberculosis PLCs, hydrolyse both ceramide and DAG containing phosphocholine phospholipids (SM and PC respectively). Moreover, as shown above, PlcHR2 is able to hydrolyse plasmenylcholine phospholipids (plasmalogens), which contain a vinyl ether bond at the sn-1 position. In this regard, it is interesting that the haemolytic PLC of P. aeruginosa has been shown to cause platelet aggregation (Coutinho et al., 1988) and suppression of the neutrophil respiratory burst (Terada et al., 1999), which correlates with the known plasmalogen content of these cells (Nagan and Zoeller, 2001).

PlcR1,2 proteins were previously reported to be required for the secretion of PlcH (Cota-Gomez et al., 1997). At that time it was proposed that PlcR1,2 either post-translationally altered the physical or biochemical nature of PlcH, or releases PlcH from a complex in the cell so that it can be secreted. Data presented here show that PlcR2 in addition to functioning as a chaperone, remains associated with PlcH after it is secreted into the culture supernatant and throughout purification. Moreover, PlcR2 affects the enzymatic and haemolytic properties of PlcH in an opposing fashion. That is, addition of PlcR2 to free PlcH results in an increase in enzymatic activity but a decrease in its haemolytic activity. At present this effect is not fully understood and awaits further experimentation.

The PlcHR1 enzyme complex was not purified in this study despite the fact that PlcR1 is secreted into culture supernatants along with PlcH and PlcR2. PlcR1 was not recovered after DEAE chromatography and a significant proportion of PLC activity (>50%) remained associated with the DEAE matrix. As described above, the mature PlcR1 is identical to PlcR2 except for the additional N-terminal 36 amino acids in PlcR1, 10 of which are prolines and 15 of which are hydrophobic amino acids. We therefore propose that the PlcHR1 complex was not recovered as a consequence of a new property imparted to the complex by the additional amino acids present in PlcR1. Cota-Gomez et al. (1997) and Kadurugamuwa and Beveridge (1997) reported that PlcH could be found in membrane vesicles (MVs), which bud from the surface of wild-type P. aeruginosa. Consequently, it is possible that the PlcHR1 complex is associated with these MVs, while PlcHR2 is secreted as a soluble protein complex.

Another aspect of the functional differences between PlcHR1 and PlcHR2 may relate to how PlcR1 and PlcR2 are secreted. PlcH is secreted via the sec-independent, Tat-secretion apparatus (Voulhoux et al., 2001). In contrast, PlcR1 possess typical sec-dependent signal se-quence while PlcR2 contain no known amino-terminal signal sequence for its secretion. One feature of the Tat-secretion system that significantly differs from the sec system is that the Tat system will secrete folded proteins across the inner membrane into the periplasm. Additionally, proteins with Tat signal sequences can serve as carriers for proteins lacking Tat or sec secretion signal sequences (Berks et al., 2000). Consequently, it is likely that the PlcHR2 complex is formed before secretion through the inner membrane, whereas PlcH and PlcR1 do not associate with each other until they both reach the periplasmic space. The different secretion pathways used by PlcR1 and PlcR2 could influence the ultimate location and properties of the PlcHR1 and PlcHR2 complexes.

It is also noteworthy that PlcR1 and PlcR2 are each predicted to contain four EF-hands (Cota-Gomez et al., 1997), but PlcH does not contain any known calcium binding motifs (e.g. C2 domain or EF-hand). As described above, both PlcH and PlcR2 bind calcium but, whereas calcium has no detectable effect on the enzymatic activity, it inhibits the haemolytic activity of both PlcH and PlcHR2. This inhibitory effect of calcium on the haemolytic activity of PlcH differs significantly from the effect of this cation on other haemolytic PLCs. In most cases calcium is required and enhances the haemolytic activity of phospholipases (Titball, 1993; Songer, 1997; Jepson and Titball, 2000). Moreau et al. (1988) demonstrated that calcium ions are required for the binding of the C. perfringensα-toxin and the B. cereus PLC with PC films. In addition, calcium ions are also required by eukaryotic lipid binding proteins and enzymes, including intracellular phospholipases C and A2 and snake venom PLA2 (Renetseder et al., 1985; Stahelin and Cho, 2001). In the case with C. perfringensα-toxin its N-terminal domain contains the active site while the C-terminal domain possesses three calcium-binding sites that allow binding to membrane phospholipids (Naylor et al., 1999; Jepson and Titball, 2000). When the N-terminal domain is isolated it retains PLC activity, but its haemolytic and cytotoxic activities are lost (Titball et al., 1991). Accordingly, it is expected that the function of the calcium ions associated with PlcH and PlcR1,2 will vastly differ from the role calcium plays in the biology of other phospholipases. Alternatively, it is possible that the calcium ions associated with PlcH and PlcR2 serve some function in their secretion via the Tat pathway. In fact, many proteins destined for secretion via this apparatus are metal co-factored enzymes (Berks, 1996; Berks et al., 2000).

Homologues of PlcH and PlcN are found in a remarkably diverse group of organisms including bacteria, fungi and plants. Accordingly, each member of this superfamily may have evolved different characteristics to suite the distinct lifestyles of each organism. PlcH and PlcN clearly play a role in the pathogenesis of P. aeruginosa in model infections (Hollsing et al., 1987; Rahme et al., 1995; Ostroff et al., 1989; Jander et al., 2000; Hogan and Kolter, 2002). PlcH could act as a nutrient scavenging mechanism in an infected host or in its natural environment. For example, P. aeruginosa can survive on PC as its sole carbon and nitrogen source (A. Barker and M. Vasil, unpublished observations). Furthermore, we have demonstrated that PlcHR2 can have a negative impact on the function of neutrophils and macrophage (Konig et al., 1997; Terada et al., 1999). In addition to its direct effect on host cells, perhaps the production of DAG and ceramide through hydrolysis of eukaryotic membrane and surfactant phospholipids by this extracellular enzyme has a deleterious effect on host cellular signalling mechanisms. It is of particular interest in this regard that the gene located immediately 5′ to plcH in P. aeruginosa encodes an extracellular ceramidase that is highly homologous to a mammalian ceramidase (Okino et al., 1998; 1999). The P. aeruginosa ceramidase could lead to the generation of sphigosine-1-phosphate, a powerful intercellular eukaryotic signalling molecule, via the ceramide generated by PlcHR2.

The diversity of this superfamily is further illustrated by the inclusion of the homologous extracellular APases. In fact, some species (e.g. Burkholderia spp.) in addition to having multiple copies of the gene encoding PlcH homologues, also carry genes encoding the phosphatase members of this family. Consequently, it appears that this novel group of enzymes, which ranges from simple phosphatases to more complex heterodimeric, multifunctional enzymes such as PlcHR2, is evolutionary dynamic. Further analysis of the molecular architecture and enzymology of these proteins will lead to a better understanding of how the members of this superfamily contribute to the biology of the diverse array of organisms which produce them.

Finally, PlcHR2 could be useful as a powerful tool to examine lipid signalling cascades in eukaryotic cells due to its more specific substrate specificity (SM, PC) in comparison to the widely used, broad spectrum B. cereus PLC that in addition to PC will also hydrolyse PE, PS and phosphatidylglycerol.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References


The pre-swollen microgranular anion exchanger diethylaminoethyl cellulose DE52 was purchased from Whatman. The synthetic substrates ρ-nitrophenyl-phosphorylcholine (NPPC) and ρ-nitrophenyl-phosphate (NPP) were pur-chased from Sigma. Bovine SM [choline-methyl-14C] (1.99 GBqmmol−1), PC l-α-dipalmitoyl [choline-methyl-14C] (5.88 GBqmmol−1) and PC l-α-dipalmitoyl [choline-methyl-3H] (1.59 TBqmmol−1) were purchased from NEN Life Science Products. L-3-phosphatidyl-l-[3-14C] serine, 1,2,-dioleoyl (2.07 Gbq mmol−1) was purchased from Amersham Biosciences. The BODIPY® labelled phospholipids N-(4,4-difluoro - 5,7 - dimethyl - 4 - bora - 3a,4a - diaza - s - indacene-3- pentanoyl) sphingosyl phosphocholine (250 µg), 2-(4,4-difluoro - 5,7 - dimethyl - 4 - bora - 3a,4a - diaza - s - indacene - 3-pentanoyl) - 1 - hexadecanoyl - sn - glycero-3-phosphoethanolamine (100 µg) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a - diaza - s - indacene - 3 - dodecanoyl) - 1- hexadecanoyl-sn-glycerol-3-phosphocholine (100 µg) were purchased from Molecular Probes. All other unlabelled phospholipids were purchased from Sigma. The short chain C6 lipids 1,2-dicaproyl-sn-glycero-3-phosphoethanolamine, 1,2-dicaproyl-sn-glycero-3-phosphocholine and 1,2-dicaproyl-sn-glycero-3-[phospho-l-serine], along with N-hexanoyl-d-erythro-sphingosylphosphorylcholine PC containing approximately 33% plasmalogen were purchased from Avanti Polar Lipids. All lipid stocks were stored in chloroform at −20°C.

Strains and plasmids

The P. aeruginosa PAO1 derivative ADD1976 was used for the overexpression of the plcHR1,2 genes. ADD1976 is identical to PAO1 except that the T7 polymerase gene under the control of the lacUV5 promoter is integrated into the chromosome. T7 expression studies in ADD1976 were performed using the plasmid pADD3268 that contains a multiple cloning site (MCS) located 3′ to the T7 promoter and several T7 transcriptional terminators located 3′ to the MCS (Brunschwig and Darzins, 1992). For overexpression and purification the haemolytic phospholipase, a 4 kb BamHI-SphI DNA fragment containing the plcHR1,2 operon was cloned into the MCS of pADD3268. Gamma irradiated M. tuberculosis H37RV was obtained from Colorado State University.

Mycobacterium tuberculosis PLC extracts

Gamma irradiated M. tuberculosis H37Rv (0.1 g) was resuspended in 300 µl buffer A (50 mM Tris-HCl, pH 7.2, 50 mM NaCl), 0.5% Triton X-100. The cell suspension was disrupted three times with a Tissue Tearor (Biospec Products, model # 985–370) at 30 000 r.p.m. for 30 s on ice. The cell debris was pelleted (10 min, 16 000 g, 4°C) and the supernatant saved. The extraction was repeated two more times carrying over the supernatant each time to achieve a highly concentrated sample.

Gel electrophoresis conditions

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (Laemmli, 1970). Non-denaturing gel conditions for resolution of PlcHR2 included: upper chamber running buffer (40 mM Tris-HCl, pH 8.89, 40 mM glycine), lower chamber running buffer (60 mM Tris-HCl, pH 7.47), separating gel (237 mM Tris-HCl, pH 8.48), and stack gel (40 mM Tris-HCl, pH 6.9). After separation, proteins were visualized by staining with SYPRO® Orange (Molecular Probes). SYPRO® Orange has been demonstrated to stain protein non-preferentially and is as sensitive as silver stain (Steinberg et al., 1996a, b).

PlcH and PlcR monoclonal antibody production

Monoclonal antibodies were produced in the Colorado University Health Science Center Monoclonal Core Laboratory following the established monoclonal preparation schedule. PlcR monoclonal antibodies were produced using a purified GST/PlcR1 preparation. PlcH monoclonal antibodies were produced using a purified denatured PlcH preparation.

Western Blot conditions

Protein samples were separated under either denaturing or non-denaturing condition and transferred onto nitrocellulose (Optitran, Scleicher and Scuell). The nitrocellulose was probed with PlcH, PlcR or a combination of both monoclonal antibodies. Horseradish peroxidase conjugated goat anti-mouse was used as the secondary antibody and bands were detected using the ECL + Plus Western blot detection kit (Amersham Pharmacia Biotech).

Protein concentration determination

Protein concentration was determined by the bicinchoninic acid method (Pierce) or SDS-PAGE using the SYPRO® Orange broad range molecular weight markers as standards (Molecular Probes).

Purification of the P. aeruginosa haemolytic PLC

Pseudomonas aeruginosa ADD1976::pADD1976 containing the plcHR1,2 operon was grown at 37°C in M9 minimal media (750 µg ml−1 carbenicillin, 75 µg ml−1 tetracycline) until the optical density at 590 nm reached 0.5–0.7. A typical purification consisted of 1.5 L of culture. The culture was then induced with isopropylthio-β-galactopyronaoside (IPTG) for 5 h at 32°C. The supernatant was harvested by centrifugation and the ionic strength reduced by addition 1.25 volumes cold ddH2O. The supernatant was then batch bound to DE52 (50 g l−1 supernatant) for 30 min. After binding the DE52 was washed with buffer A (pH 7.2) and batch eluted with 50 mM Tris-HCl (pH 7.2), 500 mM NaCl. The DE52 eluate was concentrated via 75% ammonium sulphate precipitation, dialysed extensively with buffer A, and loaded onto a 7.5% non-denaturing polyacrylamide preparative gel (BioRad model 491, 27 mM diameter). The prep cell was run at 12 watts constant power. Proteins were eluted in buffer A (pH 7.2) at a flow rate of 350 µl min−1 and 2 ml fractions were collected. All purification procedures were carried out at 4°C. Up to 2 mg of the protein of interest and its nearest neighbour can be effectively resolved using a preparative gel (Bio-Rad Model 491 Prep Cell Instruction Manual). Fractions possessing PC-PLC activity were identified using the synthetic substrate NPPC (Kurioka and Matsuda, 1976). Fractions with peak activity were pooled, aliquoted, and stored at − 80°C. The purification of free PlcH and PlcR2 consisted of pooling and concentrating via centrifugation in an Ultrafree-15 centrifugal filter devise (Millipore) with a 10 kDa MWCO those fraction identified by Western blot to contain the individual proteins. The concentrated proteins were aliquoted and stored at − 80°C.

Auto CAD (collision-activated dissociation) and database searching

Collision-activated dissociation (CAD) spectra were acquired for proteolytic fragments (Lys-C or trypsin, averaged from five spectra) and submitted to the P. aeruginosa database using the sequest algorithm (Eng et al., 1994; Yates et al., 1995). The sample was digested with Lys-C protease in 4 M urea at 37°C for 18 h. For subsequent experiments portions were diluted to reduce the urea concentration and subdigested with trypsin for an additional 8 h in order to improve the interpretability of the fragmentation spectra, see for example the R2 peptide shown in Fig. 4A and B. Two microlitres of the digested complex were then loaded onto a Monitor C18 50 µm i.d. × 10 cm HPLC column. Using an elution gradient of 1–50% acetonitrile over a 25 min period, the peptide fragments were eluted from the column and electrosprayed into a TSQ 7000 triple quadrupole mass spectrometer (Finnigan, San Jose, CA) using an inlet system described previously (Wang et al., 1997). The eluted peptides were detected by the third quadrupole (MS1 using Q3) of the mass spectrometer at a scan rate of 1.5 s per scan over a mass range 300–1400 amu. The mass spectrometer automatically switched to CAD mode (Ducret et al., 1998) when the parent ion intensity exceeded a preset threshold (30 000 counts). In the auto CAD mode, the first quadrupole (Q1) of the mass spectrometer was used as a mass filter, allowing only the parent ions of interest to be selected, and then passed into and fragmented in the RF only collision cell (Q2, which contained argon gas, 3 mTorr). The resulting fragment ions were then analysed in the third quadrupole (Q3), which was scanned from m/z 50–2000 at 1.5 s scan−1. Five CAD spectra per parent ion were acquired with either incrementally increasing or decreasing collision energy, depending on the fragmentation efficiency. The mass spectrometer then switched back to main beam mode and readied itself for the next auto CAD acquisition (Fig. 4A). The P. aeruginosa database was created by extracting the deduced amino acid sequence data for all known P. aeruginosa proteins from the larger OWL database.

The 1 : 1 PlcH/PlcR2 subunit stoichiometry was determined by averaging the signals for at least seven peptides each from the Lys-C, tryptic and Asp-N digestions and taking the ratio of the average signals observed for PlcH and PlcR2 respectively. Peptide standards with known molar response factors were used to insure that the data were not skewed by abnormally high or low absolute molar response, a precaution that was necessary given the wide variability in absolute signal intensity that one often sees in peptides analysed using electrospray ionization. The behaviour of the complex on native PAGE (Fig. 3D) and size exclusion gels (data not shown) precluded serious consideration of higher order non-covalent structures, e.g. 2 : 2, 3 : 3, etc.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was performed as previously described (Mancini et al., 1998).

PLC activity assays

Phospholipase activity was measured by a number of methods. The water-soluble C6 short chain PLC assay was performed via a sensitive assay based on the quantification of inorganic phosphate (Pi) using C6 phospholipids (Avanti Polar Lipids) (Hergenrother and Martin, 1997). Briefly, this assay consisted of 25 µl of PlcHR2 in buffer A (pH 7.2), 0.025% bovine serum albumin (BSA) or B. cereus PLC (Sigma) in buffer A (pH 7.2), 0.1 mM Zn2+ added to 50 µl of C6 phospholipid substrate in buffer A (pH 7.2). The reaction was incubated at 37°C for 20 min. All substrates were assayed at concentrations below their respective critical micelle concentrations (Hergenrother and Martin, 1997).

Enzymatic assays with the artificial substrate ρ-nitrophenyl-phosphorylcholine (NPPC) were carried out as previously described (Kurioka and Matsuda, 1976). It was determined that the optimal NPPC reaction conditions for PlcHR2 consisted of 100 mM Tris-HCl (pH 7.2), 25% glycerol at 37°C. The kinetic parameters for PlcHR2 in a NPPC assay were determined by assaying PlcHR2 (0.01 µg ml−1) with varying concentrations (1.41, 2.81, 5,63, 11.25, 22.5, 45, 67.5, 90, 112.5 and 135 mM) of NPPC.

Mixed-micelle kinetics for PlcHR2 were determined using the surface dilution kinetic model (Deems, 2000). This model uses micelles and carries out initial rate studies at varying ligand concentrations. Briefly, the surface concentrations of PC or SM were varied from 1.78 to 25.28 mole per cent phospholipid to neutral dilutor (Triton X-100). Bulk concentrations of phospholipid substrate were varied from 37.5 to 800 µM. Reaction conditions consisted of 25 mM Hepes (pH 7.2), 100 mM NaCl, and 0.1 µg ml−1 PlcHR2. Mixed micelles were formed by drying down a measured amount of phospholipid under a stream of nitrogen. Triton X-100 buffer was added and the mixture was sonicated and vortexed several times. Reactions were carried out at 37°C and quenched by transferring 50 µl of the reaction to 500 µl chloroform/methanol/HCl (50 : 50 : 0.3, v/v/v). Soluble counts were released by the addition of 200 µl of 1 M HCl, 5 mM EGTA. The mixture was vortexed and then centrifuged to facilitate phase separation. A 400 µl aliquot of the aqueous phase was used for liquid scintillation counting using a Beckman LS-7000 scintillation counter. Mixed micelle assays were also performed with BODIPY® labelled phospholipid substrates (Molecular Probes). A typical assay consisted of adding 3 µl of diluted PlcHR2 to 1 µl (0.2 µg) of BODIPY® phospholipids in 25 mM Hepes (pH 7.2), 100 mM NaCl, 0.4 mM octyl-β-d-glucopyranoside. The samples were incubated at 37°C for 1 h. At which time activity was assessed by resolving the 4 µl reaction via thin layer chromatography (TLC) using a H2O saturated ethyl acetate/iso-octane/glacial acetic acid (135 : 75 : 30, v/v/v) solvent system. BODIPY® labelled products were detected using a Bio-Rad Fluor-S MultiImager. Image analysis was carried out using Quantity One version 4.0.3 (BioRad).

The radiolabelled unilamellar vesicle assay was performed as described by others (Lopez et al., 1998). The kinetic parameters for PlcHR2 in a vesicle assay were determined by assaying PlcHR2 (0.1 µg ml−1) with varying concentration (50, 100, 200, 300, and 600 µM) of PC in 25 mM Hepes (pH 7.2), 100 mM NaCl.

Haemolytic activity

Purified PlcHR2 was added to 1% human erythrocytes in 0.85% NaCl and the reactions were incubated at 37°C for the indicated time points. Aliquots were withdrawn from the reaction at various times and the erythrocytes and cell debris were removed by centrifugation (800 g). Hot-cold haemolysis was assayed by transferring the samples to ice for the final 2 min of the time point and then pelleting at 4°C (800 g). The amount of haemoglobin released was determined spectrophotometrically at 562 nm. Erythrocytes were incubated in H2O to measure total lysis and background lysis was determined by incubating erythrocytes in 0.85% NaCl. Per cent lysis was calculated as follows: 100•(A562 sample – A562 background)/(A562 total lysis – A562 background).

Phosphatase activity

Phosphatase activity was assayed using ρ-nitrophenyl-phosphate (NPP) hydrolysis. Similar to the NPPC assay, hydrolysis of NPP by phosphatases releases inorganic phosphate and the chromogenic compound ρ-nitrophenol, which can be quantified spectrophotometrically at 410 nm. PlcHR2 was assayed for phosphatase activity at both pH 6.0 and 7.2.

Plasmalogen assay

PlcHR2 enzyme was evaluated for specificity towards the plasmalogen containing component of a PC mixture. An extract of heart PC containing approximately 33% plasmalogen was purchased from AVANTI Polar Lipids. The commercial product (25 mg ml−1 in chloroform) was diluted to a working stock solution of 1 mg ml−1 in chloroform. Aliquots of this stock solution were dried down and resolvated in 100 µl of 50 mM Hepes (pH 7.2), 80 mM KCl to a sample concentration of 150 µM. PlcHR2 (0.1 µg ml−1) was added to these preparations for 5, 10, 20 and 40 min with controls of enzyme alone in Hepes, and lipid mixture alone with no enzyme. Samples were extracted with 200 µl each chloroform/methanol (50 : 50 v/v) and the CHCl3 layer was recovered and saved for mass spectrometric analysis.

Samples were analysed by electrospray ionization mass spectrometry on an API III plus tandem mass spectrometer. Samples were analysed in positive ion mode by loop injection of 1–2 µM of sample at a flow rate of 30 µM min−1. Instrument parameters for single quad scans included an ionspray voltage of 4800 V, orifice potential of 75 V. Collisional activation in MS/MS studies used a collision gas thickness (argon) of 142 × 1013 molecules cm−2 and a relative collision energy of 15 eV. Make up solvent consisted of a 1 : 1 mixture of solvent A, methanol–water–acetonitrile (60 : 20 : 20, v/v/v) containing 1 mM ammonium acetate, and solvent B, 1 mM ammonium acetate in methanol (Khaselev and Murphy, 2000).

Calcium binding

Purified PlcHR2, PlcH and PlcR2 were separated under denaturing or non-denaturing condition and transferred to nitrocellulose (Optitran, Scleicher and Scuell). The nitrocellulose was then incubated in 25 mg ml−1 ruthenium red, 10 mM Tris-HCl (pH 6.8), 5 mM MgCl2 for 1 h. Ruthenium red binds to calcium binding proteins, staining them red on the membrane (Charuk et al., 1990).


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank the Colorado State University of the NIH, NIAID Contract NO1 AI-75320 entitled, ‘Tuberculosis Research Materials and Vaccine Testing’ for the gamma irradiated M. tuberculosis H37RV. We are also grateful to P. J. Wilderman for critical reading of the manuscript and Houle Wang for his technical assistance with the TSQ7000 mass spectrometer. This work was supported by the grant (HL62608) from the National Institute of Heart, Lung and Blood to Michael Vasil.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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