Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival.

A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.

Comparison and validation of visual assessment and image processing algorithms to quantify morphology dynamics of Euglena gracilis.

Image processing algorithms were developed and compared with visual assessment from 12 volunteers to quantify the temporal morphological structure of a single Euglena gracilis organism. Representative images of E. gracilis, showing different morphological characteristics from ovate to cylindrical and elongate, were captured with a bright-field video microscopy system. These images were ranked by the volunteers in order from ovate to elongate. The images were analyzed in the spatial and spatial frequency domain, and the order of the images from each analysis was ranked against the visual assessment. The assessment methods agreeing with the volunteer's preferred sequence were an eccentricity measurement (major axis over the sum of the minor axis at three points), the cross correlation of the image without high pass filtering or edge detection, and cross correlation of the power spectral density.

BapC autotransporter protein is a virulence determinant of Bordetella pertussis.

A protein designated Bap-5 (GenBank accession no. AF081494) or BapC (GenBank accession no. AJ277634) has been identified as a member of the Bordetella pertussis autotransporter family and the present work suggests that this protein, like the previously characterised BrkA, is a Bvg-regulated serum resistance factor and virulence determinant. B. pertussis bapC and brkA, bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA, bapC mutants, like the brkA mutant, were found to be more sensitive to the antimicrobial peptide cecropin P1 than the parent strains. In the genome sequence of B. pertussis strain Tohama, bapC is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5' end of the gene which creates a truncated BapC protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms but it appeared that all had an ORF that would be able to produce BapC.

Safety and protective efficacy of intramuscular vaccination with a live aroA derivative of Pasteurella multocida B:2 against experimental hemorrhagic septicemia in calves.

Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 10(9), 10(8), and 10(7) CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than in group V3 (P = 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected subcutaneously at 10 weeks of age with ca. 10(7) CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and safety.

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK.

To determine the value of gene markers for surveillance and to assess the genetic stability of potential acellular pertussis vaccine components, the sequence variation in ten virulence-related genes of Bordetella pertussis was investigated in strains isolated in the UK between 1920 and 2002. These genes encode: pertactin (prnA); pertussis toxin subunits S1 (ptxA) and S3 (ptxC); tracheal colonization factor (tcfA); bordetella autotransporter protein C (bapC); bordetella resistance to killing protein (brkA); fimbrial antigen 2 (fim2); outer-membrane protein Q (ompQ); virulence-activated gene 8 (vag8) and adenylate cyclase toxin (cyaA). The encoded proteins are either components of current acellular vaccines (ACVs), or potential virulence markers for B. pertussis. Three strains used in the pertussis UK whole-cell vaccine (WCV), strain Tohama-I used for production of ACV components and the type strain of B. pertussis (18323(T)) were also analysed. Several novel alleles were found. The UK isolates were assigned multi-locus sequence types (MLSTs) according to a previously described scheme for B. pertussis based on three of these genes (ptxA, ptxC and tcfA). Compared with isolates from other countries, the UK clinical strains showed a distinct distribution of MLSTs. Apart from one strain that was MLST-3, all other recent isolates (2000-2002) were identified as MLST-5. These isolates differed from the three WCV strains, which were MLST-2 or MLST-3, the Tohama-I strain (MLST-2) and the type strain of B. pertussis (MLST-9). MLST-3 and MLST-5 differ only by a single synonymous mutation, but this method does indicate that currently circulating strains of B. pertussis are not identical to the vaccine types, and they may differ in other important characteristics. Two new MLSTs were identified amongst historical UK isolates. Sequence-based typing offers a convenient method of analysing and comparing populations of B. pertussis from different time periods and from different countries. The variation exhibited by prnA and fim2 suggests that they could be useful, additional epidemiological markers in such a typing scheme.

Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity.

Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response.

Use of a dual reporter plasmid to demonstrate Bactofection with an attenuated AroA(-) derivative of Pasteurella multocida B:2.

A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA(-) derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.

Identification of immunogenic proteins associated with protection against haemorrhagic septicaemia after vaccination of calves with a live-attenuated aroA derivative of Pasteurella multocida B:2.

Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes. As a step towards the identification of individual antigens that may protect against HS, proteins present in a sonicated cell extract (SCE) and outer-membrane protein (OMP) preparation of a wild-type P. multocida serotype B:2 were investigated by immunoblotting with sera from calves which had been protected against challenge with a virulent strain of P. multocida B:2 by vaccination with a live-attenuated aroA derivative of the challenge strain. Five proteins in SCE, of approximately 50, 37, 30, 26 and 16 kDa, were recognised by the sera. In an OMP preparation, two bands, at 37 and 50 kDa, were recognised as strongly immunogenic. Mass spectrometry analysis of proteins corresponding in size to those detected by immunoblotting identified the 37 kDa band as OmpA, but the band at 50 kDa was not identified with certainty. A major 30 kDa OMP, identified as OmpH, was not strongly immunogenic.

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis.

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.

Transcriptional responses of murine macrophages to the adenylate cyclase toxin of Bordetella pertussis.

Three different recombinant forms of CyaA were used to investigate transcriptional responses of murine bone marrow-derived macrophages (BMMs) using Affymetrix Mouse Genome GeneChips. These forms were enzymically active, invasive CyaA, non-enzymically active, invasive CyaA (CyaA*) and non-enzymically active, non-invasive CyaA (proCyaA*). BMMs, treated with 20 ng/ml of CyaA for 24h, showed over 1000 significant changes in gene transcription compared with control cells. CyaA caused an increase in transcription of many inflammatory genes and genes associated with various signalling cascades such as those involved in cyclic AMP-dependent protein kinase A signalling. Most strikingly, CyaA caused down-regulation of numerous genes involved in cell proliferation. CyaA* at 20 ng/ml significantly up-regulated the transcription of only twelve genes after 24h whereas proCyaA* at this concentration significantly increased the transcription of only two genes.

Formulation of the adenylate cyclase toxin of Bordetella pertussis as protein-coated microcrystals.

Adenylate cyclase toxin (CyaA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and, in its detoxified form, a potential component of acellular pertussis vaccines. This study reports the application of a novel technology, formulation of CyaA as protein-coated microcrystals (PCMC), to improve the performance of CyaA as a vaccine component. CyaA is normally stored in a high urea concentration to prevent aggregation and to maintain stability of the protein. The aim of the work was to stabilise CyaA on a crystalline support to create a dry powder that could be reconstituted in aqueous buffer, free of urea. CyaA, formulated as PCMC with microcrystals of dl-valine, retained full adenylate cyclase (AC) and cell invasive (cytotoxic) activities after solubilistion in urea buffer. After storage as a dry powder at 37 degrees C for 2 weeks, the AC activity recovered from the CyaA-PCMC was only marginally reduced when solubilised in urea buffer. No AC activity was detected after attempts to solubilise CyaA-PCMC in aqueous buffer alone, in the absence of urea. Inclusion of various ionic, non-ionic or zwitterionic detergents in the aqueous buffer had little effect on recovery of CyaA activities. However, preparation of PCMC with CyaA plus calmodulin (CaM) or bovine serum albumin (BSA) or with both proteins allowed restoration of AC and cytotoxic activities of CyaA upon solubilisation in aqueous buffer. Incorporation of BSA and CaM with CyaA allowed essentially full recovery of AC activity but lower recovery of cytotoxicity. CyaA-CaM-BSA-PCMC, after reconstitution in aqueous buffer, induced a strong serum IgG response to CyaA when injected subcutaneously into mice.

Adjuvant effects of adenylate cyclase toxin of Bordetella pertussis after intranasal immunisation of mice.

This study examined the ability of the adenylate cyclase toxin (CyaA) of Bordetella pertussis to act as a mucosal adjuvant for other antigens when co-administered by the intranasal route in mice. Two forms of CyaA were used: the cell-invasive, enzymically active form and a cell-invasive toxin lacking adenylate cyclase enzymic activity (CyaA*). Co-administration intranasally (i/n) of CyaA or CyaA* with ovalbumin (Ova) significantly enhanced (P<0.05) anti-Ova IgG and IgA antibody responses in the serum and anti-Ova IgA responses in lung and nasal secretions compared to those generated by immunisation i/n with Ova alone. The effects were greater with CyaA*. Administration of CyaA* with Ova induced priming of Ova-specific T cells in vivo to a greater extent than that obtained after immunisation with Ova alone. Co-administration of CyaA or CyaA* with pertactin (Prn) significantly enhanced (P<0.05) the serum anti-Prn IgG responses and immunisation with Prn and CyaA* significantly increased the anti-Prn IgA responses in the lungs compared with responses after immunisation with Prn alone. Immunisation i/n with Prn alone partially protected mice (P<0.05) against challenge i/n with B. pertussis. Co-administration of CyaA or CyaA* with pertactin (Prn) significantly increased protection (P<0.05) against challenge compared to that obtained with Prn alone. These effects were particularly apparent with CyaA* as the adjuvant.

Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model.

Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis were compared for the ability to enhance protection against B. pertussis in mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P > 0.05) protection against B. pertussis in a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P < 0.05) reductions in bacterial numbers in the lungs after intranasal challenge compared with those for control mice. When administered with ACV, both CyaA and CyaA* further reduced bacterial numbers in the lungs of mice after intranasal challenge compared with those for ACV-immunized mice, but the enhanced protection was only significant (P < 0.05) with CyaA*. Coadministration of CyaA* with ACV caused a significant (P < 0.05) increase in immunoglobulin G2a antibody levels against pertactin compared with those in mice immunized with ACV alone. Spleen cells from mice immunized with ACV plus CyaA* secreted larger amounts of interleukin-5 (IL-5), IL-6, gamma interferon (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did cells from mice immunized with ACV plus CyaA or ACV alone after stimulation in vitro with a mixture of B. pertussis antigens. Spleen cells from mice immunized with ACV plus CyaA* also secreted larger amounts of IFN-gamma and GM-CSF than did cells from mice immunized with CyaA* alone after stimulation in vitro with CyaA*. Macrophages from mice immunized with ACV plus CyaA* produced significantly (P < 0.05) higher levels of nitric oxide than did macrophages from mice immunized with CyaA* alone, ACV alone, or ACV plus CyaA after stimulation in vitro with a mixture of B. pertussis antigens or heat-killed B. pertussis cells. These data suggest that the enhancement of protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses to B. pertussis antigens.

Development of a carbohydrate binding assay for the B-oligomer of pertussis toxin and toxoid.

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and, in its detoxified form PTd, is an important component of pertussis vaccines. The in vivo histamine sensitization test (HIST) is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. PTx has two functionally distinct domains: the enzymatic A-protomer and the B-oligomer that facilitates host-cell binding and entry of PTx into the cell. The development of a quantitative PTx binding assay using glycoproteins or defined oligosaccharides is reported. PTx was found to bind preferentially to multiantennary N-glycans, with the highest binding toward the fully sialylated structures. In contrast, PTd lost the ability of PTx to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. The developed assay was shown to be specific, sensitive, and robust and could be used for investigating the mechanisms of PTx detoxification and for monitoring PTx binding activity in vaccine formulations. This assay could also be used to complement a PTx-enzymatic assay, developed recently, and together they may form the basis of a potential alternative in vitro assay to replace the in vivo HIST.

Efficacy of vaccination of calves against hemorrhagic septicemia with a live aroA derivative of Pasteurella multocida B:2 by two different routes of administration.

Two groups of four calves each were immunized either intramuscularly (i.m. vaccinated) or intranasally (i.n. vaccinated) at 2 and 6 weeks of age with ca. 10(9) CFU of a derivative of P. multocida serotype B:2 strain 85020 containing a deletion in the aroA gene (strain JRMT12). Both groups of calves and three unvaccinated control calves were challenged subcutaneously at 8 weeks of age with ca. 10(7) CFU of the wild-type 85020 strain. The first and second vaccinations caused a significant pyrexia and increase in the mean demeanor score (P <0.05) in i.m. but not i.n. vaccinated calves. Serum agglutinating activity against whole cells of P. multocida strain 85020 and immunoglobulin G antibody concentrations increased after the second vaccination in i.m. but not in i.n. vaccinated animals, and this difference was statistically significant (P <0.05). Concentrations of serum amyloid A (SAA) increased significantly 3 h after both the primary (P <0.05) and booster (P <0.001) i.m. vaccinations, but not in i.n. vaccinated calves. All four i.m. vaccinated calves were solidly immune to challenge with wild-type P. multocida B:2. However, the mean rectal temperatures, demeanor scores, and serum SAA concentrations of i.n. vaccinated and control calves increased significantly (P <0.01). Three i.n. vaccinated and two control calves were killed for humane reasons within 14 h postchallenge, and postmortem examination revealed pathological lesions consistent with hemorrhagic septicemia. These data showed that the aroA mutant strain, given i.m. as two doses 4 weeks apart, acted as an effective live-attenuated vaccine strain to protect calves against challenge with the virulent parent strain.

Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussis towards co-administered antigens.

The cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis was shown to be highly antigenic in mice, stimulating serum anti-CyaA IgG antibody responses which were able to neutralise the cytotoxic effect of CyaA on J774.2 macrophage-like cells. The effect of co-administration to mice of the fully functional CyaA toxin or a toxin lacking adenylate cyclase enzymic activity (CyaA*) with other antigens from B. pertussis, namely pertussis toxin (PT) or pertussis toxoid (PTd), filamentous haemagglutinin (FHA) and pertactin (PRN), was investigated. CyaA* enhanced the serum IgG antibody responses to each of these antigens whereas, with CyaA, only anti-PRN antibody titres showed a modest increase. Peritoneal macrophages and spleen cells, collected at 2 weeks post-immunisation, were cultured and tested for nitric oxide (NO) and IFNgamma production, respectively, after stimulation in vitro with heat-killed B. pertussis cells or CyaA proteins. NO and IFNgamma production were higher in cells collected from mice immunised with CyaA or CyaA* in combination with a PT, FHA and PRN antigen mixture than from those taken from mice injected with antigen mixture alone, again with CyaA* acting as a better adjuvant than CyaA. The apparent enhancement of immune responses to the antigen mixture by CyaA* in particular was not paralleled by increased protection of mice against aerosol challenge with B. pertussis, but a statistically significant increase in protection was seen after intranasal challenge with B. parapertussis.

Protective immunity conferred by attenuated aroA derivatives of Pasteurella multocida B:2 strains in a mouse model of hemorrhagic septicemia.

Hemorrhagic septicemia (HS) is a fatal systemic disease of cattle and buffaloes. In South Asia HS is caused by infection with Pasteurella multocida serotype B:2. Some control is achieved with alum-precipitated or oil-adjuvanted killed whole-cell vaccines injected subcutaneously, but these vaccines provide only short-term immunity and require annual administration for effective use. Live attenuated vaccines have the advantage of a natural route of entry into the host, but for live strains to be used as vaccines, the mode of attenuation should be well defined. We constructed aroA attenuated derivatives of two P. multocida serotype B:2 strains by allelic exchange of the native aroA sequence with aroA sequences disrupted with a kanamycin resistance cassette or with marker-free aroA sequences containing an internal deletion. These strains were confirmed to be aroA mutants by PCR and Southern blot analysis, enzyme assay, and lack of growth on minimal medium. The aroA derivatives were highly attenuated for virulence in a mouse model of HS. Mouse challenge experiments showed that intraperitoneal or intranasal vaccination of an aroA strain completely protected mice against challenge with a high dose (>1,000 50% lethal doses) of either the parent strain or the other wild-type B:2 strain. The spread of the parent and the aroA derivatives to different organs was compared when the organisms were inoculated by different routes.

A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.

Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.