Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac.

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.

Complete switch from α-2,3- to α-2,6-regioselectivity in Pasteurella dagmatis ß-D-galactoside sialyltransferase by active-site redesign.

Structure-guided active-site redesign of a family GT-80 ß-D-galactoside sialyltransferase (from Pasteurella dagmatis) to change enzyme regioselectivity from α-2,3 in the wild type to α-2,6 in a P7H-M117A double mutant is reported. Biochemical data for sialylation of lactose together with protein crystal structures demonstrate highly precise enzyme engineering.

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis.

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His(284) in the active site replaced by Asn, Asp or Tyr showed up to 10(4)-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His(284) in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.

Characterization of a multifunctional α2,3-sialyltransferase from Pasteurella dagmatis.

A new multifunctional α2,3-sialyltransferase has been discovered in Pasteurella dagmatis. The enzyme, in short PdST, was identified from the P. dagmatis genome by sequence similarity with sialyltransferases of glycosyltransferase family GT-80. In addition to its regioselective sialyltransferase activity (5.9 U/mg; pH 8.0), purified PdST is alternatively active at low pH as α2,3-sialidase (0.5 U/mg; pH 4.5) and α2,3-trans-sialidase (1.0 U/mg; pH 4.5). It also shows cytidine-5'-monophosphate N-acetyl-neuraminic (CMP-Neu5Ac) hydrolase activity (3.7 U/mg; pH 8.0) when no sialyl acceptor substrate is present in the reaction. After sialyltransferase PmST1 from P. multocida, PdST is the second member of family GT-80 to display this remarkable catalytic promiscuity. A unique feature of PdST, however, is a naturally occurring Ser-to-Thr substitution within a highly conserved Y(112)DDGS(116) sequence motif. In PmST1, the equivalent Ser(143) is involved in binding of the CMP-Neu5Ac donor substrate. Reversion of the natural mutation in a T116S-PdST variant resulted in a marked increase in α2,3-trans-sialidase side activity (4.0 U/mg; pH 4.5), whereas the major sialyltransferase activity was lowered (3.8 U/mg; pH 8.0). The Michaelis-Menten constant for CMP-Neu5Ac was decreased 4-fold in T116S mutant when compared with wild-type PdST (KM=1.1 mM), indicating that residue 116 of PdST contributes to a delicate balance between substrate binding and catalytic activity. D-Galactose and various ß-D-galactosides function as sialyl acceptors from CMP-Neu5Ac, whereas other hexoses (e.g. D-glucose) are inactive. Structure comparison was used to rationalize the particular acceptor substrate specificity of PdST in relation to other GT-80 sialyltransferases that show strict α2,3-regioselectivity, but are flexible in using α/ß-galactosides for sialylation.

Sequential two-step multienzyme synthesis of tumor-associated sialyl T-antigens and derivatives.

A series of α2-3-sialylated ß1-3-linked galactosides, including sialyl T-antigens, 3'-sialyl galacto-N-biose, 3'-sialyl lacto-N-biose, and their derivatives containing natural and non-natural sialic acid forms have been synthesized from simple monosaccharides using an efficient sequential two-step multienzyme approach.

Monodisperse hyaluronan polymers: synthesis and potential applications.

In many cases, the cellular response to hyaluronan (HA) depends on the molecular weight (MW) of the polymer chain. Most HA preparations from Nature or its derivatives possess wide size distributions called polydisperse. New chemoenzymatic synthesis technology allows the production of very narrow size distribution polymers called monodisperse. The use of stoichiometrically controlled and synchronized polymerization reactions allows an assortment of new HA reagents in the range of 10 kDa to 2,500 kDa for answering HA biology questions or potentially treating disease.

Pasteurella multocida sialic acid aldolase: a promising biocatalyst.

Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D: -mannosamine (ManNAc). A capillary electrophoresis assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg l(-1) culture) has a higher expression level (2.5-fold) than EcNanA (94 mg l(-1) culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene.

The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodA(int)) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodA(int) sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodA(int) sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodA(int) sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (> 98% sodA(int) sequence identity) but shared only 92% sodA(int) identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.

Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers.

The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems. Long and short HA polymers often appear to have antagonistic or inverse effects. However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available. We present a method to produce synthetic HA with very narrow size distributions in the range of approximately 16 kDa to approximately 2 MDa. The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors. Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion. As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length. In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse. HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e. molar ratio of precursors and acceptor molecules). The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g. fluorescent, radioactive). In this scheme, each molecule has a single foreign moiety at the reducing terminus. Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers. Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease.

Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection.

A strain of Pasteurella trehalosi serotype 10, E(CO)-100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A (lktA) from E(CO)-100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E(CO)-100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E(CO)-100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E(CO)-100 (r=0.5, P=0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.

Genetic relationships among Pasteurella trehalosi isolates based on multilocus enzyme electrophoresis.

Genetic diversity among 60 British Pasteurella trehalosi isolates representing the four recognized capsular serotypes, T3, T4, T10 and T15, and recovered predominantly from sheep suffering from systemic pasteurellosis, was estimated by analysing electrophoretically demonstrable allelic variation at structural genes encoding 19 enzymes. Thirteen of the locl were polymorphic and 20 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The population structure of P. trehalosi is clonal and its genetic diversity is limited compared with most other pathogenic bacteria. ETs represent clones, and isolates of the same ET were generally associated with the same combination of serotype, LPS type and outer-membrane protein (OMP) type. The genetic diversity of isolates within each of the capsular serotypes varied. Serotype T10 was represented by 18 isolates in two related ETs and exhibited little diversity. By contrast, serotype T15 was represented by 18 isolates in nine ETs and was almost as diverse as the species as a whole Serotype T4 was represented by 18 isolates in five ETs and was less diverse than serotype T15. Although serotype T3 was more diverse than serotype T15 it was represented by only three isolates. With the exception of the T10 isolates and those recovered from healthy sheep, 35 disease isolates belonged to 16 ETs, each of which was represented by only one to four isolates. The fact that a high proportion of disease is caused by a relatively large number of clones suggests that P. trehalosi is essentially an opportunistic pathogen. In addition to having the same capsular structure, isolates belonging to the two T10 clones were characterized by possession of similar, if not identical, O-antigens (LPS types 2 and 4). The occurrence of 18 serotype T10 isolates in only two ETs suggests that the T10 capsule and type 2/4 O-antigen confer enhanced virulence on members of these two clones. Multilocus enzyme electrophoresis (MLEE) had greater resolving power than did capsule/LPS/OMP analysis, being able to distinguish 20 rather than 14 sub-divisions within P. trehalosi. The technique demonstrated genetic identity or non-identity among strains of the same or different serotypes from different geographic localities within the UK and was a useful epidemiological tool.

Divergent activity and function of superoxide dismutases in Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10.

Representative strains of Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were examined for the presence of superoxide dismutase. Visualisation of superoxide dismutase enzyme activity on polyacrylamide gels, and specific inhibition with potassium cyanide verified a copper/zinc (Cu/Zn) superoxide dismutase only in serotype A2 whereas serotypes A1 and T10 showed other superoxide dismutase activity. Using a simple freeze-thaw method the cellular location of superoxide dismutase enzyme activity was determined in all three serotypes. In serotypes A1 and A2 but not T10 superoxide dismutases were located in the periplasm. The viability of serotypes A2 and T10 cells in the presence of exogenous superoxide was unchanged over a 30 min period, whereas serotype A1 cells declined in viability between 15 and 30 min. Purified immunoglobulin from sheep convalescent serum did not reduce superoxide dismutase activity in the serotypes in an in vitro assay. The presence of this enzyme within the pasteurellae suggests a supportive role in the virulence of this major pathogen of ruminants.

Occurrence of [copper, zinc]-cofactored superoxide dismutase in Pasteurella haemolytica and its serotype distribution.

Fifty-two ovine strains of Pasteurella haemolytica and P. trehalosi representing serotypes 1-16 were examined for the presence of [copper, zinc]superoxide dismutase DNA sequences. This was done using a combination of polymerase chain reaction with degenerate primers based on the sequence of the [Cu,Zn]superoxide dismutase gene (sodC) in related species and Southern hybridization using a fragment of sodC from P. haemolytica A2 serotype as a probe. Both detection methods identified a fragment of the sodC gene in 9/9 strains of P. haemolytica serotype 2 examined and in 5/8 strains of serotype 7. No evidence of this gene was found in any other serotype of P. haemolytica or in any P. trehalosi serotype. Comparison of DNA sequence showed near identity between sodC from the A2 and A7 serotypes of P. haemolytica and substantial similarity (70%) to sodC previously sequenced in P. multocida, Haemophilus parainfluenzae and H. influenzae. Analysis by gel electrophoresis of the superoxide dismutase activity present in cell lysates showed that one or more superoxide dismutase is present in all serotypes. However, cyanide-inhibitable activity, corresponding to [Cu,Zn]superoxide dismutase, was detected only in those strains of serotypes A2 and A7 which showed evidence of the sodC gene fragment.

Extracellular neuraminidase production by Pasteurella species isolated from infected animals.

A total of 721 field isolates of various Pasteurella species (haemolytica, multocida, and testudinis) from various regions of the United States were examined for extracellular neuraminidase production. All strains were grown and tested in the same way. Included were 372 P. haemolytica serotype 1 isolates, 181 P. haemolytica serotype 2 isolates, 63 P. haemolytica serotype 6 isolates, 101 Pasteurella multocida isolates, and 4 Pasteurella testudinis isolates. All Pasteurella species examined produced the enzyme. The data revealed the following: (1) Several transfers of P. haemolytica strains on blood agar medium did not cause a decrease in enzyme activity. (2) P. haemolytica serotypes 2 and 6 produce more neuraminidase than P. haemolytica serotype 1, P. multocida, and P. testudinis isolates. (3) There was no apparent change in neuraminidase production by P. haemolytica serotypes 1 and 2 obtained from the same animal taken on different days in the feedyard. (4) There was no significant change in neuraminidase production by P. haemolytica serotype 2 isolates taken from the same animal at the auction market and later at the feedyard.

Occurrence of sialidase and N-acetylneuraminate lyase in Pasteurella species.

Pasteurella species and related taxa are opportunistic pathogens parasitizing on mucous membranes of higher organisms containing sialic acids. Therefore, sialidase is a virulence factor which up to now has been described to be present in P. haemolytica, P. multocida, and P. volantium. Because of some taxonomic changes and the description of many new species or still unnamed groups, the presence of sialidase and the metabolic successor enzyme, N-acetylneuraminate lyase, was investigated in 65 Pasteurella or Pasteurella-like strains. The detection of enzymes was performed by colorimetry, by paper chromatography and immunoelectrophoresis. Using bovine submaxillary mucin as substrate, sialidases were produced in all strains studied although the activities were different. Most strains but not all were positive in N-acetylneuraminate lyase, too. Taken together, the strains of Pasteurella sensu stricto showed the strongest activities of sialidase, those of the Pasteurella aerogenes complex the lowest. However, because of loss of sialidase activity during subcultivation, there is little feasibility to characterize Pasteurella species by these enzymes.

Bacterial [Cu,Zn]-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!

Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens-members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

Detection of an adenylate cyclase gene in Pasteurella species.

A Pasteurella multocida adenylate cyclase gene has been previously cloned in Escherichia coli and sequenced. A 1200 bp HpaI fragment from the coding region was used as a probe to analyse the presence of the gene in different Pasteurella species and subspecies, Actinobacillus ureae (formerly P. ureae) and group EF-4 bacteria. Thirty-seven strains were checked for the presence of the gene. It was shown that the adenylate cyclase gene was detected only in the species Pasteurella multocida.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.