Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen.

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Mannheimia haemolytica IgA-specific proteases.

Mannheimia haemolytica colonizes the nasopharynx of cattle and can cause severe fibrinous pleuropneumonia. IgA proteases are metalloendopeptidases released by bacteria that cleave IgA, enhancing colonization of mucosa. The objectives of these studies were to characterize M. haemolytica IgA1 and IgA2 proteases in vitro and in silico, to clone and sequence the genes for these proteases, and to demonstrate immunogenicity of components of the entire IgA protease molecule. Both IgA protease genes were cloned, expressed, and sequenced. Sequences were compared to other published sequences. Components were used to immunize mice to determine immunogenicity. Sera from healthy cattle and cattle that recovered from respiratory disease were examined for antibodies to IgA proteases. In order to assay the cleavage of bovine IgA with IgA1 protease, M. haemolytica culture supernatant was incubated with bovine IgA. Culture supernatant cleaved purified bovine IgA in the presence of ZnCl2. Both IgA proteases contain three domains, 1) IgA peptidase, 2) PL1_Passenger_AT and 3) autotransporter. IgA1 and IgA2 peptidases have molecular weights of 96.5 and 87 kDa, respectively. Convalescent bovine sera with naturally high anti-M. haemolytica antibody titers had high antibodies against all IgA1 & IgA2 protease components. Mouse immunizations indicated high antibodies to the IgA peptidases and autotransporters but not to PL1_Passenger_AT. These data indicate that M. haemolytica produces two IgA proteases that are immunogenic, can cleave bovine IgA, and are produced in vivo, as evidenced by antibodies in convalescent bovine sera. Further studies could focus on IgA protease importance in pathogenesis and immunity.

Plasmid-located extended-spectrum ß-lactamase gene blaROB-2 in Mannheimia haemolytica.

OBJECTIVES: To identify and analyse the first ESBL gene from Mannheimia haemolytica. METHODS: Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation. The sequence was determined by WGS and confirmed by Sanger sequencing. RESULTS: The M. haemolytica strain 48 showed high cephalosporin MICs. A single plasmid, designated pKKM48, with a size of 4323 bp, was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs of all ß-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double-disc confirmatory test for E. coli. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1, resulting in two amino acid exchanges and one additional amino acid in the deduced ß-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae. CONCLUSIONS: This study described the first ESBL gene in Pasteurellaceae, which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries.

Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles.

Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica.

X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid.

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.

A novel Erm monomethyltransferase in antibiotic-resistant isolates of Mannheimia haemolytica and Pasteurella multocida.

Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens.

A three-way comparative genomic analysis of Mannheimia haemolytica isolates.

BACKGROUND: Mannhemia haemolytica is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of M. haemolytica at molecular level and assist in devising an effective control strategy. DESCRIPTION: During our comparative genomic sequence analysis of three Mannheimia haemolytica isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors. CONCLUSIONS: During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in M. haemolytica will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.

Identification and characterization of transcriptional regulation of the Mannheimia haemolytica ferric uptake regulator.

The ferric uptake regulator (Fur) is an iron-dependent transcriptional regulator that regulates genes related to iron acquisition, oxidative stress response, and various other functions. Transcription of fur is typically self-regulating and sensitive to iron and oxidative stress. Following the identification of a fur gene in the genome of the bovine pathogen Mannheimia haemolytica, an attempt was made to characterize the transcriptional control of M. haemolytica fur. Northern blotting, RT-PCR, and primer extension were done to determine that M. haemolytica fur is transcribed using three distinct promoters, two of which are located within the upstream fldA gene. The third promoter is located upstream of a conserved hypothetical protein and drives transcription of a tricistronic message. Quantitative real time PCR experiments indicated that unlike current models of Fur regulation, M. haemolytica fur transcription is unchanged by iron depletion at logarithmic phase and repressed by iron depletion at stationary phase.

Characterization of carbohydrate structures of bovine MUC15 and distribution of the mucin in bovine milk.

The present work reports the characterization of carbohydrate structures and the distribution of the newly identified mucin MUC15, a highly glycosylated protein associated with the bovine milk fat globule membrane (MFGM). Distribution of MUC15 was investigated in various fractions of bovine milk by densitometric scanning of Western blots. In raw milk, MUC15 was shown to constitute 0.08% (wt) of the protein and approximately 1.5% (wt) of the MFGM-associated proteins. Surprisingly, this study showed that in addition to the fat-containing fractions, such as MFGM and buttermilk, MUC15 was present in nonfat-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N-linked glycans were shown to contain mainly hybrid-type N-glycans. In addition, the N-glycans were shown to be sialylated and contain terminal poly-lactosamine structures. The O-linked glycans were found to constitute some unsubstituted Core-1 structures and a substantial number of sialylated Core-1 O-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O-linked glycans is decorated with lactosamine on their terminal ends.

Production of a D-glycero-D-manno-heptosyltransferase mutant of Mannheimia haemolytica displaying a veterinary pathogen specific conserved LPS structure; development and functionality of antibodies to this LPS structure.

Previous structural studies of the lipopolysaccharides from the veterinary pathogens Mannheimia haemolytica (Mh), Actinobacillus pleuropneumoniae (Ap) and Pasteurella multocida (Pm) had identified a conserved inner core oligosaccharide structure that was present in all strains investigated. In order to examine the potential of this inner core structure as a vaccine, a mutagenesis strategy was adopted to interrupt a D-glycero-D-manno-heptosyltransferase gene (losB) of Mh. This gene encodes the enzyme responsible for the addition of a D-glycero-D-manno-heptose residue, the first residue beyond the conserved inner core, and its inactivation exposed the conserved inner core structure as a terminal unit on the mutant LPS molecule. Subsequent analyses confirmed the targeted structure of the mutant LPS had been obtained, and complementation with losB in trans confirmed that the losB gene encodes an alpha-1,6-D-glycero-D-manno-heptosyltransferase. Monoclonal antibodies raised in mice to this LPS structure were found to recognise LPS and whole-cells of the truncated mutant and wild-type Mh. The antibodies were bactericidal against a wild-type Mh strain and were able to passively protect mice in a model of Mh disease. This illustrates that it is possible to raise functional antibodies against the conserved inner core LPS structure.

Efficacy of recombinant sialoglycoprotease in protection of cattle against pneumonic challenge with Mannheimia (Pasteurella) haemolytica A1.

Secreted recombinant sialoglycoprotease fusion protein (Gcp-F) of Mannheimia (Pasteurella) haemolytica A1 was examined for its ability to protect cattle from experimental challenge with M. haemolytica A1. Five M. haemolytica vaccines were compared including Gcp-F, logarithmic phase culture supernate (Presponse) and Presponse enriched with Gcp-F, recombinant leukotoxin (rLkt) or both. All calves receiving Gcp-F had significant serum antibody responses to this antigen, measured by ELISA, prior to challenge. Those vaccinated with Gcp-F alone had significantly lower percent pneumonic tissue than unvaccinated controls and a trend (P=0.085, one-tailed test) to lower clinical scores. Calves receiving Presponse with Gcp-F and rLkt had lower percent pneumonic tissue than those receiving Presponse alone, and calves receiving Presponse enriched with Gcp-F and/or rLkt had lower mean clinical scores, but the differences were not significant. This trial demonstrates the protective capacity of sialoglycoprotease. While, remarkably, recombinant Gcp-F provided some protection alone the results support its practical potential as a component of a multiple antigen vaccine.

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.

Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2.

Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43 kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153 m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77 mM for CTP and 1.82 mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.

Responses to aggregating agents after cleavage of GPIb of human platelets by the O-sialoglycoprotein endoprotease from Pasteurella haemolytica- potential surrogates for Bernard-Soulier platelets?

Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave other glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectively cleaves the heavily O-glycosylated GPlb, but does not cleave N-linked glycoproteins or unglycosylated proteins. Isolated, [14C]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 microg/mL endoprotease for 60 minutes at 37 degrees C. These platelets did not release [14C]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and release of [14C]serotonin by the endoprotease-pretreated platelets were inhibited in response to low concentrations of thrombin, SFLLRN (the PAR-1-activating peptide), collagen, and U46619 (a thromboxane A(2) mimetic); aggregates were smaller in size. The presence of fibrinogen overcame the inhibition of responses induced by SFLLRN, collagen, and U46619. With fibrinogen, primary ADP-induced aggregation was scarcely affected by pretreatment with the endoprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collagen, thromboxane A(2), fibrinogen (GPIIb/IIIa), and ADP appear to function normally on the endoprotease-pretreated platelets. Since only GPIb is cleaved by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet functions in this condition.

In vivo production of neuraminidase by Pasteurella haemolytica in market stressed cattle after natural infection.

Pasteurella haemolytica (Ph) is the most important cause of the bovine acute fibrinohemorrhagic pneumonia that occurs in market stressed calves after shipment to feedyards. Recent characterization of neuraminidase production by these organisms has shown that all 16 serotypes produce an immunologically similar form of the enzyme. Anti-neuraminidase antibody against PhA1 and PhA6 was determined in 101 2- to 5-month-old calves, on their farms of origin, at the order buyer barn (OBB), and through 28 days in the feedyard. Half of the calves were vaccinated with a killed Ph serotype-A1 (PhA1) product. Nasal secretion and tonsil wash specimens were cultured for Ph and Pasteurella multocida (Pm). Serum antibody against PhA1 and PhA6 was measured by indirect hemagglutination (IHA), and anti-neuraminidase antibody was determined by the neutralization assay. At the feedyard, 73 calves had respiratory tract disease. IHA values ranged between 1:2 and 1:1024 for PhA1 and between 1:2 and 1:512 for Ph serotype A6 (PhA6). Forty-two, 24, and 28% of the calves were infected with PhA1, PhA6, and Pm, respectively. Ninety-six percent of the calves experienced an increase in anti-PhA1 neuraminidase antibody when sera drawn on feedyard day 28 were compared with sera drawn on the farm. These data demonstrate that the enzyme neuraminidase is produced in vivo in market stressed cattle after a natural Ph infection.

Bovine platelet adhesion is enhanced by leukotoxin and sialoglycoprotease isolated from Pasteurella haemolytica A1 cultures.

Platelet and fibrin deposits are among characteristic changes observed in lung alveoli of cattle with pasteurellosis induced by Pasteurella haemolytica (biotype A, serotype 1). To determine whether the platelet function could be directly affected by protein products produced by the bacterium, the effects of leukotoxin and O-sialoglycoprotease, culture supernatant antigen secreted by Pasteurella haemolytica A1, on bovine platelet activation were examined by evaluating the enhancement of platelet adhesion to a negatively charged surface relative to untreated control samples. The glycoprotease, or the leukotoxin, was added to plasma free suspensions of bovine platelets and platelet adhesion assessed by two parameters: (i) the number of 3H-adenine-labeled adherent platelets and (ii) the morphology of unlabeled platelets adhering to the charged surface under scanning electron microscopy (SEM). In the presence of calcium, the glycoprotease produced a dose-dependent increase in adhesion. At a concentration of 4.0 micrograms glycoprotease extract protein per 10(7) platelets, a 2-fold increase in adhesion was observed which was similar to the increase in adhesion induced by 0.10 units of thrombin, a known platelet agonist. Both increased platelet adhesion and platelet aggregation were observed with 0.8 microgram glycoprotease extract protein in the presence of calcium. The response of the bovine platelet suspensions to leukotoxin extract protein was dependent on the dosage of the leukotoxin. Adhesion was enhanced at dosages of 25 micrograms leukotoxin protein per 10(7) platelets and below, while at dosages of 50 micrograms and above adhesion was suppressed. Thus, the two proteins secreted by P. haemolytica may interact directly with bovine platelets to initiate platelet aggregation and fibrin formation in alveolar tissue in pneumonic pasteurellosis.

Membrane protein proteolysis assayed by fluorescence quenching: assay of O-sialoglycoprotein endopeptidase.

The assay of the O-sialoglycoprotein endopeptidase of Pasteurella haemolytica has previously used the cleavage of 125I-labeled glycophorin A, measured by SDS-PAGE, autoradiography, gel-slicing, and scintillation counting. A new assay is based on the increased fluorescence which results from proteolytic cleavage of a fluorescence-quenched micellar substrate, 4,4-difluor-5,7-dimethyl-4-bora-3 alpha, 4 alpha-diaza-s-indacene-3-propionic acid conjugated to glycophorin A (BODIPY-FL-glycophorin A). Micellar association of glycophorin A molecules results in 97% fluorescence quenching despite a low molar ratio of BODIPY-FL-glycophorin A. Proteolysis of the membrane protein causes greatly enhanced fluorescence which is used for a rapid one-step proteolysis assay. Direct monitoring of proteolysis in microcuvettes, or routine assay in microtiter plates can be used. Reproducibility is higher than with the radiolabeled substrate and the K(m) values for the two substrates are similar. The assay is suitable for the O-sialoglycoprotein endopeptidase activity of chromatographically purified enzyme or unpurified bacterial culture supernatants and can be used to monitor inhibition of the O-sialoglycoprotein endopeptidase by neutralizing antibodies. The O-sialoglycoprotein endopeptidase assay employing BODIPY-FL-glycophorin A provides a rapid and nonradioactive method for the assay of this highly specific enzyme.

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. When the bacterium was grown at 37 degrees C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 microg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40 degrees no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of Nacetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 microM.