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D G Pritchard

Publications and source records attributed to D G Pritchard.

At least 19 recordsLinked to original sources
PubMed2000

Characterization of the active site of group B streptococcal hyaluronan lyase.

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.

Calcium↗
PubMed2000

Action pattern and substrate specificity of the hyaluronan lyase from group B streptococci.

The hyaluronan lyase of group B streptococci rapidly cleaves hyaluronan by an elimination mechanism to yield the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose. Additionally, it has been shown that the enzyme has limited specificity for achondroitin sulphate and cleaves the chain at unsulphated sites [Baker,Yu, Morrison, Averett and Pritchard (1997) Biochem. J. 327,65-71]. In the present extension of that study it was found that 6-sulphated regions of chondroitin sulphate are also susceptible to cleavage by this hyaluronan lyase. Of the four 6- and/or 4-sulphated tetrasaccharides which can be isolated from testicular hyaluronidase digests of chondroitin sulphate, only those two tetrasaccharides with a6-sulphated disaccharide at the reducing end were cleaved. From thisand other data, a model is proposed for the cleavage specificity of hyaluronan lyase on a chondroitin sulphate. Evidence is presented in support of an action pattern for hyaluronan lyase which involves aninitial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharide. Since the on lyoligosaccharides which tend to accumulate in near-complete digests of hyaluronan are unsaturated, it is argued that the processive cleavage occurs from the non-reducing to the reducing end of a hyaluronan chain. This detailed knowledge of substrate specificity contributes to our understanding of the enzyme's role in Group B streptococcal pathogenesis. In addition, the hyaluronan lyase may find application in sequence studies of chondroitin sulphates.

Animals↗
PubMed1997

Specificity of the hyaluronate lyase of group-B streptococcus toward unsulphated regions of chondroitin sulphate.

The purification and properties of a hyaluronate lyase secreted by Streptococcus agalactiae, which is believed to facilitate the invasion of host tissues by the organism, have been described previously [Pritchard, Lin, Willingham and Baker (1994) Arch. Biochem. Biophys. 315, 431-436]. The specificity of the limited cleavage of chondroitin sulphate by the enzyme is the subject of this report. To simplify the task, a chondroitin sulphate from the Swarm rat chondrosarcoma, which contains only 4-sulphated and unsulphated disaccharide repeats, was used in this study. Tetrasaccharides from an ovine testicular hyaluronidase digest of the chondroitin sulphate were isolated, identified and tested as substrates of the streptococcal hyaluronate lyase. Only tetrasaccharides with an unsulphated disaccharide at the reducing end were cleaved (by elimination at the N-acetylgalactosaminidic bond). Thus chondroitin sulphate chains are cleaved by the action of this lyase at every unsulphated disaccharide repeat, but release of unsaturated unsulphated disaccharides only occurs from sites where two or more sequential unsulphated disaccharide repeats are present. Analysis of the chondrosarcoma chondroitin sulphate showed that of approximately five unsulphated disaccharide repeats per chain, two are clustered. The ability of group-B streptococcal hyaluronate lyase to cleave chondroitin sulphate may allow the organisms to invade tissues more efficiently. The demonstrated specific and highly limited cleavage of chondroitin sulphate by this bacterial lyase promises to be a useful tool in the determination of chondroitin sulphate structure and variability.

Animals↗
PubMed1997

Identification of a histidine residue essential for enzymatic activity of group B streptococcal hyaluronate lyase.

Hyaluronate lyase produced by group B streptococci (GBS) degrades hyaluronan completely to unsaturated disaccharide units and also cleaves unsulfated regions of chondroitin sulfate. The enzyme is rapidly inactivated by diethyl pyrocarbonate and enzymatic activity is restored by treatment with hydroxylamine, suggesting that a histidine residue is present in the active site. Amino acid sequence comparisons of GBS hyaluronate lyase and four other related enzymes revealed that one of the 16 histidine residues of the enzyme (His-479) is present in a highly conserved region. Conversion of His-479 to a glycine by site-directed mutagenesis resulted in a complete loss of enzymatic activity of the modified protein. We propose that His-479 is in the active site of GBS hyaluronate lyase and participates in the initial abstraction of hydrogen ions from the glucuronic acid residues of hyaluronan.

Amino Acid Sequence↗
PubMed1996

Characterization of PepB, a group B streptococcal oligopeptidase.

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of the gene for group B streptococcal hyaluronate lyase.

Group B streptococci (GBS) are a major cause of serious human perinatal infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.

Amino Acid Sequence↗
PubMed1994

Characterization of the group B streptococcal hyaluronate lyase.

Hyaluronate lyase is one of several proteins secreted by group B streptococci which are believed to contribute to strain virulence. Characterization of the purified enzyme revealed that it degrades hyaluronan by a mechanism different from that of other previously studied hyaluronidases. Instead of randomly cleaving hyaluronan chains leading to a continuous decrease in average chain size, the group B streptococcal enzyme initially yields primarily unsaturated disaccharides. The observation that most of the free reducing ends generated during group B streptococcal hyaluronate lyase digestion are present in the unsaturated disaccharide units supports the conclusion that they are released primarily from the ends of the hyaluronan chains. Furthermore, the experimental evidence is consistent with a mode of action by which the enzyme initially makes a random cut in a hyaluronan chain and then processively moves along the chain releasing disaccharide units. Group B streptococcal hyaluronate lyase also slowly degrades chondroitin sulfate, and its desulfation greatly increases the reaction rate. A preferential cleavage of unsulfated residues is consistent with the observed extensive release of free chondroitin sulfate chains following very limited digestion of aggrecan from bovine nasal cartilage.

Animals↗
PubMed1993

Group B streptococcal neuraminidase is actually a hyaluronidase.

The extracellular group B streptococcal enzyme described in numerous reports as a neuraminidase is really a hyaluronidase. Over the past 25 years, the enzyme was routinely assayed with bovine submaxillary mucin as the substrate and by the thiobarbituric acid procedure to measure released sialic acid. Characterization of the actual compound released by the enzyme revealed it to be an alpha,beta-unsaturated derivative of hyalobiuronic acid that was derived from hyaluronic acid contaminating the mucin preparation. Previous reports describing an association of elevated levels of extracellular neuraminidase with virulent strains of group B streptococci must be reevaluated with the recognition that the enzyme is really a hyaluronidase.

Amino Acid Sequence↗
PubMed1993

Capsular type-specific polysaccharide partially inhibits group B Streptococcus-induced pulmonary hypertension.

Capsular type-specific polysaccharide is thought to be an important pathogenetic factor in Group B streptococcus (GBS) sepsis. To determine the effects of capsular type-specific polysaccharide on GBS-induced hemodynamic responses, anesthetized infant piglets were infused for 3 h with three related GBS Type lb strains that express different amounts of capsular type-specific polysaccharide. A larger capsule strain and a smaller capsule strain were isolated from an infected infant and its mother, respectively. A capsule-deficient mutant was then made from the larger capsule strain by transposon insertion mutagenesis. The smaller capsule strain and capsule-deficient mutant caused similar elevations in mean pulmonary artery pressure and pulmonary vascular resistance index and reductions in cardiac index. The larger capsule strain caused moderate pulmonary hypertension, but this response was smaller than for the other two GBS strains. Further comparisons in responses between the large capsule strain and its capsule-deficient mutant were then performed using unanesthetized piglets. The mutant caused significantly greater pulmonary hypertension and arterial plasma thromboxane B2 levels than the large capsule strain. The pulmonary hypertension induced by both strains was reversed by dazmegrel, a thromboxane A2 synthase inhibitor. These results suggest that (1) capsular type-specific polysaccharide is not an essential component in the generation of acute hemodynamic responses; (2) expression of large amounts of capsular type-specific polysaccharide on the organism surface partially inhibits GBS-induced pulmonary hypertension; and (3) the inhibition of the pulmonary responses is due to reduced thromboxane A2 release.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides.

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates.

Antibodies, Monoclonal↗
PubMed1992

Role of capsule in pulmonary hypertension induced by group B streptococcus.

The type-specific polysaccharide capsule of group B streptococcus (GBS) is thought to be an important factor in the pathogenesis of disease. We used an acutely instrumented piglet model to assess the hemodynamic effects of rapid infusions of two heat-killed GBS type Ib strains isolated from the spinal fluid of an infant with late-onset meningitis and from the vaginal culture of his mother. These strains expressed different amounts of capsule, as determined by buoyant density centrifugation and electron micrographs, and they produced different hemodynamic effects in the piglets. The mother's strain, which had a smaller capsule, caused significantly higher increases in pulmonary artery pressure and vascular resistance than did the infant's strain, which had a larger capsule. Transposon mutants were then made from the infant's isolate to further study the role of capsule in pulmonary hypertension. Two mutants lacking detectable capsular type-specific polysaccharide were compared with the original isolate and with an isogenic mutant containing transposons but having a large capsule. The nonencapsulated mutants caused significantly higher changes in pulmonary artery pressure and resistance than did the encapsulated strains. Pulmonary hypertension may play a role in the pathophysiology of GBS sepsis, but the presence of a large capsule may partially cloak the hemodynamically active component(s) of the bacteria. The lower initial host response to heavily encapsulated GBS may play a role in pathogenesis by helping the organisms avoid host defense mechanisms.

Animals↗
PubMed1991

Structure of the type-specific polysaccharide antigen of Streptococcus rattus.

The structure of the type-specific polysaccharide antigen of Streptococcus rattus was determined by methylation analysis, periodate oxidation, and by 2D-1H- and 13C-n.m.r.-spectroscopy. The polysaccharide was found to possess the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Galp-(1----3)]-alpha-L-+ ++Rhap- (1----.

Carbohydrate Sequence↗
PubMed1990

Effect of fibronectin on IgA-mediated uptake of type III group B streptococci by phagocytes.

Previous studies have shown that a type-specific IgA monoclonal antibody alone or in combination with fibronectin (Fn) enhances protective efficacy in two animal models of group B streptococcal infection. To investigate the mechanisms by which IgA mediates protection, the effects of Fn on phagocytosis of group B streptococci (GBS) opsonized with a type III-specific IgA monoclonal antibody were examined. Specific IgA alone or in combination with Fn did not promote the phagocytosis of GBS by polymorphonuclear leukocytes (PMNL). Fibronectin also had no significant effect on phagocytosis of IgA-opsonized GBS by monocytes. Specific IgA alone promoted phagocytosis of GBS by culture-derived macrophages in a dose-dependent fashion. Fibronectin enhanced macrophage uptake of the GBS opsonized in a suboptimal concentration of specific IgA (phagocytic index = 2.32 +/- 0.56 vs. 3.26 +/- 0.48 with Fn; P less than .05). These data suggest that protection against GBS in neonatal rats by a combination of Fn and specific IgA is mediated by macrophages rather than by PMNL or monocytes. Fibronectin may have a critical role in host defense at sites where IgA and macrophages predominate.

Antibodies, Monoclonal↗
PubMed1989

An IgA monoclonal antibody directed against type III antigen on group B streptococci acts as an opsonin.

We have investigated the mechanisms by which a murine IgA mAb directed against the type III Ag (IgA anti-III mAb) of group B streptococci (GBS) protects neonatal rats from lethal infection with these organisms. Purified IgA anti-III mAb enhanced phagocytosis of type III GBS by rat peritoneal macrophages in vitro by fourfold compared with phagocytosis of buffer-treated GBS. In the absence of antibody, neonatal rat serum did not promote phagocytosis, but addition of neonatal rat serum to GBS opsonized with IgA anti-III led to a sevenfold increase in phagocytosis. Heat inactivation of C destroyed the ability of neonatal rat serum to enhance phagocytosis in the presence of IgA. C3 deposition was observed when GBS coated with IgA anti-III mAb were incubated in untreated neonatal rat serum or in serum treated with Mg/EGTA. This latter observation suggested that C3 deposition occurred through activation of the alternative pathway. The control IgA mAb MOPC 315 did not enhance GBS ingestion or C3 deposition on GBS. Depletion of C in vivo by using cobra venom factor abolished the protective effect of IgA anti-III mAb in the neonatal rat model. These data suggest that the ability of this IgA to activate C further enhances its opsonic activity and may be essential for its protective effect in vivo.

Animals↗
PubMed1989

Severe disease in a dairy herd associated with acute infection with bovine virus diarrhoea virus, Leptospira harjo and Coxiella burnetii.

Between November 1984 and February 1985, a serious outbreak of pyrexia, diarrhoea, agalactia, metritis and severe weight loss affected most of the recently calved cows in a 183-cow dairy herd in Norfolk. Fifteen cows died and 20 were culled. Forty cows aborted during or after the outbreak, and many of them produced mummified fetuses; 18 calves were stillborn and three others died soon after birth. Necropsy of three affected cows revealed ulceration of the gastrointestinal tract similar to that seen in cases of mucosal disease. Bovine virus diarrhoea virus was isolated from the intestines of one cow that died soon after the onset of illness. The virus was also isolated from the blood of four acutely ill cows and seroconversion was demonstrated in three of those that survived. The virus was isolated from three aborted fetuses, a stillborn calf and a live neonatal calf. Body fluids from two aborted fetuses were seropositive for the virus as were sera from all the aborting cows tested. In addition to widespread seroconversion to bovine virus diarrhoea virus during the outbreak, there was serological evidence of recent infection with Leptospira interrogans serovar hardjo and Coxiella burnetii in a high proportion of cows. It was concluded that this was primarily an acute outbreak of bovine virus diarrhoea but its unprecedented clinical severity was probably associated with the concurrent introduction of L hardjo and C burnetii into an immunologically naive herd during the main calving period. Epidemiological analysis suggested that the source of the virus and L hardjo was down-calving heifers returning from communal marsh grazing.

Abortion, Veterinary↗
PubMed1988

Structure of the group G streptococcal polysaccharide.

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.

Carbohydrate Conformation↗