Potent inhibition of bacterial neuraminidase activity by pterocarpans isolated from the roots of Lespedeza bicolor.

Bacterial neuraminidase has been highlighted as a key enzyme for pathogenic infection and sepsis. Six pterocarpans displaying significant levels of neuraminidase inhibitory activity were isolated from the root bark of Lespedeza bicolor. The isolated compounds were identified as three new pterocarpans (1-3) together with known compounds erythrabyssin II (4), lespebuergine G4 (5), and 1-methoxyerythrabyssin II (6). The new compounds were characterized as bicolosin A (1), bicolosin B (2), and bicolosin C (3). All compounds inhibited bacterial neuraminidase in a dose-dependent manner with significant inhibition (IC(50)=0.09-3.25 µM). All neuraminidase inhibitors screened were found to exhibit noncompetitive kinetics. The three most potent neuraminidase inhibitors (1, 3 and 6) feature a methoxy substitution on C-1.

Fatal myelotoxicity after azathioprine treatment.

Azathioprine and 6-mercaptopurine have been used for many years in the treatment of inflammatory bowel disease. Approximately 0.3% of the population are homozygous for variant alleles associated with extremely low thiopurine S-methyltransferase enzyme activity. We describe the case of a young patient with ulcerative colitis, homozygous for TPMT*3A alleles, who suffered fatal azathioprine-induced myelotoxicity after standard dosing with azathioprine. Screening for decreased activity of TPMT in patients prior to azathioprine treatment is advised to minimize the risk of drug-induced toxicity.

The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97A resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens.

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.

Early diagnosis of clostridial gas gangrene using sialidase antibodies.

In order to improve the diagnosis of gas gangrene, especially at an early stage of infection, new ways for the detection of the responsible Clostridia were investigated. Sialidase, known to be excreted in large amounts by the most frequently occurring myonecrotizing clostridial species, Clostridium perfringens, Clostridium septicum, and Clostridium sordellii, was isolated. With polyclonal antibodies raised against these enzymes, two immunological assays were established, which are directed against the sialidase activity (sialidase inhibition test) and the enzyme protein ('sandwich'-ELISA), respectively. Using these assays, species-specific information about the presence of clostridial sialidase was obtained within 50 min or 6 h. Animal tests revealed that both assays are applicable 8-12 h after clostridial infection, using resected tissues or wound fluids for estimations. The assays allow specific, sensitive, and quantitative measurement of clostridial sialidases, and no significant interference by sialidases from other microbes or from host tissues occurred. The applicability of the new assays for an early diagnosis of gas gangrene in human patients is discussed.

Sialidase activity in the sera of patients and rabbits with clostridial myonecrosis.

The origin and nature of gas gangrene can be diagnosed exactly only by time-consuming bacteriological tests. In order to improve the diagnostic procedures, rabbits were infected with strains of Clostridium perfringens, Clostridium septicum or Clostridium sordellii. Sialidase activity was found to increase rapidly in serum; elevated creatine kinase activities were observed, too. High sialidase concentrations were found in sera (up to 1.6 mU/ml) and in tissues of wounded regions (up to 110 mU/g) of patients diagnosed to be infected with C. perfringens. By inhibition of enzyme activity with antibodies specific for the sialidase from this Clostridium species, it was possible to identify the clostridial origin of the sialidase activities. In the same material from other patients supposed to suffer from gas gangrene, but where no Clostridia could be detected, significant sialidase activity was not found. Thus, sialidase may be a useful tool for the diagnosis of myonecrosis due to clostridial infection.

Histochemical and electron microscopical studies of skeletal muscle affected by gas gangrene.

Ultrastructural and histochemical changes of skeletal muscle were studied in three patients affected with gas gangrene. There was complete lack of the phosphorylase, succinate dehydrogenase and adenosine triphosphatase activities in the affected muscles of all the patients. In unaffected muscles these enzymes showed weaker activities than in norm. The lactate dehydrogenase activity, especially the heart type isozyme (LDH-1 or H4) proved less sensitive to the effect of clostridial toxin. A general increase in the acid phosphatase activity was found both in affected and in unaffected muscles. On electron microscopic examination damage to sarcolemmal membrane and disintegration of myofilaments was seen. The mitochondria were swollen and their cristae distorted and fragmented.