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Homogenates of HeLa cells contain neuraminidase activity. This enzyme is particle-bound, and it has a pH optimum of 4.2. Hydrocortisone-regulated cells contain two to three times as much neuraminidase as the corresponding controls. The hydrocortisone treatment also causes an increase in the cell content of beta-glucuronidase and acid deoxyribonuclease.
Firmly established methylcholanthrene fibrosarcomas in syngeneic mice will totally disappear if the hosts are treated with living tumor cells that have been exposed to Vibrio cholerae neuraminidase in vitro. The effect is magnified by the simultanieous injection of a nonspecific immunostimulant, BCG. The rejection of the methylcholanthrene tumor is immunospecific and can be induced only with tumor cells, treated with Vibrio cholerae neuraminidase, identical in type with the growing tumor.
A specific inhibitor of the neuraminidase of the protozoan parasite Trypanosoma cruzi was isolated recently and named cruzin. It is now shown that cruzin is similar to high-density lipoprotein by amino acid homology, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by immunoblot analysis, and by isoelectric focusing. Cruzin purified by ion exchange chromatography and high-density lipoprotein isolated by density gradient ultracentrifugation inhibited Trypanosoma cruzi neuraminidase to the same extent. Cruzin or high-density lipoprotein restores to normal the decreased multiplication rate of Trypanosoma cruzi epimastigotes grown in a medium depleted of lipoproteins, suggesting that it may be important for survival of the parasite in nature.
The human pathogen Trypanosoma cruzi (Y strain) contains a neuraminidase activity that varies widely in the different developmental stages of the parasite. The specific neuraminidase activity of infective trypomastigotes obtained from tissue culture and from the bloodstream of infected mice is 7 to 15 times higher than that of the acellular culture forms. Amastigotes were devoid of enzyme activity. The enzyme has a pH optimum of 6.0 to 6.5. Live trypanosomes released sialic acid from human erythrocytes and plasma glycoproteins. Several sialyl compounds were hydrolyzed by the parasite, but the best substrate was the protein orosomucoid. Erythrocytes from infected mice with T. cruzi parasitemia were agglutinated by peanut lectin and the hemagglutination titer was correlated with the degree of parasitemia.
The low activity of liver neuraminidase that is characteristic of mouse strain SM/J is inherited as a single gene on chromosome 17, near the major histocompatibility complex. This gene, neuraminidase-1 (Neu-1), is represented by the low activity allele Neu-1s in SM/J and the high activity allele Neu-1b in C57BL/6J and most other strains. Previously described variations in the posttranslational processing of acid phosphatase, alpha-mannosidase, arylsulfatase-B, and alpha-glucosidase are attributed to pleiotropic effects of this gene.
A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A (H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascertained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations (EC(50)) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91, EC(50)s (determined by neutral red assay) of the novel compounds were < or =1.5 microM. Twelve strains of H3N2 and two strains of avian H5N1 viruses were inhibited at <0.3 microM. Influenza B/Beijing/184/93 and B/Harbin/07/94 viruses were inhibited at <0.2 microM, with three other B virus strains inhibited at 0.8 to 8 microM. The novel inhibitors were comparable in potency to (or slightly more potent than) zanamivir and oseltamivir carboxylate. No cytotoxicity was seen with the compounds at concentrations of < or =1 mM in cell proliferation assays. The antiviral activity of RWJ-270201, chosen for clinical development, was studied in greater detail. Its potency and that of oseltamivir carboxylate decreased with increasing multiplicity of virus infection. Time-of-addition studies indicated that treatment with either compound needed to begin 0 to 12 h after virus exposure for optimal activity. Exposure of cells to RWJ-270201 caused most of the virus to remain cell associated, with extracellular virus decreasing in a concentration-dependent manner. This is consistent with its effect as a neuraminidase inhibitor. RWJ-270201 shows promise in the treatment of human influenza virus infections.
The cyclopentane influenza virus neuraminidase inhibitor RWJ-270201 was evaluated against influenza A/NWS/33 (H1N1), A/Shangdong/09/93 (H3N2), A/Victoria/3/75 (H3N2), and B/Hong Kong/05/72 virus infections in mice. Treatment was by oral gavage twice daily for 5 days beginning 4 h pre-virus exposure. The influenza virus inhibitor oseltamivir was run in parallel, and ribavirin was included in studies with the A/Shangdong and B/Hong Kong viruses. RWJ-270201 was inhibitory to all infections using doses as low as 1 mg/kg/day. Oseltamivir was generally up to 10-fold less effective than RWJ-270201. Ribavirin was also inhibitory but was less tolerated by the mice at the 75-mg/kg/day dose used. Disease-inhibitory effects included prevention of death, lessening of decline of arterial oxygen saturation, inhibition of lung consolidation, and reduction in lung virus titers. RWJ-270201 and oseltamivir, at doses of 10 and 1 mg/kg/day each, were compared with regard to their effects on daily lung parameters in influenza A/Shangdong/09/93 virus-infected mice. Maximum virus titer inhibition was seen on day 1, with RWJ-270201 exhibiting the greater inhibitory effect, a titer reduction of >10(4) cell culture 50% infective doses (CCID(50))/g. By day 8, the lung virus titers in mice treated with RWJ-270201 had declined to 10(1.2) CCID(50)/g, whereas titers from oseltamivir-treated animals were >10(3) CCID(50)/g. Mean lung consolidation was also higher in the oseltamivir-treated animals on day 8. Both neuraminidase inhibitors were well tolerated by the mice. RWJ-270201 was nontoxic at doses as high as 1,000 mg/kg/day. These data indicate potential for the oral use of RWJ-270201 in the treatment of influenza virus infections in humans.
The influenza virus neuraminidase (NA) inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world between 1999 and 2002. In order to monitor the potential development of resistance, the Neuraminidase Inhibitor Susceptibility Network was established to coordinate testing of clinical isolates collected through the World Health Organization influenza surveillance network from different regions of the world (M. Zambon and F. G. Hayden, Antivir. Res. 49:147-156, 2001). The present study establishes the baseline susceptibilities prior to and shortly after the introduction of the NA inhibitors. Over 1000 clinical influenza isolates recovered from 1996 to 1999 were tested. Susceptibilities were determined by enzyme inhibition assays with chemiluminescent or fluorescent substrates with known NA inhibitor-resistant viruses as controls. The 50% inhibitory concentrations (IC(50)s) depended upon the assay method, the drug tested, and the influenza virus subtype. By both assays, the mean zanamivir IC(50)s were 0.76, 1.82, and 2.28 nM for the subtype H1N1 (N1), H3N2 (N2), and B NAs, respectively, and the oseltamivir IC(50)s were 1.2, 0.5, and 8.8 nM for the N1, N2, and B NAs, respectively. The drug susceptibilities of known zanamivir- and oseltamivir-resistant viruses with the NA mutations E119V, R292K, H274Y, and R152K fell well outside the 95% confidence limits of the IC(50)s for all natural isolates. Sequence analysis of the NAs of viruses for which the IC(50)s were above the 95% confidence limits and several control isolates for which the IC(50)s were in the normal range revealed variations in some previously conserved residues, including D151, A203, T225, and E375 (N2 numbering). Known resistance mutations are both influenza virus subtype and drug specific, but there was no evidence of naturally occurring resistance to either drug in any of the isolates.
Dimeric derivatives (compounds 7 to 9) of the influenza virus neuraminidase inhibitor zanamivir (compound 2), which have linking groups of 14 to 18 atoms in length, are approximately 100-fold more potent inhibitors of influenza virus replication in vitro and in vivo than zanamivir. The observed optimum linker length of 18 to 22 A, together with observations that the dimers cause aggregation of isolated neuraminidase tetramers and whole virus, indicate that the dimers benefit from multivalent binding via intertetramer and intervirion linkages. The outstanding long-lasting protective activities shown by compounds 8 and 9 in mouse influenza infectivity experiments and the extremely long residence times observed in the lungs of rats suggest that a single low dose of a dimer would provide effective treatment and prophylaxis for influenza virus infections.
Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK(2) cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 micro M in HA inhibition assays and from 0.02 to 20 micro M in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 micro M. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.
Streptococcus pneumoniae frequently colonizes the upper respiratory tract of young children and is an important cause of otitis media and invasive disease. Carriage is more common than disease, yet the genetic factors that predispose a given clone for disease are not known. The relationship between capsule type, genetic background, and virulence is complex, and important questions remain regarding how pneumococcal clones differ in their ability to cause disease. Pneumococcal neuraminidase cleaves sialic acid-containing substrates and is thought to be important for pneumococcal virulence. We describe the distribution of multilocus sequence types (ST), capsule type, and neuraminidase genes among 342 carriage, middle ear, blood, and cerebrospinal fluid (CSF) pneumococcal strains from young children. We found 149 STs among our S. pneumoniae isolates. nanA was present in all strains, while nanB and nanC were present in 96% and 51% of isolates, respectively. The distribution of nanC varied among the strain collections from different tissue sources (P = 0.03). The prevalence of nanC was 1.41 (95% confidence interval, 1.11, 1.79) times higher among CSF isolates than among carriage isolates. We identified isolates of the same ST that differed in the presence of nanB and nanC. These studies demonstrate that virulence determinants, other than capsule loci, vary among strains of identical ST. Our studies suggest that the presence of nanC may be important for tissue-specific virulence. Studies that both incorporate MLST and take into account additional virulence determinants will provide a greater understanding of the pneumococcal virulence potential.
Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.
Neuraminidase activities of Pasteurella haemolytica cell suspensions were measured. Type isolates of the 12 established serotypes and bovine and ovine field isolates were included in the study. Neuraminidase activities ranged from 0 to 0.87 U per mg (dry weight) of P. haemolytica. Activity levels among the isolates studied were serotype associated.
A polyclonal antibody obtained against neuraminidase purified from Trypanosoma cruzi was used for the localization of the protein in whole cells by immunofluorescence microscopy and in thin sections of parasites (epimastigote, amastigote, and trypomastigote forms) embedded at a low temperature in Lowicryl K4M resin. The intensity of labeling, as evaluated by the number of gold particles associated with the parasite, varied according to the protozoan developmental stage. In the noninfective epimastigote forms, labeling of the cell surface was very weak. However, an intense labeling of some cytoplasmic vacuoles was observed. Labeling of the surfaces of most of the trypomastigote forms was weak, while gold particles were seen in association with the flagellar pockets of these forms, which suggests that the enzyme is secreted through this region. Intense labeling of the surfaces of many, but not all, transition forms between trypomastigote and amastigote forms was observed. Amastigote forms found in the supernatant of infected cell cultures had their surfaces intensely labeled, while few particles were seen on the surfaces of intracellular amastigotes. The results obtained are discussed in relation to the role played by T. cruzi neuraminidase in the process of parasite-host cell interaction.
The properties of an extracellular neuroaminidase produced by a Pasteurella haemolytica A1 strain (isolated from a case of bovine pneumonia) during growth in a defined medium were examined in this investigation. This enzyme, isolated from concentrated culture supernatants of P. haemolytica A1, was active against N-acetylneuramin lactose, human alpha 1-acid glycoprotein, fetuin, and bovine submaxillary mucin. Neuraminidase production paralleled bacterial growth in a defined medium and was maximal in the stationary phase of growth. The enzyme was purified to homogeneity by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 100.62 mumol of sialic acid released per min per mg of protein against human alpha 1-acid glycoprotein. The Km value for this enzyme with human alpha 1-acid glycoprotein as the substrate was 1.1 mg/ml, and the enzyme possessed a pH optimum of 6.5. The P. haemolytica A1 neuraminidase had a molecular weight of approximately 150,000 as estimated by gel filtration and approximately 170,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at 4 degrees C for 3 h. At 37 degrees C for 3 h, 25% of enzymatic activity was lost. Approximately 55% of the enzyme activity was lost within 30 min at 50 degrees C, with greater than 70% of the enzyme activity being destroyed within 10 min at temperatures of > or = 65 degrees C.
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.
The infectivity of trachoma-inclusion conjunctivitis (TRIC) organisms (TW-5) was enhanced by pretreatment of HeLa cell monolayers before inoculation with diethylaminoethyl (DEAE)-dextran (30 mug/ml) and poly-l-lysine (10 mug/ml) and inhibited by dextran sulphate (250 mug/ml), fetuin (4%), ovomucoid (5%), N-acetyl neuraminic acid (0.5%), and Cholera vibrio neuraminidase (100 U/ml). The infectivity of lymphogranuloma venereum organisms (434) was not affected by DEAE-dextran, fetuin, and neuraminidase, was slightly inhibited by poly-l-lysine, and was inhibited by dextran-sulphate, ovomucoid, and N-acetyl neuraminic acid. The study suggested that sialic acid residues on the cell surface may be specific receptors for TRIC organisms. The receptors for TRIC organisms (TW-5 and TW-3) could be specifically blocked with inactivated (56 C for 30 min) TRIC organisms at the ratio of one live to 100 inactivated TRIC organisms, but not by inactivated lymphogranuloma venereum (434) or influenza virus (A(2)/Jap 305).
In Corynebacterium diphtheriae and closely related neuraminidase-producing corynebacteria, we have found an N-acetylneuraminate (NAN) lyase activity which cleaves NAN into N-acetyl-d-mannosamine and, presumably, pyruvate. In vitro, these lyases can be shown to synthesize NAN. A survey of representative corynebacteria, "plant pathogenic corynebacteria," mycobacteria, and nocardias revealed that only those corynebacteria closely related to C. diphtheriae exhibited both neuraminidase and NAN lyase activities.