Purification and properties of a neuraminidase from Streptococcus K 6646

A neuraminidase was purified from the culture filtrate of Streptococcus 6646 (group K) by means of ammonium sulfate fractionation and successive column chromatographies on N-(p-aminophenyl)oxamic acid-substituted Sepharose derivative and p-aminophenyl-2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside-substituted Sepharose derivative. The former adsorbent was found to bind a β-galactosidase and a β-N-acetylhexosaminidase in addition to the neuraminidase, and the latter adsorbent bound the β-galactosidase in addition to the β-d-N-acetylhexosaminidase. These adsorbents effectively eliminated the contaminating glycosidase activities and a 1,500-fold purification of the neuraminidase was achieved by this procedure.The neuraminidase thus purified was homogeneous by electrophoresis on polyacrylamide gel, and its molecular weight was estimated to be 110,000 by gel filtration on Biogel P-200. The activity of the purified neuraminidase was slightly stimulated by Ca²⁺, Mg²⁺, Mn²⁺, and Co²⁺, and strongly inhibited by heavy metals. The specificity of the purified neuraminidase was almost the same with Vibrio cholerae or Clostridium perfringens neuraminidase. It completely hydrolyzes sialic acid residues in neuraminyl lactose and porcine thyroglobulin, but it liberates only 50% of sialic acid residues from porcine submaxillary mucin and ganglioside GD_1a.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.     

Prevalence of Bacterial Vaginosis in Women Attending Family Planning Clinics

Prevalence of bacterial vaginosis in 242 women attending family planning clinics in Concepción, Chile was studied using the Nugent criteria. The syndrome was present in 33.1% of women. No statistical difference was found between the occurrence of bacterial vaginosis and the of oral contraceptives (31.9%) or intrauterine devices (33.1%). The sensitivity and specificity of the Nugent criteria compared with the Amsel criteria were 86.7% and 91.0%, respectively. Vaginal fluid pH and appearance had low specificity in this population. Microorganisms associated with bacterial vaginosis were Gardnerella vaginalis (96.3%), Prevotella/Porphyromonas (70%),Mobiluncus spp. (48.8%), Mycoplasma hominis (21.3%) and Ureaplasma urelyticum 21.3). These results suggest that preventive actions and management of clinical infection to prevent complications are needed.     

Correlation between Either Cupriavidus or Porphyromonas and Primary Pulmonary Tuberculosis Found by Analysing the Microbiota in Patients’ Bronchoalveolar Lavage Fluid

Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB’s secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research.     

I-cell disease Desialylation of β-hexosaminidase and its effect on uptake by fibroblasts

The pinocytosis of fibroblasts of β-hexosaminidase (EC 3.2.1.30) excreted by cultured skin fibroblasts from a patient with I-cell disease was not enhanced by neuraminidase treatment of the enzyme. The uptake of sialic acid-rich normal plasma β-hexosaminidase was minimal and neuraminidase treatment did not appreciably enhance uptake. In contrast, sialic acid-rich normal seminal fluid β-hexominidase was readily pinocytosed regardless of neuraminidase treatment. Thus the presence of sialic acid on β-hexosaminidase does not influence uptake and a neuraminidase deficiency in I-cell disease may not be directly responsible for excessive extracellular enzyme.     

Effects of Enzymes,Chemicals, and Tempertaure on Steinernema carpocapsae Attraction to Host Plasma

Migration of exsheathed infective juveniles of Steinernema carpocapsae to plasma of the host insect Spodoptera litura was not affected by treatments with the lectins concanavalin A, soybean agglutinin, or wheat germ agglutinin; with the enzymes neuraminidase, α-mannosidase, lipase, pronase, or phospholipase C; or with cetyl trimethylammonium bromide or spermidine. Treatment with sodium metaperiodate or sodium hypochlorite inhibited nematode attraction towards insect plasma; numbers of randomly wandering nematodes increased. Nematode migration towards the source of attraction was unaffected by temperatures below 33 C but was impaired at 35 and 37 C. The adverse effect of 5 mM and 10 mM NaIO₄ on migratory behavior was reversed 24 hours after rinsing with buffered saline. The effect of NaOCl on nematode behavior was slightly reversible at concentrations of 0.2 and 0.4% (v/v) but apparently irreversible at 0.6 and 1.0%. The effect of heat treatment at 35 and 37 C was reversible.     

In vitro activity of HMR 3004 (RU 64004) against 502 strains of anaerobic bacteria

The in vitro activity of HMR 3004 (RU 64004) was compared to azithromycin (AZ), clarithromycin (CL), erythromycin (ER) and roxithromycin (RO). Anaerobes (n =512) were tested using the NCCLS-approved Wadsworth brucella laked blood agar dilution method. Breakpoints for the inclusion of strains into the susceptible/intermediate categories were 4 μg/mL for all agents. Organisms tested included Bacteroides fragilis group species (206), Campylobacter gracilis (13), Bacteriodes species (13), Fusobacterium species (540 K), Bilophila wadsworthia (27),Peptostreptococcus (35), Porphyromonas sp. (12), Prevotella sp. (41), Sutterella wadsworthensis (16), Clostridium sp. (56) and Gram-positive non-sporeforming rods (29). HMR 3004 inhibited 78% of B. fragilis and 69% of B. fragilis groups species, respectively. Ninety-three percent of otherBacteroides were inhibited by HMR 3004, compared to 80% for AZ, 87% for ER, and 93% for CL and RO. HMR 3004 and AZ inhibited all Porphyromonas species, compared to 93–94% for the other agents. HMR 3004 inhibited 98% of Prevotella compared to 91%, 93%, 93% and 95% for ER, AZ, RO and CL, respectively. Activity against Biophila wadsworthia (85–96%) was excellent compared to poor or no activity for the other macrolides. All agents inhibited 100% of Clostridium perfringens and 50% of C. difficile and C. ramosum . None of the agents had activity against the Fusobacterium mortiferum/varium group. Approximately 90% of non-sporeforming Gram-positive rods were inhibited by all of the agents. Overall, HMR 3004 had the best activity against anaerobes, inhibiting 77% compared to AZ (46%), CL (73%), ER (47%) and RO (46%).     

Accessibility of plasma membrane sialoglycoconjugates of novikoff tumor cells to exogenous neuraminidase and proteases

Plasma membrane glycoconjugates of Novikoff tumor cells were radioactively labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄ Submission of the radioactively labeled glycoconjugates to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by fluorography revealed the presence of at least ten major glycoproteins and a glycolipid fraction. The glycolipid fraction contained 34% of the cell-surface radioactive label. Pretreatment of cells with neuraminidase from Vibrio cholerae reduced radioactive labeling of the glycoproteins by 71% and that of the glycolipids by 39%. Sequential treatment of cells with papain and neuraminidase further reduced radioactive labeling of the glycolipid fraction, indicating that resistance of this fraction to the hydrolytic action of neuraminidase was determined, at least in part, by steric factors. Incubation of cells with papain resulted in extensive degradation of most of the radioactively labeled glycoproteins with the exception of a subset of glycoproteins having apparent molecular weights of $\text{48 000 ± 5000}$. Trypsin was more selective, degrading three glycoproteins having apparent molecular weights of 200 000, 140 000 and 37 000.     

患子宫内膜炎母猪产道致病菌谱分析及卟啉单胞菌的分离鉴定

【背景】子宫内膜炎是规模化养殖场母猪常患的生殖道疾病之一,对养殖业造成的经济损失较大,细菌感染是常见的病因之一,但具体病原及致病机制尚未完全明确。【目的】探究产道菌群对母猪子宫内膜炎的影响。【方法】采用第三代细菌16S rRNA基因全长高通量测序技术进行对比,研究健康和患有子宫内膜炎母猪产道菌群差异,并依据菌群分析结果对子宫内膜炎母猪产道分泌物进行细菌分离。建立荧光定量PCR计数方法测定母猪产道样品中的卟啉单胞菌数。【结果】健康和患子宫内膜炎母猪产道菌群的丰富度和多样性差异显著;与健康组母猪相比,患子宫内膜炎母猪产道菌群变形菌门(Proteobacteria)、拟杆菌门相对丰度显著增加(P<0.05)。在属分类水平上,健康母猪产道的主要优势菌属为金黄杆菌属、芽孢杆菌属、毛螺菌属等,这些优势属在患子宫内膜炎母猪产道丰度降低,患子宫内膜炎母猪产道丰度最高的菌属为埃希氏菌属(Escherichia)、Rodentibacter和卟啉单胞菌属(Porphyromonas)。在种水平上,Porphyromonas somerae是患子宫内膜炎母猪产道的主要条件性致病菌。依据菌群分析结果,本研究从子宫内膜炎母猪产道分泌物中分离到一株卟啉单胞菌,经鉴定该菌16S rRNA基因与Porphyromonas somerae DSM 23386 strain JCM 13867(NR_113090.1)相似性达99.04%。荧光定量PCR计数方法测定,子宫内膜炎母猪产道卟啉单胞菌数量显著高于健康母猪(P<0.05),推测卟啉单胞菌与该批次母猪子宫内膜炎病因有关。【结论】本研究为子宫内膜炎母猪的病因和发病机制奠定基础,并为其治疗提供理论依据。     

Crystal structures of respiratory pathogen neuraminidases

Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.     

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

BackgroundSialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.MethodsNeuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.ResultsClostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.ConclusionsImmobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.General significanceImmobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

Km values of influenza virus neuraminidases for a new fluorogenic substrate, 4-methylumbelliferone N-acetyl neuraminic acid ketoside.

A fluorogenic substrate of neuraminidase, 4-methylumbelliferone N-acetylneuraminic acid ketoside (MUN), was synthesized. K_m values were obtained for Clostridium perfringens neuraminidase and strains of A- and B-type influenza neuraminidase from chicken red blood cell influenza virus eluates.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Enzymatic composition of mitogen-induced lymphoblasts and untreated lymphocytes fractionated by rate-zonal centrifugation

Treatment of rat lymph node cells with neuraminidase plus galactose oxidase leads to blast transformation of T lymphocytes. Rate-zonal centrifugation was used to separate both untreated lymph node cells and neuraminidase plus galactose oxidase-treated lymph node cells cultured for 2 days. The majority of untreated lymph node cells were small lymphocytes with a mean size of 130 μm³ as determined by electronic sizing. Only 3% of the cells sedimented rapidly enough to reach fractions distal from the axis of rotation. In contrast, 20% of the cells in cultures of the neuraminidase plus galactose oxidase-treated lymph node cells sedimented rapidly and these were almost exclusively large, transformed lymphoblasts (mean size 300 μm³). The most slowly sedimenting cells in these cultures were small, untransformed lymphocytes. Rate-zonal centrifugation can by used to separate and recover in high yield mitogen-induced lymphoblasts from lymphocyte cultures and thus allows the direct comparison of their biochemical properties with those of the small precursor cells. After neuraminidase plus galactose oxidase-induced blastogenesis, the amount per cell of DNA, cytochrome oxidase, β-galactosidase. cathepsin D, and arylsulfatase in whole cultures were similar to those of untreated lymph node cells. In contrast, the levels of protein, RNA, 5′-nucleotidase, γ-glutamyltranspeptidase, alkaline phosphatase, N-acetyl-β-glucosaminidase and N-acetyl-β- galactosaminidase increased 2–3-fold. Moreover, separation of these cells revealed that for each of these latter constituents there was a progressive increase in the amount per cells as the mean size increased. Thus, three plasma membrane-associated enzymes and two lysosomal acid hydrolases increased markedly in mitogen-induced blast cells as compared to the small precursor cells, but these increases were quntitatively similar to the increases in mean cellular volume and protein content.     

The role of anaerobic bacteria in chronic suppurative otitis media in children: Implications for medical therapy

This review describes the microbiology, diagnosis and medical management of chronic suppurative otitis media (CSOM) in children highlighting the role of anaerobic bacteria. In studies that employed adequate method for recovery of anaerobic bacteria polymicrobial aerobic and anaerobic flora was isolated from over half of the children with CSOM. The predominant aerobic isolates were Staphylococcus aureus and Pseudomonas aeruginosa and the most frequently isolated anaerobic organisms were Peptostreptococcus, Fusobacterium spp. and pigmented Prevotella and Porphyromonas spp. Several studies illustrated the efficacy of anti-infective agents effective against anaerobic bacteria in the treatment of CSOM. The medical therapy of CSOM should be directed at the eradication of the pathogenic aerobic and anaerobic organisms.     

Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations

Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.     

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).     

Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.