The influenza virus neuraminidase inhibitor produced by Staphylococcus aureus

The glycoproteinic complex has been isolated from the Staphylococcus aureus culture fluid possessing an activity inhibiting influenza virus neuraminidase. Two fractions have been further purified containing different monosugars components, the first one has been shown to contain mannose, glucose, rhamnose, glucosamine, and galactosamine, while rhamnose is absent in the second fraction. Л component of fraction J inhibits the neuraminidase activity and at the same time enhances the virus hemagglutinating activity more strongly comparing to the fraction 2. Some chemical modifications of the substances of the fractions studied (periodate oxidation, protease pretreatment, and delipidization) prove the carbohydrate component of the complex to be responsible for its antineuraminidase activity. The protease pretreatment decreases twice the antineuraminidase activity of the fraction 2 having no influence on this activity of the fraction J.

Introduction.Enzymes of virions become one of the most attractive fields of scientific research during last two decades.The neuraminidase being an integrative part of orthomyxoviruses is among the best in vestigated viral enzymes.It participates in the cle avage of mucine layers and neuraminic acid residues from cell walls causing in such a way virus particles desaggregation.It is very probable that this enzyme prevents virions adsorption on mucines, a process of the great importance for influenza infection patho genesis permitting virions to infect new sensitive cells.
So the neuraminidase inhibitors described beco me now especially interesting for a lot of research workers.A series of specific inhibitors being Nacetylneuraminic acid derivatives have been synthe sized.Some data have been also published concerning isolation of inhibitors produced by bacteria and being glycoprotein substances [1].
We have isolated a neuraminidase inhibitor from .S. aureus culture fluid.The aim of the present paper is to describe both this substance purification and the © S. L. RYBALKO, A V SHAPIRO, L I).VARBANETS, 1999 data concerning its chemical properties, composition, and some biological characters.Materials and Methods.Neuraminin-producing S, aureus strain and nutrient medium.The neuraminin preparations used in this investigation have been purified from the culture fluid of the .S. aureus strain 392, this strain having been isolated from a sputum sample of a patient with acute pneumonitis and deposited in the All-Union Antibiotics Research Ins titute (Moscow) in 1988.This strain is found to be sensible to lysis by phages belonging to the III phage group, 53/85 phagomosaic, and to the type 18 accor ding to the Pillet classification.
Isolation of neuraminidase inhibitor.The neura minidase inhibitor has been obtained from the culture fluid using ethanol fractionation approach (30-50 %) and boiling during 10 min followed by gel filtration on the Sepharose 4B columns (2.2 * 60 cm) equilibrated with 0.1 M ammonium acetate.In the fractions collected (2.5 ml) the protein content has been determined according to Lowry et al. [2], the carbohydrates have been assessed using phenol and sulfuric acid approach [31 Analytical methods.Analytical disk electropho resis has been run in 5.6 % polyacrylamide gel in the presence of 0.2 % sodium laurylsulfate, the current being 5 mA per tube.The samples have been prepa red in the Tris-acetate buffer, pH 7.4, containing sodium laurylsulfate (1 %) and /i-mercaptoethanol (0.001 %).
The process was usually stopped when the mar ker dye (bromephenol blue) formed a line situated at the distance about 1 cm from the lower edge of each tube.To detect the proteins separated, the gels have been stained using the Coomassie R-250 solution, the carbohydrates having been visualized by the Schiff reagent.
The molecular masses of the components sepa rated have been calculated from the disk electro phoresis data [4 J using several standard marker proteins -hexokinase, bovine serum albumin, and trypsin, their molecular masses being 96000, 68000, and 23000 Da, respectively.Amino acids and hexosamines have been deter mined with the automatic amino acids analyzer KLA («Hitachi», Japan) after the hydrolysis in 6 N HCI (20 hs, 110 °С).
Monosaccharides composition of our preparations has been determined after the acid hydrolysis (using 2 N HCI treatment during 20 hs at 100 °С) by paper chromatography method in a solvent system contai ning butanol, pyridine, and water (6:4:3); our develo pers were acidic phtalate-aniline, alkaline caustic silver, and alcoholic ninhydrin solutions [5 ].Another method of monosaccharides determination was gasliquid chromatography permitting to obtain acetic polyol products with the «Chrom-5» device.
Sialic acids determination has been performed with resorcinol method [5].
To determine fatty acids, the neuraminin inhibi tor preparations have been hydrolyzed by 2 N HCI (the acid is to be diluted by methanol) at 100 °С during 2 hs.The fatty acids have been then extracted by chloroform and analyzed at the «Zwiet-100» chromatograph.
The periodate oxidation of S. aureus neuraminin has been performed as follows.The aqueous neura minin solution (0.1 %) has been added to 0.2 M sodium periodate, the mixture has been kept at 5 °С during 72 hs, periodate excess being usually dest royed with ethylene glycol.The preparation dialyzed overnight has been then dried, treated with sodium borhydride, the excess of the last reagent being taken off using KY-2(H + ) resin, and lyophilized [5].
To separate phospholipids, the preparation has been treated by methanol (1 ml per 10 mg of preparation) during 10 min at 55 °С The mixture has been then cooled and treated with chloroform (2 ml per 10 mg), shaken overnight at 20 °С and centrifugated.Such a treatment has been repeated four times.Some neuraminin modifications have been obtai ned using a proteolytic complex of wide specificity isolated from Bacillus mesentericus, the reaction be ing stopped by the protease inhibitor added to the mixture.
Virus strains.The following influenza virus stra ins have been used in this research: 4/1, a highly immunogenic variant of A/Khabarovsk/77/HlNl strain; 4/2, a variant of the same strain possessing low immunogenity; 2/1, a highly immunogenic va riant of A/Victoria/75/H3N2; 5/2, a variant of this strain with low immunogenity; T5 and T14 strains isolated from mice lung tissues at the second and at the 14th days of infection; strains 38 and 44 isolated from mice lung materials at the third month after infection; an isolate of the A/Kiev/6/84/H3N2 virus from a patient serum; a clinical isolate of the А/Le ningrad/13/85/H1N1 virus; B/Kiev//l/84/ virus iso lated from cerebrospinal fluid of a meningoence phalitis patient; X-97, a recombinant of A/Leningrad/9/46/Но/ and А/Victoria/72 strains; A/Port-Chalmers/73/H3N2.
Study of biological activity.The antineuramini dase activity of preparations has been tested from the data concerning the inhibition of virus particles neu raminidase activity after the neuraminin treatment.The neuraminidase activity has been determined according to the Aminoff approach [6 J, the hemagglutinating activity of all the virus preparations being assessed after Gorbunova and Bokolova [7 ].
Results.As mentioned above the purification of neuraminidase inhibitor produced by S. aureus has been performed using the Sepharose 4B gel filtration (Fig. 1); it is seen that we have successfully purified this product from some contaminating protein sub stances.The fractions 40-50 have been further purified by centrifugation (4 hs at 144 000 g).The neuraminidase inhibiting activity has been detected in the supernatant material.
The results of our disk electrophoresis expe riments have not permitted to detect any covalent protein-carbohydrate links because no protein line corresponding to carbohydrate substances mobility has been found.At the same time our gel filtration data also suggest there is no coincidence of protein and carbohydrate peaks, so the bacterial inhibitor is not proved to be a glycoprotein; it seems more probably to be a glycoprotein complex (Fig. 2).Our study of the inhibitor chemical composition shows the presence of both protein and carbohydrate (1:10).Besides, the neuraminidase inhibitor of Staphylo coccus origin contains also fatty acids carrying 11-20 C-atoms in their chains.

Our study of the inhibitor's ammo acids content proves the presence of almost all of them with the only exception of arginine (see Table 1).
The determinations concerning monosaccharides composition of neuraminidase inhibitor show mannose to be the main monosugar in this compound, glucose content being rather poor; the ratio of these two neutral monosaccharides is 24:1 (Fig. 3, a, b).Addi tionally, we have also detected two hexosaminesgalactosamine and glucosamine, their relative contents being 1:3.5 (Fig. 4).

Sialic acid has not been found in the neura minidase inhibitor preparations.
The specificity of inhibitory action of Staphylocot't^s-produced substance and its effect on the influenza virus neuraminidase activity have been studied using different virus strains and isolates with different antigenic properties, the results reflecting the changes in the hemagglutinative and neura minidase activities of virus preparations studied (Ta ble 2).The Staphylococcus-produccd neuraminin has been shown to inhibit strongly the neuraminidase activity of all the influenza virus strains studied here, the level of inhibition being from.71 up to 94.4 % (Table 2).
The gel filtration on the Sephadex G-200 column has separated two fractions of the bacterial inhibitor, fraction 1 and fraction 2 (see Fig. 5).The inves tigation of their monosaccharides content has shown the substance in the fraction 2 to have monosugar composition identical with the original preparation.The substance in the fraction 1 has rather different The investigation of fractions effect on the neura minidase and hemagglutinating activity of influenza virus strains (Table 3) shows the substance in the fraction 1 possesses higher antineuraminidase activity and simultaneously causes two-fold increase of the hemagglutinating activity comparing to the fraction 2.
To answer the questions concerning molecular compo nents responsible for the activities mentioned above, we have modified the purified substances by periodate oxidation, proteases treatment, and delipidization process.These modifications are shown to influence differently on the neuraminidase inhibitor activity (see Table 3).The elimination of fatty acids has no effect on the antineuraminidase activity of the sub stance 1 and increases such activity of the substance 2. The carbohydrate modification causes a total elimination of substances 1 and 2 antineuraminidase activities.The elimination of proteins has no effect on the antineuraminidase activity of the substance 1 but destroys, however, totally the same activity of the substance 2. The delipidization procedures do not influence at all on the hemagglutinating activity of both substances; at the same time, the periodate oxidation as well as the use of the proteolytic complex cause the loss of the hemagglutinating activity.
Discussion.To our knowledge, the specific neu raminidase inhibitors described in the scientific litera ture and being both natural and synthesized sub stances may be attributed to two groups.The first one contains neuraminic acid analogues and derivatives, the second group contains the substances having no direct binding with any enzyme substrate or with its product.The Staphylococcus-produccd inhibitor be longs probably to the second group because of no sialic acids having been detected in this substance.
Comparative analysis of this inhibitor and also of the neuraminidase inhibiting substance isolated from Streptomyces [1 ] shows mannose and glucose to be the main neutral monosaccharides in both inhibitors, their ratios being, however, quite different.The Staphylococcus-produced inhibitor contains two aminosugars -glucosamine and galactosamine, the neu- Information concerning strains origin and some properties of these strains is given above (see «Materials and Methods).Viral hemagglutinative activities are expressed as inverse values of hemagglutination titers.Neuraminidase activities are given as optical density units at 549 nm (OD549). 4NIT -virus samples are treated with neuraminidase inhibitor preparations. 5Control -control variants without inhibitor preparations.6PI -per cent of inhibition after neuraminidase inhibitor treatment.
raminidase inhibiting substance isolated from Streptomyces contains only the first of them.No sialic acids has been found in both substances.Two inhibitors are also different in their amino acids content: the inhibitor of Staphylococcus origin contains methionine and no arginine.But the striking difference of these two substances is that, according to the data of Lin et al. [1], the Streptomyces-productd inhibitor is a glycoprotein.
Our results obtained with the disk electrophoresis approach show no covalent binding between protein and carbohydrate moieties in the neuraminin mole cule because no line has been found corresponding to carbohydrate electrophoretic mobility; the gel filtra tion data on the Sepharose 4B column proving no coincidence of protein and carbohydrate peaks permit to conclude that the neuraminidase inhibitor isolated from S. aureus is not glycoprotein.Our point of view is that this inhibitor is more probably a glycolipoprotein complex.
The carbohydrate content of the Staphylococcus inhibitor including also its amino sugars resembles strongly influenza virus glycoproteins.Taking into consideration such a multicomponent patterns of inhi bitor compounds -carbohydrates, proteins, and fatty acids -it would be of great interest to investigate the nature of the Staphylococcus neuraminin active group responsible for its antineuraminidase activity in our experiments with influenza virus preparations.To answer this question, two inhibitor preparations have been modified using periodate oxidation, proteolytic complex treatment, and delipidization.As a result we have found the inhibitory activities of preparations 1 and 2 to be different.The splitting of fatty acids has no effect on the antineuraminidase activity of the preparation I and increases markedly such an activity of the preparation 2. The carbohydrate modification eliminates totally neuraminidase inhibiting activity.The protein elimination has no effect on the antineu raminidase activity of the preparation 1, but destroys

Table 3 Responsibility of different components of the neuraminidase inhibitor isolated from Staphylococcus aureus on the neuraminidase and on the inhibition of the liemagglutinative function found for the influenza virus AiHongkong!'68/H3N21 strain
The OD549 values below 0.100 are thought to be negative.
completely such an activity of preparation 2 (see Table 3).Berezin et al. [8 ] have studied the participation of carbohydrate moiety in the influenza virus neura minidase activity; they have found that the elimi nation of the carbohydrate component of the viral enzyme with glycosidase does not change markedly the enzyme activity, i. e. this component has no effect on the enzymatic activity.Our results show the enzyme activity to be decreased during the viral neuraminidase interaction with the carbohydrate com ponent of Staphylococcus preparation; the complete inhibitor inactivation has been observed after the carbohydrate modification by periodate oxidation.A similar mechanism of inhibitory action has been previously described for lectins -concanavalin A and phytohemagglutinin.These lectins are sensitive to the presence of glucose, mannose and sterically similar monosaccharide residues; so Lis and Sharon [9 ] and Rott [101 suppose that the interactions between lectins and the viral neuraminidase are realized due to the carbohydrate residues.
Because of the remarkable similarity of mono saccharides content in the influenza virus neura minidase and in the Staphylococcus-produced enzyme inhibitor we postulate their interaction to be con nected with the defense of the enzyme active center during its interaction with substrate molecules.