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Earlier studies are extended to the higher-numbered K-types of Klebsiella, several of which may now be fitted into the previously found groups. Additional correlations between chemical structure and serological specificity are given, not only among the higher K-types, but also for several which were previously uninterpretable because structures were not known at the time. K1, K5, K6, K7 and K56 are shown to form a related group, although the reasons for their cross-reactivities are not always the same.

An earlier study of more than 60 Klebsiella K-polysaccharides in anti-pneumococcal sera of many capsular types provided the impetus for the present work. With anti-Klebsiella sera, some of these cross-reactions are now shown to be reciprocal. In most instances in which several or many K-substances precipitated antipneumococcal sera of a given type, these same K-polysaccharides were cross-reactive with one or more anti-Klebsiella sera belonging to the same group. For example, the polysaccharides K1, 15, 24, 31, 39, 42, and 59 precipitated an equine antipneumococcal type X serum. Pneumococcal capsular polysaccharide type X and Klebsiella polysaccharides K15, 24, 31, 39, 42, and 59 precipitated one or both of the equine anti-Klebsiella K1 sera available. Similar results with anti-Klebsiella rabbit sera to other K-types show the validity of this approach in revealing hitherto unknown serological, and hence chemical, relationships between the K-types.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.

Burgia malayi and B. pahangi microfilariae were isolated from the blood of infected Mastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thr-rich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis- and trans-splicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Ls-shp3 and their orthologues in Brugia, implicating conserved regulatory elements.

A 120 kDa antigen produced by juvenile female Litomosoides sigmodontis (Juv-p120) was isolated and purified. The amino acid composition of the molecule was determined. Juv-p120 was shown to be highly modified with N,N-dimethyl-aminoethanol (28.4 mol%). Treatment of Juv-p120 with potassium hydroxide (beta-elimination) or with sodium m-periodate leads to the destruction of epitopes recognized by antibodies immune affinity-purified with isolated Juv-p120. Juvenile L. sigmodontis were shown to release Juv-p120 into the pleural cavity of infected Mastomys coucha before the onset of patency.

A microfilarial sheath protein gene (shp2) coding for the major constituent of the insoluble, cross-linked sheath remnant (SR) from Brugia malayi, Brugia pahangi and Litomosoides carinii has been cloned and sequenced, based on peptide partial amino-acid sequences. All three closely related single-copy shp2 genes in the two genera carry a single intron in identical position; shp2 mRNAs are post-transcriptionally modified by both cis-splicing and trans-splicing. In accordance with their extracellular destinations the encoded proteins include signal peptide sequences; molecular masses of approx. 23 kDa are hence predicted for the mature secreted polypeptides. In their structures sheath matrix proteins shp2 may be regarded as extreme cases of a modular constitution, since these proteins largely consist of two different segments of multiple sequence repetitions, PAA and QYPQAP (or QYPQ), separated by elements of unique sequence. Extreme insolubility and cross-linking are likely to originate from these repetitive sequences within shp2, and to constitute the basic properties of a microfilarial matrix largely consisting of an shp2 network.

There is a glycanase activity associated with the particles of Klebsiella bacteriophage No. 13 which catalyzes hydrolysis of O - β - d -glucopyranosyl-(1 → 4)- β - d -mannopyranose linkages in Klebsiella serotype 13 capsular polysaccharide. The initial glycanase reaction velocity (at 37° and the optimum pH of 7–8) exhibits hyperbolical dependence on low concentrations of substrate, with K m = 170 μ m (in repeating units), and V max = 140 nmol/min (glucose equivalents, for 10 10 plaque-forming units). The reaction is inhibited by reaction products (type-13 penta- and decasaccharides, one and two repeating-units), and also by the substrate at concentrations above ∼ 1m m . Of seventy-four heterologous Klebsiella , and two other bacterial capsular heteropolysaccharides, as well as two substrate derivatives tested, only the Klebsiella serotype-2, -22, and -37 glycans were also depolymerized by the phage-13 enzyme. The reacting polysaccharides consist of repeating units, characteristically containing (a) chain 3 eq ,1 eq → 4 eq ,1 eq dihexopyranosyl sequences around the susceptible linkages, with equatorial hydroxyl and hydroxymethyl groups at C-2 and C′-5 next to them, and ( b ) branch carboxyl groups that may have to be located within a certain distance from these linkages.

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.

Fibre X-ray diffraction patterns have been obtained from oriented, semi-crystalline films of the Klebsiella capsular polysaccharide secrotype K57. K57 is a polytetrasaccharide that contains galactosyluronic acid, mannosyl, and galactosyl residues in the backbone, and an additional mannosyl group as a side-appendage. The simplest interpretation of the diffraction pattern is that the molecule crystallizes as a three-fold helix with an axially projected repeat of 1.143 nm which correlates directly with the chemical repeat. The chain is highly extended, even though it incorporates a 1,2-diaxial linkage in the main backbone. Molecular models have been built using least-squares techniques to minimise interatomic compression and simultaneously meet the observed helical parameters. These models have been compared with the experimental data by using cylindrically averaged, Fouriertransform calculations.

Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.

Virus-associated glycanases acting on bacterial capsular polysaccharides or on the 0-specific side chains of cell wall lipopolysaccharides, carboxylic ester and amide hydrolases removing 0— and N—acetyl residues from these glycans, as well as lyases for bacterial 2xopolysaccharides are known to occur on bacteriophages for different bacterial species, notably for many belonging to the family of Enterobacteriaceae. A review of the current knowledge on this group of enzymes is presented, covering the following aspects: (i) Isolation and selection of appropriate bacteriophages, (ii) purification of bacteriophage particles and of the virus organelles ('spikes" or thick "fibers") carrying the active center(s) , (iii) electron microscopy of the viruses, (iv) molecular properties of the virus organelles, (v) enzymology (pH dependence, kinetics, substrate specificity etc.) of the reactions as catalyzed by complete bacteriophage particles, (vi) applications of the viral glycanases, which allow the preparative isolation of oligosaccharides consisting of one and more repeating units of the host surface heteropolysaccharides.

Summary Using pairs of smooth and rough forms of Enterobacteriaceae, six smooth-specific bacteriophages were isolated from sewage and another was obtained from Dr Hedda Milch, Budapest. Upon incubation of the individual extracted (smooth) host cell wall lipopolysaccharides with the homologous purified viruses, liberation of reducing groups (i.e. of about di- to nonasaccharides) was observed in four cases, indicating the action of glycanases — but no liberation of acetic acid, indicating the absence of esterase activity. Under the electron microscope, all phages were seen to exhibit Bradley group B or C morphology and to carry tail spikes.

Two bacteriophages degrading 3-deoxy-D-manno-2-octulosonic acid-(KDO)-containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage phi 95 catalyzes the hydrolysis of beta-octulofuranosidonic linkages of the K95 glycan; and phi 1092, the alpha-octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross-reactivity of the phage enzymes with other KDO-containing capsular polysaccharides was observed.

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α- D -Glc p -(1/ar4)β- D -GlcA p -(1/ar4)-α- L -Fuc p -(1/ar chain with a β- D -Gal p -(1→ branch at position 4 of the D -glucosyl residue.

Purified particles of two small bacteriophages, active on Klebsiella K20 or K24 strains, were found to exhibit galactosidase or glucosidase activities, respectively, that cause an extensive depolymerization of isolated host capsular polysaccharides.

The primary structure of the Klebsiella serotype 6 capsular polysaccharide has been shown to consist of →3)-α- l -Fuc p -(1→3)-β- d -GlC p -(1→3)-β- d -Man p -(1→4)-α- d -GlcA p -(1 → repeating-units. substituted by pyruvate acetal on positions 4 and 6 of the mannosyl residue.

Fibre type X-ray diffraction patterns have been obtained from oriented, semi-crystalline films prepared from the sodium salt of the capsular polysaccharide of Klebsiella serotype K25. This molecule has a tetrasaccharide repeating structure consisting of a disaccharide backbone and a disaccharide side chain. The backbone contains a di-equatorially 1,4 linked β- d -glucose residue followed by a di-equatorially 1,3 linked β- d -galactose residue. The side chain is attached to the axial O(4) position of the galactose residue and consists of a di-equaltorially 1,2 linked β- d -glucoronic acid with a β- d -glucose residue attached terminally. An interesting feature of the backbone linkage geometry of this polysaccharide is its similarity with those of the animal connective tissue polydisaccharides. Analysis of diffraction patterns gives rise to an extended three fold helical conformation with an axially projected advance per chemical repeat of 0.97 nm. Molecular models have been computer generated using least squares techniques to optimize interatomic contacts and simultaneously meet the observed helical parameters. A left handed helix with inter-residue stabilizing hydrogen bonds was found to be most favourable and comparison of this model with other relevant polysaccharide structures is male.

Using periodate oxidation, methylation analysis, the characterization of oligosaccharides obtained by partial acid hydrolysis, p.m.r. spectroscopy, and analytical ultracentrifugation, the structure of the (mildly alkali-treated) Klebsiella serotype 11 capsular polysaccharide has been elucidated. The tetrasaccharide repeating-unit comprises the sequence ₒ3)-β- D -Glc p -(1ₒ3)-β- D -GlcUA p -(1ₒ3)-α- D -Gal p -(1→ with a 4,6- O -(1-car☐yethylidene)-α- D -galactosyl residue linked to O-4 of the glucuronic acid residue. The structural basis for some serological cross-reactions of the Klebsiella K11 antigen is discussed, and it is shown that rabbit antisera against the Klebsiella K11 test-strain predominantly contain K agglutinins specific for branch-terminal 4,6- O -(1-car☐yethylidene)- D -galactose.

As previously shown, there is a host capsule deploymerase activity associated with the spikes of Klebsiella bacteriophage No. 11 (Bessler et al. , 1973). Purified particles of phage No. 11 were now subjected to a variety of denaturing conditions, and it was found that mild acid treatment (3 to 10 min at pH 3.5 and 20°) causes a pronounced decay of the viruses into their structural elements, but a comparatively moderate loss of depolymerase activity. From batches of phage fragments obtained in this manner, (I) detached pairs of spikes, (II) spike subunits still exhibiting host capsule depolymerase activity, and (III) head capsomeres were isolated. Electron optical visualization and SDS PAA gel electrophoresis of the preparations showed that the enzymatically active spike subunits are small (possibly hollow) particles of about 50–60 A diameter consisting of one 62,500-dalton polypeptide only, and that the head capsomeres are doughnut-shaped structures of approximately 80 A diameter, containing polypeptides of about 41,000 daltons. Previous results concerning the morphology and polypeptide composition of the whole phage spikes were verified.

The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues.

Using 74 serological test strains for different Klebsiella K antigens as propective hosts, 50 new bacteriophages were isolated, the particles of which catalyze host capsular glycan depolymerization. As seen under the electron microscope, these viruses most often belong to Bradley's morphology group C (36 cases), occasionally to group B (11), and rarely to group A (3); they all carry tail spikes (or thick fibers) with which, presumably, the glycanase activity is associated. Employing the same set of Klebsiella strains, the host range of a total of 55 such bacteriophages was tested and 28 cross-reactions (host capsule depolymerization and plaque formation at a relative efficiency of plating ⪕ 10 5 ) were detected; in addition, one abortive infection (host capsule depolymerization without plaque formation) was found. The viral depolymerases thus proved to be very specific (33 cross-reacting with no, 18 with one, 2 with two, and 1 each with three or four heterologous polysaccharides). For further characterization of the over 80 phage-catalyzed depolymerization reactions thus identified, the purified virus particles were incubated with the isolated bacterial capsular polysaccharides, and the oligosaccharides produced were analyzed for reducing end sugar (glucose, galactose, mannose, fucose, or rhamnose), and for size (mainly one to three repeating unit oligosaccharides in about 85% of the cases). In those 49 phage-host systems, where the primary structures of the substrate polysaccharides were known, the reducing end sugar analyses led to unequivocal (36 reactions) or tentative (13) identifications of the phage enzyme susceptible glycosidic linkages, and hence to the following general conclusions: (I) in most cases cleavage occurs on either side of the (a) sugar unit carrying the negative charge(s), but reducing glycuronic acids are not produced; (II) most often, the reducing end sugar formed is substituted at position 3; (III) in the majority of cases, β-glycosidic linkages are hydrolyzed; (IV) in most polysaccharides which are acted upon by several phage enzymes, the same glycosidic bonds are split by the different agents.

The preparation and use of particles of Klebsiella bacteriophage No. 11 are described. A glycanase activity associated with the viruses catalyses the depolymerization of (alkali-treated)Klebsiella serotype 11 capsular polysaccharide, ultimately to a mixture of oligosaccharides consisting of one or two repeating units. Mainly glucosidic bonds are hydrolysed. The substrate specificity of the viral enzyme has been characterized by using derivatives of serotype-11 polysaccharide, as well as 81 heterologous, bacterial, capsular glycans. It is concluded that the glycanase will (at least) also depolymerize all polysaccharides containing the unsubstituted chain-trisaccharide repeating-unit of its natural substrate.

Using periodate oxidation, methylation analysis, characterization of oligosaccharides by Smith degradation or partial acid hydrolysis, as well as proton magnetic resonance, the primary structure of the Escherichia coli serotype 29 capsular polysaccharide (the receptor of E. coli K phage 29) was reinvestigated. The polymer was found to consist of hexasaccharide repeating units of the following structure: (see article).

The surface glycoprotein (mixture of isoglycoproteins with Mr 69 000 and 71 000) was isolated from the particles of Friend murine leukemia virus, and was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. Roughly 20% (w/w) of the carbohydrates in this glycoprotein were thus released, and they were fractionated by high-performance liquid chromatography after reduction with KB3H4/NaBH4. The radioactive oligosaccharitol fractions obtained were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by fast atom-bombardment mass spectrometry, as well as by capillary gas-liquid chromatography/mass spectrometry following hydrolysis, reduction and peracetylation. Around 85% (mol/mol) of the endo-H-sensitive viral glycans were thus found to be oligomannosidic oligosaccharitols of size classes Man5GlcNAcOH, Man6GlcNAcOH, Man8GlcNAcOH, Man7GlcNAcOH, and Man9GlcNAcOH (in order of prevalence), and the major structural isomers of each size class were identified. About another 15% (mol/mol) of the oligosaccharitols were shown to be of the 'mixed type', comprising mainly four species in which the Man(alpha 1----6)-branch of the Man alpha 1----6 (Man alpha 1----3) Man beta 1----4GlcNAcOH core is substituted by one or two additional alpha-mannoses, while the Man(alpha 1----3)-branch carries an N-acetyllactosamine unit substituted by sialic acid, or by Gal(alpha 1----3).

Depolymerization of bacterial, capsular polysaccharides by phage enzymes is a convenient method of preparing oligosaccharides that correspond to one, or several, repeating unit(s). Thus, the capsular polysaccharide from Klebsiella K21 yields a linear pentasaccharide, and that from Klebsiella K32, a linear tetrasaccharide. Both oligosaccharides contain acetal substituents, but, whereas the 4,6- O -(1-carboxyethylidene)- D -galactosyl residue in the K21 structure is relatively acid-stable, the corresponding 3,4- O -(1-carboxyethylidene)- l -rhamnosyl residue in K32 is extremely acid-labile. Phage degradation may, therefore, be the only way by which an oligosaccharide corresponding to an intact repeating-unit may be obtained in such circumstances.

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli , all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella ) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.

A capsule depolymerase induced by bacteriophage No. 11 on Klebsiella 390 (03:K11), has been isolated. The material was essentially homogeneous in analytical ultracentrifugation and immunoelectrophoresis. It catalyzes the hydrolysis mainly of β- d -glucopyranosido-(1 → 3)- d -glucuronic acid, but also that of a few (1 in about 20) d -galactopyranosido-(1 → 3)- d -glucose bonds in K11 capsular polysaccharide. The depolymerase consists of dimers of club- or drop-shaped particles of a molecular weight of 155,000 ± 10,000 containing one polypeptide chain of about 62,500 and one of 94,000 dalton. Klebsiella bacteriophage No. 11 is an isometric virus; its head has a diameter of about 450 A. It bears a base plate of about 265 A diameter which has the shape of a six-pointed star with a central hole or prop, carrying two spikes for each of its six points. The following observations support the view that the depolymerase consists of pairs of free spikes of phage 11: (I) The depolymerase monomers and phage spikes in situ have the same dimensions. (II) Polypeptide chains of the same size as the two in the depolymerase also occur in whole virus. (III) The free depolymerase shows approximately the same enzymatic activity as do phage spikes in situ. Purified depolymerase and bacteriophage particles catalyze the hydrolysis of the same bonds in Klebsiella K11 capsular polysaccharide, ultimately forming oligosaccharide fragments of one, two, and three repeating units of the (alkali-treated) polymer. (IV) Phage 11 and the depolymerase strongly cross-react serologically, and anti-depolymerase IgG antibodies specifically adhere to the spike region of whole virus.

Some Escherichia coli K bacteriophage particles, capable of interacting specifically with bacterial polysaccharide capsules, carry an endo-glycosidase activity, probably located in the spikes.

A glycanase activity associated with the particles of Klebsiella bacteriophage No. 6 catalyses cleavage of O-beta-D-glycopyranosyl-(1 leads to 3)-4,6-O-(1-carboxyethylidene)-beta-D-mannopyranose linkages in Klebsiella serotype-6 capsular polysaccharide. Of 74 heterologous Klebsiella polysaccharides and two derivatives of the type-6 glycan, only the type-1 and type-57 polymers were additionally degraded by the phage-6 enzyme. The repeating units in the three substrates have a 1ax leads to 3eq, 1eq leads to eq-linked chain D-gluco- or D-galacto-pyranosyl residue in common (which constitutes the reducing end after glycanase action), and a carboxyl group on the next hexopyranosyl residue. Of the 72 polysaccharides not affected by the viral enzyme, at least the type-11 and type-21 glycans also contain the same homology of primary structure. This indicates that the conformation at the glycanase recognition-site also constitutes an important feature of the substrates.

During the past few years, the structures have been determined of many more immunologically specific extracellular polysaccharides of the more than 80 serotypes of Klebsiella. Structures of several additional pneumococcal type-specific polysaccharides have also been elucidated. The new information is used to check inferences made earlier and to trace new correlations between chemical structure and immunological specificity.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one‐ and two‐dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N‐Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N‐acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N‐acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three‐dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.

Periodate oxidation and Smith degradation, methylation analysis including uronic acid degradation, partial hydrolysis with acid, bacteriophage degradation, and p.m.r. spectroscopy have been used to elucidate the primary structure of the Klebsiella serotype-13 capsular polysaccharide. The polymer consists of pentasaccharide repeating-units comprising a 4)-beta-D-Manp-(1 leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to chain with a 3,4-O-(1-carboxyethylidene)-beta-D-Galp-(1 leads to 4)-alpha-D-GlcAp-(1 leads to branch at position 3 of the mannose. It is shown that there is a glycanase activity associated with particles of Klebsiella bacteriophage No. 13, which catalyses hydrolysis of chain beta-D-Glcp-(1 leads to 4)-beta-D-Manp linkages in the type-13 polysaccharide. The chemical basis of some serological cross-reactions of the Klebsiella K13 antigen is discussed.

Four neutal fraction glycosphingolipids, designated components 4-7, were purified from the pupae of Calliphora vicina and isolated by the use of high performance liquid chromatography. Their chemical structures were determined to be: GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; and GlcNAC(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer. By the use of specific exoglycosidases, it was possible to assign anomeric configurations to all the sugar residues present. Analysis of the ceramide moiety by electron-impact mass spectrometry revealed the dominant fatty acid and sphingoid to be arachidic acid (C20:0) and tetradecasphing-4-enine, respectively.

† Mutants of Escherichia coli B with increasing structural defects in the cell wall lipopolysaccharide (above) were tested for adsorption resistance to bacteriophages T3 and T4, and the lipopolysaccharide extracted from these mutants was assayed for loss of ability to inactivate the viruses. Absence of terminal glucose, or of any of the substituents (branch heptose, phosphate and pyrophosphorylethanolamine) on the two chain heptoses was found to have little effect on T3 and T4 susceptibility. However, all mutants lacking both glucoses were resistant to both phages.

The Klebsiella serotype-51 (K51) capsular polysaccharide consists of a tetrasacchariderepeating-unit comprising a → 3)- α - d -Gal p -(1 → 3)- α - d -Gal p -(1 → chain substituted by an α - d -G1c p A-(1 → 6)- α - d -Glc p -(1 → branch at position 4 of one galactosyl residue.

Publisher Summary Glycan glycanohydrolase activities catalyzing the depolymerization of bacterial surface polysaccharides are associated with the tail parts of many bacteriophages. During the infective process, they help the viruses to penetrate the outer layers of their host cells. These bacterioviral hydrolases are highly specific for one or a few structurally related heteroglycans; therefore, they contribute to the restriction of bacteriophage host range. This chapter describes the propagation and purification of Escherichia coli bacteriophage ϕ92,7 the depolymerization of colominic acid, [-α-NeuAc-(2 → 8)-]n, by the virus particles, and the isolation of the oligosialic acid products. The chapter discusses the process of propagation and purification of bacteriophage. There is a discussion on the materials used, bacteriophage stock, large-scale propagation of bacteriophage, and purification of bacteriophage particles. The chapter describes the procedures of depolymerization of colominic acid and isolation of sialic acid oligomers and their principles, substrates, and reagents. The chapter examines how colominic oligosaccharides may also be separated by gel filtrations and how larger sialic acid oligomers are obtained after short periods of incubation with the virus.

The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.

A set of five Escherichia coli K phages has been isolated. These phages are adsorbed to and lyse the capsular forms of the host bacteria, whereas their spontaneous, acapsular mutants are not affected. All host strains are heavily encapsulated test strains for E. coli K antigens of the thermostable A type and they readily segregate acapsular mutants. In four of the phage-host systems, all secondary growth obtained was found to be acapsular. When tested for host-range mutants on 38 strains of E. coli and Klebsiella , less than one mutant per 10 5 plaque-forming units was found. No cross-reacting neutralizing antibodies were obtained when rabbits were immunized with the K phages. The latent periods (between 16 and 30 min) and average burst sizes (between 145 and 580) were determined by one-step growth experiments.