Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

The influence of the O-antigen and the capsular polysaccharide on the establishment of PlCmts lysogeny in Klebsiella pneumoniae

The O-antigen of the lipopolysaccharide in Klebsiella pneumoniae caused a significant reduction in the frequency of establishment of PlCmts lysogeny, while the capsular polysaccharide showed no effect on this frequency. The bacterial receptor for PlCmts are the lipopolysaccharide-core oligosaccharides, the results suggest that K. pneumoniae strains with an O-antigen in their lipopolysaccharide have a poorly accessible lipopolysaccharide-core (the PlCmts bacterial receptor), while K. pneumoniae strains lacking the O-antigen have a highly accessible lipopolysaccharide-core. The accessibility of the receptor is independent of the K antigen (capsular polysaccharide).     

Escherichia coli capsule bacteriophages. VII. Bacteriophage 29-host capsular polysaccharide interactions.

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.     

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage φR1-37 when grown at 37°C but not when grown at 22°C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22°C. The transposon library of YeO3-c was grown at 37°C and screened for phage φR1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the trs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarity to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus ; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage φR1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk—hemH and galE—gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage φR1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Isolation and characterization of a bacteriophage polysaccharide.

A high molecular weight heteropolysaccharide, composed of glucose, glucuronic acid, N-acetylglucosamine, and mannose in an approximate molar ratio of 1:2:2:5, respectively, was isolated from phage K-2 and from the soluble fraction of phage-infected Aerobacter aerogenes lysates. Treatment of pure phage with 8 M urea at 4 degrees quantitatively solubilizes the bound polysaccharide and capsular polysaccharide (Yurewicz, E.C., Ghalambor, M.A., Duckworth, D.H., and Heath, E.C. (1971) J. Biol. Chem. 246, 5607-5616) with the release of only traces of other phage constituents; on this basis, it was concluded that the polysaccharide, like the the glycanohydrolase, is externally localized in the phage structure. Phage polysaccharide and glycanohydrolase fractionate similarly on ion exchange resins and gel electrophoresis in sodium dodecyl sulfate, but each may be purified to homogeneity by the procedures employed. The biosynthesis of the polysaccharide was shown to be uniquely dependent upon phage K-2 infection by: (a) absence of the polysaccharide in cells, the culture filtrate, or sonicated extracts of uninfected cells; (b) kinetics of polysaccharide synthesis following phage infection; and (c) isotopic double-labeling experiments that demonstrated the synthesis of polysaccharide only after initiation of phage replication in infected cells.     

Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel-filtration chromatography to give an OPS-rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [-->4)alpha-L-Rhap(1-->3)beta-D-Manp(1-->].     

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.     

Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

Background

Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed.

Methodology/Principal Findings

To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS⁻ mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis.

Conclusions/Significance

Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.     

Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage.     

Physicochemical surface properties ofKlebsiella pneumoniae

The following cell surface physicochemical characteristics were investigated inKlebsiella pneumoniae: surface charge, surface hydrophobicity by different methods, and accessibility of the lipid fraction of the outer membrane. The capsular polysaccharide, as well as the O-antigen repeating units of the lipopolysaccharide (LPS), conferred a hydrophilic, negatively charged surface to the bacterium, and a barrier to the dye congo red, which binds sites within the lipid fraction of the outer membrane (OM).     

A high molecular weight lipopolysaccharide specific bacteriophage for Klebsiella pneumoniae

High-molecular weight lipopolysaccharide (O antigen enriched fraction) from Klebsiella pneumoniae was determined to be the receptor for bacteriophage FC3-1. A methodology for the identification of the lipopolysaccharide component involved in FC3-1 bacteriophage reception was used that is suitable for other phages and host bacteria.     

H protein of bacteriophage 16-3 and RkpM protein of Sinorhizobium meliloti 41 are involved in phage adsorption

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.     

Preparation of branched hexasaccharides by the action of a viral lyase on Klebsiella K14 polysaccharide

Klebsiella K14 capsular polysaccharide was degraded by a bacteriophage-borne enzyme to afford oligosaccharides A-C which were studied by one- and two-dimensional n.m.r. spectroscopy. A and B were the repeating-unit hexasaccharide and pyruvylated hexasaccharide, respectively, while C was a dodecasaccharide. Each oligomer was terminated by a reducing mannose and a non-reducing 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid residue, indicating that the phage enzyme had cleaved the beta-D-Manp-(1----4)-beta-D-GlcpA linkages in the polysaccharide by a lyase, rather than the more common glycosidase, activity found with other Klebsiella bacteriophages. In this respect, the depolymerisation resembles those reported for the capsular polysaccharides of Klebsiella K5 and K64     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.     

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.     

Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-d-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.     

Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant

A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil. Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm). An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R. japonicum 3I1b110 that were tested, except one, mutant strain HS123. Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots. Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface. Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin. Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1. A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not. Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage. Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition. These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.     

Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant.

A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil. Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm). An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R. japonicum 3I1b110 that were tested, except one, mutant strain HS123. Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots. Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface. Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin. Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1. A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not. Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage. Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition. These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.     

Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing

Coliphage K30 lysates contain free and phage-associated forms of a bacteriophage-encoded capsule depolymerase (glycanase) enzyme, active against the serotype K30 capsular polysaccharide of Escherichia coli. The free glycanase has been purified to apparent homogeneity. The molecular weight of the enzyme was estimated at 450,000, and when heated in SDS at 100 degrees C, the enzyme dissociated into two subunits of 90,000 and 52,000. The glycanase enzyme was used as a reagent to reversibly degrade the capsular layers on cells of Escherichia coli O9:K30 and Klebsiella O1:K20. This treatment rendered these bacteria sensitive to their respective lipopolysaccharide-specific bacteriophages, coliphage O9-1 and Klebsiella phage O1-3. This novel approach facilitated isolation of lipopolysaccharide O antigen side chain deficient mutants which retained the ability to synthesize the capsule. The response of defined mutants, O+:K-, O-:K+, and O-:K-, to exposure to nonimmune rabbit serum was measured. Results showed that the primary barrier against complement-mediated serum killing in both Escherichia coli O9:K30 and Klebsiella O1:K20 was the O antigen side chains of the lipopolysaccharide molecules. In both strains, the capsule played no role in the determination of serum resistance.