Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination

Hänfling, Peter; Shashkov, Alexander S.; Jann, Barbara; Jann, Klaus

doi:10.1128/jb.178.15.4747-4750.1996

Cited by 26 publications

(24 citation statements)

References 28 publications

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“…The ltf gene codes for a 148-kDa protein (27), and in agreement with the cleavage site proposed here, processing of a 150-kDa precursor protein into a 125-kDa protein was reported (30). The presence of a common cleavage site is also supported by results obtained for the recombinantly expressed lyase of E. coli K5 which depolymerizes the capsular polysaccharide composed of repeating units of 4-linked ␣-N-acetylglucosamine and ␤-glucuronic acid (29,31). In addition to full-length protein of 89 kDa, a 70-kDa fragment lacking the C terminus was observed (29), corresponding to the calculated fragment sizes of 73 and 16.7 kDa.…”

Section: Discussionsupporting

confidence: 64%

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Mühlenhoff

Stummeyer

Grove

et al. 2003

Journal of Biological Chemistry

105

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Bacteriophages infecting the neuroinvasive pathogenEscherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host. Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages K1E, K1F, and 63D, respectively. The cloned sequences share a highly conserved catalytic domain but differ in the length of the N-and C-terminal parts. Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF. Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation. By reinvestigation of the respective gene locus in the K1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein.Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone. Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain. Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the Cterminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF.

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Section: Discussionsupporting

confidence: 64%

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Mühlenhoff

Stummeyer

Grove

et al. 2003

Journal of Biological Chemistry

105

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“…In this case, ⌽K5 encodes a tailassociated K5 specific lyase protein that is also responsible for attachment to the cell surface and degradation of the K5 polysaccharide capsule (12,14). Phage specific for other E. coli polysaccharide antigens, including K3, K7, K12, K13, and K20 (26, 27), have also been found; all probably possess specific polysaccharide depolymerization activities as part of the phage particle.…”

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confidence: 99%

Bacteriophage K1-5 Encodes Two Different Tail Fiber Proteins, Allowing It To Infect and Replicate on both K1 and K5 Strains of Escherichia coli

Scholl

Rogers

Adhya

et al. 2001

J Virol

169

115

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A virulent double-stranded DNA bacteriophage, ⌽K1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of ⌽K5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of ⌽K1E, which allows this phage to specifically infect K1 strains of E. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of ⌽K5, ⌽K1E, and ⌽K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.Escherichia coli capsular polysaccharides (K antigens) have often been associated with increased virulence (17). The K1 antigen in particular increases the invasiveness of E. coli, and these strains are often involved in cases of meningitis and septicemia (32). These polysaccharide coats also act as recognition sites for bacteriophages, which often carry tail spikes that contain polysaccharide depolymerization activities. Several K1-specific phages have been described (10), one of which, ⌽K1E, was found to possess N-acetylneuraminidase (endosialidase) as a part of the tail fiber protein (37). This enzyme catalyzes the cleavage of ␣-2,8-linked poly-N-acetylneuraminic acid carbohydrate polymer of the K1 capsule. It has been suggested that the tail fiber protein is involved in both adsorption to the cell surface and penetration into the cell by enzymatically degrading the polysaccharide capsule. The ⌽K1E endosialidase gene has been cloned and sequenced (20). A similar gene has been cloned and sequenced from ⌽K1F (29).⌽K5 is a related bacteriophage specific for E. coli strains that display the K5 antigen, a polymer consisting of a repeating structure of 4-linked ␣-N-acetylglucosamine and ␤-glucuronic acid (N-acetyl heparosin). In this case, ⌽K5 encodes a tailassociated K5 specific lyase protein that is also responsible for attachment to the cell surface and degradation of the K5 polysaccharide capsule (12,14). Phage specific for other E. coli polysaccharide antigens, including K3, K7, K12, K13, and K20 (26, 27), have also been found; all probably possess specific polysaccharide depolymerization activities as part of the phage particle.Both ⌽K5 and ⌽K1E have a Salmonella phage SP6-like promoter upstream of their tail proteins as well as a region of homology which is just downstream of the...

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“…This bacteriophage contains a lyase which degrades the K5 polysaccharide randomly throughout the polymer by a ␤ elimination reaction. The final reaction products of this bacteriophage lyase consist of hexa-, octa-, and decasaccharides (8,12). In addition to the bacteriophage-borne K5 lyase, a chromosomally encoded K5 lyase enzyme has been described for E. coli SEBR 3282 (16).…”

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confidence: 99%

Cloning, Expression, and Purification of the K5 Capsular Polysaccharide Lyase (KflA) from Coliphage K5A: Evidence for Two Distinct K5 Lyase Enzymes

2000

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The Escherichia coli K5 capsular polysaccharide [-4)-␤GlcA-(1,4)-␣GlcNAc-(1-] is a receptor for the capsulespecific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF L ) located 5 of the endosialidase gene of the E. coli K1 capsulespecific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF L in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF L is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His) 6 -KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.At least 80 distinct capsular polysaccharides (K antigens) have been described previously for Escherichia coli (26). Expression of a polysaccharide capsule on the cell surface confers resistance to host immune defenses and other adverse environmental conditions (3,29). In addition, many of these structurally distinct capsules act as receptors for bacteriophages, and variation of capsular structure may be a mechanism for evasion of bacteriophage infection. Due to their specificity, capsule-specific bacteriophages can be used for identification of numerous E. coli K serotypes. Integral to the tail structure of many capsule-specific bacteriophages are enzymes which degrade the bacterial polysaccharide and provide the bacteriophages access to receptors on the cell surface (5, 32-35).The E. coli K5 capsular polysaccharide is a polymer of -4)-␤GlcA-(1,4)-␣GlcNAc-(1-(40). This structure is identical to N-acetylheparosan, the precursor polymer of heparin and heparan sulfate (17). Coliphage K5A, a bacteriophage specific for E. coli K5 capsule-expressing strains, has be...

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Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination

Cited by 26 publications

References 28 publications

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Bacteriophage K1-5 Encodes Two Different Tail Fiber Proteins, Allowing It To Infect and Replicate on both K1 and K5 Strains of Escherichia coli

Cloning, Expression, and Purification of the K5 Capsular Polysaccharide Lyase (KflA) from Coliphage K5A: Evidence for Two Distinct K5 Lyase Enzymes

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