Enhancement of Streptococcal Bacteriophage Lysis by Hyaluronidase

Maxted, W. R.

doi:10.1038/1701020b0

Cited by 45 publications

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“…, 1940 and Evans & Sockrider (1942). In preliminary work with phages obtained from sewage and sent to us by Professor Boulgakov of Paris, it was found that lysis was often much more readily obtained when the test was carried out in the presence of hyaluronidase (Maxted, 1952). It was also noted that the colonies of the secondary growth that appeared after vigorous lysis had taken place were often very mucoid when plated on to glucose serum agar, even when the parent strain had never shown mucoid variants upon preliminary plating.…”

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The Influence of Bacteriophage on Streptococcus pyogenes

Maxted

1955

Journal of General Microbiology

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SUMMARY: By the selective action of certain bacteriophages on cultures of susceptible streptococci it is possible to alter or enhance certain of their characteristics. The extractable M antigen may be greatly increased ; non-mucoid strains may become completely mucoid with or without the increase in M antigen. Some phageselected cultures have shown a marked increase in mouse virulence and some an ability to survive in normal human blood not possessed by the parent strain.Despite the great amount of work published on bacteriophages, the only extensive studies on those active against group A haemolytic streptococci are those by Evans (e.g. 1933Evans (e.g. , 1934Evans (e.g. , 1940 and Evans & Sockrider (1942). In preliminary work with phages obtained from sewage and sent to us by Professor Boulgakov of Paris, it was found that lysis was often much more readily obtained when the test was carried out in the presence of hyaluronidase (Maxted, 1952). It was also noted that the colonies of the secondary growth that appeared after vigorous lysis had taken place were often very mucoid when plated on to glucose serum agar, even when the parent strain had never shown mucoid variants upon preliminary plating. The secondary growth from one strain also showed increased M antigen content, and an ability to survive in human blood not possessed by the parent strain. A more detailed investigation was therefore made of the changes in antigenic structure, colonial appearance and virulence that are produced in cultures by passage through phage-containing media. MATERIAL AND METHODS Preparation of hyaluronidase.Minced bull testicle was extracted overnight in the cold with ~/ 1 acetic acid, neutralized, and the supernatant fluid Seitzfiltered.Source and propagation of phages. The phages used were derived from those isolated by Professor Boulgakov from Paris sewage; they were propagated in nutrient broth containing hyaluronidase, each on its particular strain of streptococcus. The phages are designated A l , As, A12 and A25. The letter indicates the Lancefield group to which the propagating strain belongs and the number the type. The phages are not specific for their propagating type strain.For the phage passage experiments with types 3 and 27 the phage A1 was adapted to these strains; for the experiments with the type 5 strain phage A6 was similarly adapted. A mixture of all four of the original phages was used for the serial transfers of strain 1506 type 30.

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The Influence of Bacteriophage on Streptococcus pyogenes

Maxted

1955

Journal of General Microbiology

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“…The hylP2 gene of bacteriophage 10403 of Streptococcus pyogenes encodes an enzyme hyaluronate lyase (HylP2) 3 whose primary function is to degrade hyaluronan of the bacterial capsule (19). We carried out biophysical characterization (20) and x-ray data analysis of HylP2 (Protein Data Bank code 2DP5), which demonstrated that the C-terminal domain of the protein predominantly consisted of ␤-sheets harboring the active site residues (20).…”

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Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

Mishra

Bhakuni

2009

Journal of Biological Chemistry

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The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from prefibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular ␤-sheet formation by the ␤-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar filmlike structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure.

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“…Hence, their likely function could be to degrade HA of the streptococcal capsule and allow the phage access to the cell surface, so that it can infect the encapsulated cells (20,21). The hyaluronate lyase genes found in the bacteriophage genomes show a high degree of similarity to each other with one major difference being the deletion (or addition) of a 102-bp fragment that consists of a region that encodes a collagen-like motif, Gly-X-Y repeating units (6,20). Based on this difference, they have been grouped into two types: the hylP-type, which contains a collagen-like repeat sequence and the hylP2-type, which lacks this repeat sequence.…”

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Unusual Structural Features of the Bacteriophage-associated Hyaluronate Lyase (hylp2)

Mishra¹,

Akhtar

Bhakuni

2006

Journal of Biological Chemistry

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Hyaluronate lyases are a class of endoglycosaminidase enzymes, which are of considerable complexity and heterogeneity. Their primary function is to degrade hyaluronan and certain other glycosaminoglycans and facilitate the spread of disease. Among hyaluronate lyases, the bacteriophage-associated enzymes are unique as they have the lowest molecular mass, very low amino acid sequence homology with bacterial hyaluronate lyases, and exhibit absolute specificity for one type of glycosaminoglycan, i.e. hyaluronan. Despite such unique characteristics significant details on structural features of these lyases are not available. The Streptococcus pyogenes bacteriophage 10403 contains a gene, hylP2, which encodes for hyaluronate lyase (HylP2) in this organism. HylP2 was cloned, overexpressed, and purified to homogeneity. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa, under physiological conditions. Limited proteolysis and guanidine hydrochloride denaturation studies demonstrated that the N-terminal region of the protein is flexible, whereas the C-terminal portion has a compact conformation. The enzyme shows sequential unfolding, with the N-terminal unfolding first followed by the simultaneous unfolding and dissociation of the stabilized trimeric C-terminal domain. We isolated a functionally active C-terminal fragment (Ser 128 -Lys 337 ) of the protein that was stabilized in a trimeric configuration. Comparative functional studies with full-length protein, N:C complex, and isolated C-terminal domain demonstrated that the active site of HylP2 is present in the C-terminal portion of the enzyme, and the N-terminal portion modulates the substrate specificity and enzymatic activity of the C-terminal domain.

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Enhancement of Streptococcal Bacteriophage Lysis by Hyaluronidase

Cited by 45 publications

References 2 publications

The Influence of Bacteriophage on Streptococcus pyogenes

The Influence of Bacteriophage on Streptococcus pyogenes

Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

Unusual Structural Features of the Bacteriophage-associated Hyaluronate Lyase (hylp2)

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