Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: evidence for a third hyaluronidase involved in extracellular enzymatic activity

Abstract

The hyaluronidase gene (hylP2) from a second group A streptococcal bacteriophage was isolated from ATCC T-type-22 hyaluronidase-producing strain 10403, a strain known to produce increased amounts of extracellular hyaluronidase. Sequence analysis of hylP2 and alignment with the previously described bacteriophage hyaluronidase gene (hylP) showed a high degree of similarity; however, hylP2 had deletions of regions specifying 34 amino acids. Twenty-eight of the deleted amino acids were in a region of HylP containing a series of collagen-like Gly-X-Y repeating units. By employing primers for both hylP and hylP2, PCR amplification resulted in fragments of appropriate sizes in 97% of the strains tested, with some strains producing two fragments, indicating the presence of at least two phages. When the hylP2 gene was introduced via a plasmid vector into a non-hyaluronidase-producing Streptococcus pyogenes strain, this strain was still unable to produce extracellular hyaluronidase, although intracellular hyaluronidase was present. These results, along with the absence of a typical N-terminal signal peptide, indicate that HylP2 is unable to be secreted into the extracellular milieu. Examination of more than 100 strains for production of hyaluronidase showed that only 23% of the strains produced extracellular hyaluronidase. One of these strains (strain 10403) contains a single bacteriophage hyaluronidase gene (hylP2) which, when inactivated by allelic replacement, still produces large amounts of extracellular hyaluronidase. These results suggest the presence of a different hyaluronidase gene encoding a protein that is actively secreted into the extracellular milieu.

The extracellular hyaluronidase gene (hylA) of Streptococcus pyogenes

“…Comparison of the deduced amino acid sequence of the S. pyogenes hyaluronidase with that of other hyaluronidases indicates a high degree of similarity to deduced se-quences from Streptococcus pneumoniae [6], Staphylococcus aureus [4] and Streptococcus agalactiae [5] with 49, 44 and 40% similarities, respectively, over the entire gene. Similarities with other Gram-positive organism hyaluronate lyases are weaker (21^31% similarities), with little or no homology to the streptococcal bacteriophage hyaluronidases [11,23].…”

Hyaluronidases of Gram-positive bacteria

“…A hyaluronic acid capsule can surround the host cells of both of these phages; hence the bacteriophage may require hyaluronidase for penetration of this capsular material. No hyaluronidase activity was detected in the extracellular milieu of either S. pyogenes or S. equi infected with the temperate bacteriophages [18,19], suggesting the enzymes may be part of the bacteriophage particle.…”

“…The bacterial hyaluronidases, along with their accession number, are from S. aureus (U21221) [20], Streptococcus agalactiae (Y15903) [21], S. pneumoniae (L20670) [7], S. griseus (AB028210), S. coelicolor (AL031124), P. acnes (U15927) [22], C. perfringens (P26831) [12], Proteus vulgaris (1095454) [23], along with a partial sequence of the lyase from Bacteroides thetaiotaomicron (L42367) [24]. Two sequenced bacteriophage hyaluronidases are both derived from temperate phages that infect group A streptococci (M19348 and U28144) [18]. Sequence analysis of the hyaluronidase genes provides important information on the hyaluronidases.…”

Polysaccharide binding sites in hyaluronate lyase – crystal structures of native phage–encoded hyaluronate lyase and its complexes with ascorbic acid and lactose

“…The bound hyaluronidase produced by the bacteriophage is not secreted from the cell and is a part of the bacteriophage particle. Its main function is to assist the phage in the penetration of the HA capsule that surrounds the host cells of this phage and hence gain access to the cell surface of the host Streptococcus [4]. Apart from this, an indirect role of the bacteriophage-encoded hyaluronidase in streptococcal disease has also been indicated where it transforms the nonvirulent streptococcal strains into virulent strains.…”