Extracellular Hyaluronidase Research Articles

Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because of the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in the medical field. Here, we realized the active expression of recombinant hPH20 by Pichia pastoris under a methanol-induced promoter PAOX1. By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fusion mutant AP2-△491C with the extracellular hyaluronidase activity of 258.1 U·L−1 in a 3-L bioreactor, the highest expression level of recombinant hPH20 produced by microbes. Furthermore, we found recombinant hPH20 hydrolyzed the β-1,4 glycosidic bonds sequentially from the reducing end of o-HAs, with HA6 NA as the smallest substrate. The result will provide important theoretical guidance for the directed evolution of the enzyme to prepare multifunctional o-HAs with specific molecular weights.

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM

BackgroundRecombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described. MethodsThe specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA–aggrecan complex, and in vivo, by means of a diffusion assay in nude mice. ResultsDepolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20. ConclusionsrHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA–aggrecan complex. General significancerHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.

Cloning and expression of the enzymatic region of Streptococcal hyaluronidase.

Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein) plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair) was inserted correctly into the recombinant vector. The expressed protein (65 KDa) was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient's sera. In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

Two Novel Functions of Hyaluronidase from Streptococcus agalactiae Are Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.

Cloning, expression, purification and antigenic evaluation of hyaluronidase antigenic fragments recombinant protein of streptococcus pyogenes

Streptococcus pyogenes produce an extracellular hyaluronidase which is associated with the spread of the organism during infection. Enzyme hyaluronidase is capable of degrading hyaluronic acid. The aim of the present study was to clone and express antigenic regions of the hylA of S.pyogenes in Escherichia coli. The antigenic regions of hylA gene was inserted in pGEX-4T-1 vector. E. coli BL21 was transformed with hylA_pGEX-4T-1 and gene expression was induced by isopropyl-beta- Dthiogalactopyranoside (IPTG). The expressed protein was purified by affinity chromatography with GST sepharose resin. The integrity of the product was confirmed by western-blot analysis using sera of infected individual. Sera reactivity of infected individuals was further analyzed against the recombinant hyaluronidase protein. Our data shows production of recombinant hyaluronidase improved by pGEX4T-1 in Ecoli .

Optimization of physical and nutritional parameters for hyaluronidase production by <i> Streptococcus mitis</i>

The effect of some physical and nutritional parameters were studied for the optimum production of extracellular enzyme hyaluronidase employing Streptococcus mitis MTCC*2695 by submerged fermentation. The effects of initial pH, incubation temperature and time, inoculum level and age of inoculum were studied. The maximum enzymatic activity was obtained with an initial pH 5.8, incubation temperature 37°, incubation time for 48 h and inoculum level 6% with inoculum age 24 h. The effect of different carbon and nitrogen sources and antibiotics were studied. The results indicated that sucrose and ammonium chloride showed the highest enzymatic activity among various carbon and nitrogen sources. Antibiotic clarithromycin showed strong inhibitory effect on hyaluronidase production.

Role of Hyaluronidase in Subcutaneous Spread and Growth of Group A Streptococcus

Group A streptococcus (GAS) depends on a hyaluronic acid (HA) capsule to evade phagocytosis and to interact with epithelial cells. Paradoxically, GAS also produces hyaluronidase (Hyl), an enzyme that cleaves HA. A common assumption is that Hyl digests structurally identical HA in human tissue to promote bacterial spread. We inactivated the gene encoding extracellular hyaluronidase, hylA, in a clinical Hyl(+) isolate. Hyl(+) and an isogenic Hyl(-) mutant were injected subcutaneously into mice with or without high-molecular-weight dextran blue. The Hyl(-) strain produced small lesions with dye concentrated in close proximity. The Hyl(+) strain produced identical lesions, but the dye diffused subcutaneously. However, Hyl(+) bacteria were not isolated from unaffected skin stained by dye diffusion. Thus, Hyl digests tissue HA and facilitates spread of large molecules but is not sufficient to cause subcutaneous diffusion of bacteria or to affect lesion size. GAS capsule expression was assayed periodically during broth culture and was reduced in Hyl(+) strains relative to Hyl(-) strains at the onset and the end of active capsule synthesis but not during peak synthesis in mid-exponential phase. Thus, Hyl is not sufficiently active to remove capsule during peak synthesis. To demonstrate a possible nutritional role for Hyl, GAS was shown to grow with N-acetylglucosamine but not d-glucuronic acid (both components of HA) as a sole carbon source. However, only Hyl(+) strains could grow utilizing HA as a sole carbon source, suggesting that Hyl may permit the organism to utilize host HA or its own capsule as an energy source.

The presence of a novel extracellular hyaluronidase in squid cranial cartilage

A new type of hyaluronidase was isolated from squid cranial cartilage. The enzyme seems to be localised extracellularly, since it is extracted from the tissue by 0.5 M sodium acetate, pH 7.0, in the presence of proteinase inhibitors. Degradation studies suggest that the enzyme belongs to the family of endoglycosidases generating oligosaccharides of rather large size. The best activity of the enzyme was observed at pH 7.0 and 37 °C and the optimum buffer for digestion was 0.15 M Tris acetate. It is inactive in sodium phosphate, morpholine acetate and HEPES buffers. The enzyme degrades aggrecan, hyaluronan, chondroitin sulphate and oversulphated chondroitin sulphate.

Molecular characterization and allelic distribution of the phage-mediated hyaluronidase genes hylP andhylP2 among Group A streptococci from western Norway

Forty-two isolates of group A streptococcus from patients with invasive and non-invasive diseases in western Norway, belonging to the emm sequence types emml, emm3, emm6, emm22,emm28 , emm75 and emm78 were screened by PCR for the phage-mediated hyaluronidase genes hylP andhylP2 . The amplified genes were characterized by nucleotide sequencing and/or by PCR-RFLP, with the objective of looking for possible associations between alleles of these two genes and invasiveness. The hylP was amplified from all isolates and two main alleles were found hylP-emm3 in all emm3 isolates and hylP-emm6A in allemm6 isolates, the latter possibly generated by an intergenic recombination between hylP and hylP2. The isolates of the other sequence types had either of these two alleles, or both. Only 27 isolates gave amplicons of the appropriate size with the primers targeting hylP2. Sequencing of these amplicons showed two main types: one was similar to the published hylP2 and the other (hylP-emm6B) was probably a variant of hylP. PCR-RFLP revealed the presence of both hylP-emm6B and hylP2 in at least six of the emm6 isolates. The alleles of bothhylP and hylP2 seemed to have emm sequence type preferences. No association between invasiveness and specific phage-mediated hyaluronidase genes/alleles or the production of extracellular hyaluronidase was observed.

The extracellular hyaluronidase gene (hylA) of Streptococcus pyogenes.

Group A streptococci produce an extracellular hyaluronidase (hyaluronate lyase) which may be associated with the spread of the organism during infection. The gene for this hyaluronidase (hylA) encodes an 868 amino acid protein with a molecular size of 99636 Da. Cleavage of the proposed signal peptide results in an extracellular protein of 95941 Da. Comparison with other bacterial hyaluronidases indicates strong similarities to the genes from Streptococcus pneumoniae, Streptococcus agalactiae and Staphylococcus aureus. A region internal to the hylA gene was amplified from all 175 strains of Streptococcus pyogenes tested suggesting a widespread distribution of the gene.

The extracellular hyaluronidase gene ( hylA) of Streptococcus pyogenes

Group A streptococci produce an extracellular hyaluronidase (hyaluronate lyase) which may be associated with the spread of the organism during infection. The gene for this hyaluronidase ( hylA) encodes an 868 amino acid protein with a molecular size of 99 636 Da. Cleavage of the proposed signal peptide results in an extracellular protein of 95 941 Da. Comparison with other bacterial hyaluronidases indicates strong similarities to the genes from Streptococcus pneumoniae, Streptococcus agalactiae and Staphylococcus aureus. A region internal to the hylA gene was amplified from all 175 strains of Streptococcus pyogenes tested suggesting a widespread distribution of the gene.

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

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.

Hyaluronic Acid Synthesis Operon (has) Expression in Group A Streptococci

The has operon is composed of three genes, hasA, hasB, and hasC that encode hyaluronate synthase, UDP-glucose dehydrogenase, and presumptively UDP-glucose pyrophosphorylase, respectively. Expression of the has operon was shown to be required for the synthesis of the hyaluronic acid capsule in group A streptococci. Previous studies indicated that some group A and group C streptococcal strains produce the hyaluronic acid capsule, while others do not. In addition, it was observed that encapsulated strains cultured in stationary phase of growth lose the hyaluronic acid capsule. Therefore, the molecular mechanisms controlling the expression of the hyaluronic acid capsule in group A streptococci was investigated. In this study, it was determined that all encapsulated and unencapsulated strains of group A streptococci as well as encapsulated group C streptococci analyzed possess the has operon locus. The acapsular phenotype was accounted for by the absence of hyaluronate synthase activity in the membrane and not the production of extracellular hyaluronidase. A has operon mRNA transcript was not expressed by unencapsulated strains of group A streptococci, whereas encapsulated strains of group A streptococci grown to mid to late exponential phase produced the hyaluronate capsule, as well as has operon mRNA. However, as the streptococci entered the stationary phase of growth, they became acapsular and this was concomitant with the loss of has operon mRNA transcript. These results were confirmed by primer extension analyses of RNA isolated from encapsulated and unencapsulated strains of group A streptococci as well as RNA prepared from encapsulated strains cultured in exponential and stationary phases of growth. Thus, the loss of has operon mRNA in unencapsulated group A streptococci, as well as growth phase regulation occurs at the previously mapped has operon promoter. These data suggested that the synthesis of the hyaluronic acid capsule for group A streptococci may be controlled by transcriptional mechanisms.

Degradation of hyaluronate by Streptococcus intermedius strain UNS 35.

Streptococcus intermedius strain UNS 35, a brain abscess isolate, produced extracellular hyaluronidase when grown in brain heart infusion broth. Chemical assays with this enzyme indicated that hyaluronate depolymerisation resulted in the formation of carbohydrate moieties with N-acetylglucosamine at the reducing terminal and containing an unsaturated carbon-carbon double bond. The nature of the products of this hyaluronidase were investigated further by high-field (400 MHz) proton (1H) NMR spectroscopy. Treatment of hyaluronate with the enzyme resulted in a series of new, sharp resonances in spectra (acetamido methyl group singlets located at 2.03 and 2.07 ppm, sugar ring proton multiplets in the 3.5-4.2 ppm chemical shift range, and doublets at 5.16 and 5.87 ppm) characteristic of low-M(r) oligosaccharide species, predominantly those containing glucuronosyl residues with delta 4,5-carbon-carbon double bonds. Comparison of spectra acquired from hyaluronidase-treated samples with that of an authentic sample of 4-deoxy-L-threo-hex-4-enopyranosyluronic-acid-N-acetylglucosamine (delta UA GlcNAc) indicated that this disaccharide was a major product arising from the actions of this enzyme. When used in minimal media, hyaluronate supported growth of S. intermedius, with lactate as the major metabolic end-product.

Enzyme activities from eight small-sized oral spirochetes.

The purpose of the present investigation was to detect strains of small-sized oral spirochetes isolated from subgingival plaque for protease, peptidase, lipase, glycosidase, phosphatase, hyaluronidase and chondroitinsulfatase activities. The analyses were routinely carried out with cultures in the early stationary phase of growth after 4 days incubation. Both culture media and harvested spirochete cells were examined for the different enzyme activities. The enzymes were assayed by use of the API ZYM system, by p-nitroanilide derivatized peptides, and by hydrolyzing of mucopolysaccharides incorporated in solid bacterial medium. Relatively strong activities of trypsin-like enzymes, mainly bound to the cells, were observed in all strains. Similarly all strains showed acid phosphatases bound to the cells, too. Extracellular hyaluronidase- and chondroitinsulfatase activities were detected qualitatively in all strains after 7 days growth. The activities of the two mucopolysaccharide degrading enzymes almost disappeared after 10 subcultivations. Weak lipase (butyrate), higher lipase (caprylate), and weak phosphoamidase activities were observed in all cell pellets. No glycosidase activities were found. The observations are discussed by regarding the spirochetal enzymes as potential virulence factors for the development of marginal periodontitis.

Phase variation of hyaluronidase-producing peptostreptococci associated with periodontal disease.

Peptostreptococci isolated from diseased periodontal pockets produced large amounts of extracellular hyaluronidase throughout their growth cycle. Phase variants, obtained by re-streaking aged colonies of the wild types, had different colonial morphology and shorter generation times. These variants produced less hyaluronidase, and enzyme production occurred only during the early stationary phase.

Das vorkommen extrazellulärer stoffwechselprodukte bei streptokokken der gruppen C und G

Abstract 24 group C and 31 group G streptococcal strains were studied examining the production of extracellular nucleases, NADase, Hyaluronidase and streptolysin O. 5 mucous strains were characterized by diminished growth and lack of hyaluronidase and NADase-production. Regarding the other strains of group C, one half produced nuclease-activity whereas only one out of ten group G streptococci did. Group G streptococci were more active in the production of NADase and hyaluronidase than group C streptococci. The levels of streptolysin O in groups C and G are comparable.

Effect of subminimal inhibitory concentrations of penicillin on hyaluronidase production by group a streptococci

A sensitive dye-binding assay was employed to study the extracellular hyaluronidase produced by three penicillin-sensitive strains of group A streptococci belonging to M types 4, 24 and 28. Enzyme was produced by all strains at appreciable levels. Subminimal inhibitory concentrations of penicillin in growing cultures increased enzyme production by M types 4 and 24 strains. Growth of the M type 28 R strain with added antibiotic was accompanied by a reduced production of hyaluronidase.

Immunological properties of hyaluronidases associated with temperate bacteriophages of group A streptococci.

The antigenic relationships of hyaluronidases, bound and free, associated with temperate bacteriophages of group A streptococci were examined with antibody against purified whole phage and with antibody against phage-bound enzyme released by urea and purified to homogeneity. Studies performed by double diffusion in agar (ouchterlony) with antibody against the homologous purified enzyme from a temperate phage of a type 49 streptococcus indicated that the bound and free enzyme gave a single line of identity and that the free hyaluronidase activities in induced lysates of four strains of M type 49 streptococci were immunologically indistinguishable but different from the enzyme in induced lysates of a heterologous type. The four M type 49 strains were from widely different geographical or temporal sources and of different phage subtypes as determined by lyxic patterns. These findings were confirmed in studies that employed a functional assay of enzyme neutralization. An immunoglobulin preparation of antiserum against the purified enzyme as well as one against homologous purified whole phage neutralized the hyaluronidase activity produced by induction of the M type 49 strains and present either phage-bound or soluble in phage-free lysates. These immunoglobulin preparations had little effect on the hyaluronidase activities present in phage-lysates of other M types of group A streptococci. Inhibition of propagation of temperate phages by antibody against the purified phage hyaluronidase paralleled the neutralization of phage-associated enzyme activity by this antibody, indicating that antibody to the purified enzyme can inhibit phage infection. Antibody preparations against the purified phage-bound enzyme or against purified whole phage did not neutralize the extracellular hyaluronidase in the supernate of an uninduced culture of M type 4 streptococci. A human serum strongly inhibitory for the extracellular enzyme of this strain or on the purified phage enzyme from an M type 49 strain. The results support the view that the hyaluronidases associated with the temperate bacteriophages from various M types of group A streptococci do not share common antigenic determinants but that an immunological specificity exists that parallels the serologic specificity of the M protein of the host strains.

An investigation into hyaluronidase as a factor in the mechanism of tissue invasion by Endamoeba histolytica.

SummaryNeither intra- nor extracellular hyaluronidase was produced by E. histolytica in vitro when amebae were cultured aerobically or anaerobically. No specific hyaluronidase inhibitor was produced by the flora of any strains of ameba cultured in this laboratory. Production of hyaluronidase was not induced when cultures of amebae were seeded with hyaluronate. Amebae, isolated from liver abscesses in hamsters, failed to produce hyaluronidase under the conditions used.