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J Microbiol Methods. 2015 Dec;119:163-7. doi: 10.1016/j.mimet.2015.10.019. Epub 2015 Oct 28.

A new mass spectrometry based bioassay for the direct assessment of hyaluronidase activity and inhibition.

Author information

1
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA.
2
Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
3
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, USA. Electronic address: nbcech@uncg.edu.

Abstract

The development of drug resistance by bacterial pathogens is a growing threat. Drug resistant infections have high morbidity and mortality rates, and treatment of these infections is a major burden on the health care system. One potential strategy to prevent the development of drug resistance would be the application of therapeutic strategies that target bacterial virulence. Hyaluronidase is virulence factor that plays a role in the ability of Gram-positive bacteria such as Staphyloccus aureus and Streptococcus agalactiae to spread in tissue. As such, this enzyme could be a target for the development of future anti-virulence therapies. To facilitate the identification of hyaluronidase inhibitors, quantitative and reproducible assays of hyaluronidase activity are required. In the present study, we developed a new mass spectrometry based bioassay for this purpose. This assay directly measures the quantity of a degradation product (3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine) produced by the hyaluronidase enzyme. Validation parameters for the new assay are as follows: repeatability, <7%; intermediate precision, <10%; range, 0.78-50 μM; limit of detection, 0.29 μM; and limit of quantification, 0.78 μM. Using the new assay, the IC50 value for a published inhibitor of S. agalactiae hyaluronidase, ascorbic acyl 6-palmitate, was 8.0±1.0 μM. We also identified a new hyaluronidase inhibitor, n-cyclohexanecarbonylpentadecylamine, with an IC50 of 30.4±9.8 μM. In conclusion, we describe a new, direct, and reproducible method for assessing hyaluronidase activity using mass spectrometry that can facilitate the discovery of inhibitors.

KEYWORDS:

Bioassay; Hyaluronidase; Inhibitor; Mass spectrometry; Streptococcus agalactiae

PMID:
26519769
PMCID:
PMC4663139
DOI:
10.1016/j.mimet.2015.10.019
[Indexed for MEDLINE]
Free PMC Article

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