Abstract
Hyaluronidase enzyme (HysA) is an extracellular enzyme that is considered to be an important virulence factor for Staphylococcus aureus. We screened the production of HysA enzyme in the spent media of Egyptian clinical isolates (32 isolates) via phenotypic plate assay. We found that 75% of the isolates (24 isolates) were able to produce HysA enzyme. We designed primers for qPCR analysis of hysA mRNA expression that was derived from the alignment of hysA gene sequences of 41 strains of S. aureus. The designed primers could be used for the amplification of hysA in 79.2% of the isolates (19 isolates) that were positive for HysA production as demonstrated by phenotypic plate assay. A significant positive correlation, as indicated by Pearson correlation analysis (r = 0.84 at P < 0.001), was found between phenotypic plate assay and qPCR of mRNA expression of hysA in the investigated isolates of S. aureus. In conclusion, we analyzed for the first time hysA mRNA expression via qPCR in S. aureus. Additionally, our work showed a good agreement between the phenotypic assay of HysA production via plate assay and hysA expression in S. aureus. The qPCR analysis of this study could be used as a more reliable quantitative method for hysA expression analysis particularly in infected animal models of S. aureus.
Highlights
For Quantitative Real-Time-PCR (qPCR) analysis of hysA mRNA expression, the RNA was isolated from the subcultures of the HysA producing isolates of S. aureus after 3 h cultivation
The fold of change (FC) in expression of hysA in different clinical isolates relative to hysA expression by Newman is shown as mean ± SD of three independent experiments (Table 2)
We established for the first time a protocol and primers for the qPCR analysis of the hysA mRNA expression in S. aureus
Summary
HA is a high molecular-weight polymer composed of alternating units of D-glucuronic acid and N-acetylglucosamine [9] This polymer is a major component of the extracellular matrix of human tissues and it is abundant in the skin, skeletal tissue, umbilical cord, lungs, heart valves, brain, blood, liver and a number of other tissues [10]. It plays important roles for inflammation, water homeostasis, in addition to being an immune regulator [11] [12]. Hyaluronidases have been found to be virulence factors that are involved in the invasion, and penetration of tissues in many of Gram positive bacteria [9]
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