ELISA for Alpha-hemolysin (Hla) in Methicilin-resistant Staphylococcus aureus (MRSA)

Khodaverdian, Varandt Y.; Shoham, Menachem

doi:10.21769/bioprotoc.1118

Cited by 1 publication

(3 citation statements)

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Section: Effects Of Tf3 On Hla-induced Skin Barrier Disruptionsupporting

confidence: 45%

“…To explore this matter, we co-treated human primary keratinocytes with native Hla and either 25 μg/ml or 50 μg/ml concentration of TF3, checking the status of E-cadherin, ZO-1, and claudin after 24h. We noticed that claudin is not affected by Hla or TF3, and others have reported that Hla also does not negatively affect β-catenin level [ 55 , 56 ]. However, we noticed about 99% decrease in E-cadherin levels after treatment with native Hla only, in contrast to either co-treatment of native Hla with both concentrations of TF3, or controls (i.e., untreated cells or cells treated with TF3 only), which showed no significant changes in its levels.…”

Section: Resultsmentioning

confidence: 51%

“…Hla was quantified using ELISA assay performed according to previous report [ 55 ]. Briefly, 96-well plates were coated with 1 mg/ml Hla antibody and incubated overnight at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of α-hemolysin activity of Staphylococcus aureus by theaflavin 3,3’-digallate

Goc,

Sumera,

Rath

et al. 2023

PLoS ONE

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The ongoing rise in antibiotic resistance, and a waning of the introduction of new antibiotics, has resulted in limited treatment options for bacterial infections, including these caused by methicillin-resistant Staphylococcus aureus, leaving the world in a post-antibiotic era. Here, we set out to examine mechanisms by which theaflavin 3,3’-digallate (TF3) might act as an anti-hemolytic compound. In the presented study, we found that TF3 has weak bacteriostatic and bactericidal effects on Staphylococcus aureus, and strong inhibitory effect towards the hemolytic activity of its α-hemolysin (Hla) including its production and secretion. A supportive SPR assay reinforced these results and further revealed binding of TF3 to Hla with KD = 4.57×10−5 M. Interestingly, TF3 was also able to protect human primary keratinocytes from Hla-induced cell death, being at the same time non-toxic for them. Further analysis of TF3 properties revealed that TF3 blocked Hla-prompting immune reaction by inhibiting production and secretion of IL1β, IL6, and TNFα in vitro and in vivo, through affecting NFκB activity. Additionally, we observed that TF3 also markedly attenuated S. aureus-induced barrier disruption, by inhibiting Hla-triggered E-cadherin and ZO-1 impairment. Overall, by blocking activity of Hla, TF3 subsequently subdued the inflammation and protected the epithelial barrier, which is considered as beneficial to relieving skin injury.

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Section: Effects Of Tf3 On Hla-induced Skin Barrier Disruptionsupporting

confidence: 45%

Section: Resultsmentioning

confidence: 51%