Novel role for human beta defensin-2 as a protease inhibitor in protecting HaCaT cells monolayer against S. aureus secreted proteases
Qureshi, Ayub Ahmad
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Atopic dermatitis (AD) patients’ exhibit increased susceptibility to viral and microbial infections, especially Staphylococcus aureus (S. aureus). Evidence suggests that S. aureus secreted proteases are responsible for increased breakdown of junctional and desmosomal adhesions, that help to maintain skin barrier function. I therefore examined the effect of S. aureus secreted exotoxins (proteases), on an intact monolayer of HaCaT cells, a keratinocyte cell line. Damage to the HaCaT monolayer was observed as a loss in tight junction and desmosomal contacts, which were visualized throughout the monolayer when I stained them (after staining) with specific antibodies. I quantitated those effects by Western blotting and by measuring transepidermal electrical resistance (TEER). S. aureus secreted serine protease (V8) was the key agent responsible for the gross effects on HaCaT monolayers. I also discovered that pre-treatment of HaCaTs cells with both IL-1β and LTA protected monolayer from S. aureus protease and this could be attributed to increase secretion of a novel peptide. I further demonstrate that hBD2 a novel peptide act as an ant-protease that inhibit both V8 & staphopain B. Importantly, novel peptide could reduce the deleterious effect of S. aureus conditioned media, which contain higher concentration of bothV8 and staphopain B proteases. For further specification I compared S.aureus parent strain 8325-4 and their mutant strains L17 (SspB), L22 (SspA) and (L27) Scpa and SH1000. S. aureus proteases effects were observed in the presence or absence of recombinant, synthetic and linear scrambled novel peptides. The protective role played by skin commensal was tested by heat killing S. Epidermidis. I found HaCaT monolayer was protected from S. aureus secreted proteases when pretreated with heat killed S. Epidermidis. Thus, I concluded that V8 protease may be the most important factor involved in breaking skin barrier of atopic dermatitis patients.