The anti-biofilm activity of D-LL-37 was very similar to that of LL-37, showing ~40% inhibition at 10 μg/ml (Figure Fer-1 2d). In other experiments, D-LL-37 at 26 μg/ml was able to inhibit as much as ~80% of the biofilm formation (data not shown). This strong anti-biofilm effect of D-LL-37 was surprising, as it was categorized as an ineffective AMP (Table 2), and was 10 fold less effective than LL-37. This result suggests that anti-microbial activity and anti-biofilm activity of peptides may be due to different mechanisms. For example, the anti-microbial activity could be direct physical interaction of the peptide on the bacterial

membrane, while anti-biofilm could be mediated by alteration of bacterial gene expression [32]. The scrambled version of LL-37, having the same charge and net amino-acid composition as LL-37, but lacking significant helical character, showed no inhibition of biofilm formation at any concentration tested (Figure 2e), thus demonstrating sequence specificity of the anti-biofilm effect. 2.4 D- and L-LL-37 effect S. aureus biofilm attachment The attachment of Staphylococcus spp. to solid surfaces is largely seen as an essential step in the formation of biofilm. Since most of the peptides tested in our biofilm TPCA-1 mw assays were capable of inhibiting biofilm formation (selleck except for scrambled

LL-37), we investigated a possible mechanism for this action. We incubated scrambled LL-37 (negative control), LL-37, D-LL-37, NA-CATH, and NA-CATH:ATRA1-ATRA1 peptides with S. aureus in a 1 hr attachment assay at peptide concentrations of 1 ug/ml, examining for the initial adherence to the wells of the 96 well tissue-culture treated plate [32]. For LL-37 and D-LL-37, the measured attachment to the polystyrene wells was significantly

decreased (P < 0.01, Student's t test) (Figure 3). Scrambled LL-37, NA-CATH, and NA-CATH:ATRA1-ATRA1 did not decrease S. aureus adherence. Thus, both D- and L-forms of the LL-37 peptide were equally effective at inhibiting attachment, which may contribute to their inhibition of biofilm formation. However, the most effective anti-biofilm peptide, NA-CATH:ATRA1-ATRA1 did not inhibit attachment, suggesting that this peptide inhibits biofilm formation through a different mechanism. Figure 3 Attachment assay of S. aureus in the presence of peptide. We tested Fluorouracil scrambled LL-37 (negative control), LL-37, D-LL-37, NA-CATH, and NA-CATH:ATRA1-ATRA1 against S. aureus (1 h, 37°C) at 1 μg/ml, only allowing for the initial adherence to the wells. For LL-37 and D-LL-37, the measured attachment to the polypropylene wells was significantly decreased (P < 0.01, Student’s t test). Scrambled LL-37, NA-CATH, and NA-CATH:ATRA1-ATRA1 did not decrease S. aureus adherence. 2.5 CD Spectral analysis of peptides Circular dichroism (CD) spectra of the peptides were obtained. Pronounced dichroic minima at 222 and 208 nm are traits of helical peptides (Figure 4a).