Anti-Fungal Activity of Cathelicidins and their Potential Role in Candida albicans Skin Infection  Belén López-García, Phillip H.A. Lee, Kenshi Yamasaki, Richard L. Gallo  Journal of Investigative Dermatology  Volume 125, Issue 1, Pages 108-115 (July 2005) DOI: 10.1111/j.0022-202X.2005.23713.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 In vitro growth inhibition of Candida albicans by cathelicidin peptides. (A) Cathelicidin anti-fungal activity was assessed using a broth microdilution assay in 20% mDixon pH 5.5. 2.5-5 × 104 colony-forming units per mL yeast was incubated for 48 h with increasing concentrations of peptides LL-37 (triangle), mCRAMP (square), and PR-39 (circle). (B) LL-37 activity was assessed in different conditions: 20% mDixon, pH 4.5, 20% mDixon, pH 5.5, 20% mDixon, pH 7.2, 100% mDixon, and 100% Sabouraud dextrose broth (SDB). C. albicans was incubated in the absence or presence of 15 μM of LL-37 and the percentage of growth was calculated as the ratio between A600 with peptide/A600 without peptide. Data represent the mean±(SD) of triplicate determinations from a single experiment representative of at least two experiments. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 In vitro fungicidal activity of cathelicidin peptides against Candida albicans. Yeast was incubated with different concentrations of peptides LL-37 (black triangle), mCRAMP (black square), PR-39 (black circle), KS-30 (white circle), and RK-31 (white diamond) in 20% mDixon medium (pH 5.5). After 24 h, yeast was plated onto mDixon agar plates to count cells. Initial number of yeast was 2.5-5 × 104 colony-forming units (CFU) per mL. Data represent the mean±(SD) of triplicate determinations from a single experiment representative of at least two experiments. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Kinetics of cathelicidin activity against Candida albicans. Yeast (106 colony-forming units per mL, initial number) was incubated with 0 μM (black diamond), 25 μM LL-37 (black square), or 8 μM RK-31 (white triangle). (A) Percent of remaining cells was calculated as the colony-forming units per milliliter of C. albicans after incubation with peptide compared with colony-forming units per milliliter of the yeast without peptide, determined at the indicated time points. (B) Percent of permeable cells determined by uptake of fluorescent Sytox green. Data represent the mean of triplicate determinations from a single experiment representative of two experiments. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunohistochemical analysis of mCRAMP expression in Candida albicans-infected mouse skin. (A) Normal uninfected skin and (B–D) C. albicans-infected skin. (A–C) Stained with antibody to mCRAMP. (D) Stained with rabbit pre-immune serum used as a control. Brown staining in (B) shows increased expression of mCRAMP in C. albicans-infected skin compared with uninfected skin in (A). (C) High power (scale bar=10 μm) of dermis from (B) shows evidence of multiple C. albicans organisms indicated by arrows and mCRAMP containing neutrophils (hematoxylin counterstain; scale bar=40 μm in (A, B, D)). Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Contribution of cathelicidin to inhibition of Candida albicans in mouse blood and skin. (A) Group A Streptococcus (GAS) and C. albicans killing activity of whole mouse blood. Bacteria and yeast were incubated 1 and 4 h, respectively, in fresh blood from wild-type (black bars) and mCRAMP knockout mice (white bars). Data are expressed as the mean growth index (the ratio of colony-forming units (CFU) per milliliter after incubation in blood to initial colony-forming units per milliliter)±SD of three mice tested in each group. GAS data are statistically different (Student t test, p-value≤0.001). (B) C. albicans cultured from tissue biopsies of wild-type and mCRAMP knockout mice 3 d after subcutaneous injection of 150 μL of 108 CFU per mL C. albicans. Data are shown as mean values (±SD) of colony-forming units per milliliter. Data represent the mean of triplicate determinations from a single experiment of two experiments. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Detection, dependence on proteolysis, and potency of cathelicidin peptides in sweat. (A) Analysis of normal human sweat by reverse-phase high-pressure liquid chromatography (RP-HPLC). Elution positions of cathelicidin peptides are indicated and have been confirmed by independent Maldi-MS analysis. Peptide concentration estimated based on comparison of peak areas with known amounts of synthetic peptide standards. (B) 2.5-5 × 104 CFU per mL of C. albicans was incubated for 24 h in normal sweat (1 ×), sweat concentrated by evaporation 4 ×, in salt solution that mimics the ionic composition of 4 × sweat (SwB). Also, 2.5 × 5 × 104 CFU per mL of C. albicans was incubated for 24 h in 1 × sweat with 25 μM LL-37 or 8 μM RK-31 in the absence or presence of protease inhibitor cocktail. Data are shown as the mean value (±SD) of colony-forming units per milliliter and represent the mean of triplicate determinations from a single experiment representative of three experiments. *Statistically different compared with dilute sweat alone (Student t test, p-value<0.05). (C) 2.5-5 × 104 CFU per mL of C. albicans was incubated for 24 h with different concentrations of peptides LL-37 (black triangle), KS-30 (white circle), and RK-31 (white diamond) in salt solution that mimics the ionic composition of 1 × sweat (SwB). Data represent the mean±(SD) of triplicate determinations from a single experiment. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of increasing salt concentration on activity of cathelicidin peptides. Cathelicidin peptides RK-31, KS-30, and KR-20 were combined and added to sweat buffer (A), or sweat (B) at initial concentrations of 0 μM (black bars), 2 μM (gray bars), or 10 μM (white bars). To mimic the effect of evaporation on the skin surface, each sample was then concentrated by lyophilization and redissolved in water such that the final concentration was 1 ×, 2 ×, and 5 × of the original. C. albicans was incubated with each sample for 6 h. Data show the mean±(SD) of triplicate determinations from a single experiment representative of three experiments. Journal of Investigative Dermatology 2005 125, 108-115DOI: (10.1111/j.0022-202X.2005.23713.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions