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Anti-Fungal Activity of Cathelicidins and their Potential Role in Candida albicans Skin Infection
See related Commentaries on pages v, viii and x
Anti-Fungal Activity of Cathelicidins and their Potential Role in Candida albicans Skin Infection Bele´n Lo´pez-Garcı´a, Phillip H. A. Lee, Kenshi Yamasaki, and Richard L. Gallo Division of Dermatology, University of California San Diego, and VA San Diego Healthcare Center, San Diego, California, USA
Cathelicidins have broad anti-microbial capacity and are important for host defense against skin infections by some bacterial and viral pathogens. This study investigated the activity of cathelicidins against Candida albicans. The human cathelicidin LL-37, and mouse cathelicidin mCRAMP, killed C. albicans, but this fungicidal activity was dependent on culture conditions. Evaluation of the fungal membrane by fluorescent dye penetration after incubation with cathelicidins correlated membrane permeabilization and inhibition of fungal growth. Anti-fungal assays carried out in an ionic environment that mimicked human sweat and with the processed forms of cathelicidin such as are present in sweat found that the cleavage of LL-37 to forms such as RK-31 conferred additional activity against C. albicans. C. albicans also induced an increase in the expression of cathelicidin in mouse skin, but this induction did not confer systemic or subcutaneous resistance as mCRAMP-deficient mice were not more susceptible to C. albicans in blood-killing assays or in an intradermal infection model. Therefore, cathelicidins appear active against C. albicans, but may be most effective as a superficial barrier to infection.
Key words: anti-microbial peptides/defensins/infection/innate immunity/LL-37/mCRAMP/sweat J Invest Dermatol 125:108 – 115, 2005
Cationic peptides play important roles in the defense systems of many organisms from insects to humans (Rao, 1995; Zasloff, 2002) and have been isolated from diverse organisms (Andreu and Rivas, 1998; Hancock and Chapple, 1999). A number of these have received attention because of their low toxicity against mammalian cells (Steiner et al, 1988). In particular, mammalian skin and other epithelial surfaces are a major producer of functional cathelicidins (Gallo et al, 1994; Frohm et al, 1997; Bals et al, 1998). LL-37 and mCRAMP belong to the cathelicidin family and are present in humans and mice, respectively. They are constitutively expressed, or can be induced, at the interface with the external environment where they act together with other anti-microbial molecules such as defensins (Harder et al, 1997), select proteases (Shafer et al, 1991), cytokines (Wu et al, 2000; Erdag and Morgan, 2002), and protease inhibitors (Bjorck et al, 1989; Zaiou et al, 2003). Cathelicidins have been shown to contribute to defense against invasive bacterial infection by Group A Streptococcus (GAS) (Dorschner et al, 2001; Nizet et al, 2001) and vaccinia virus (Howell et al, 2004). Whereas cathelicidins are typically expressed at low levels in normal keratinocytes, they are induced during inflammation and increase locally because of release from neutrophils and mast cells (Frohm et al, 1997). Recently, it has been demonstrated that cathelicidins are produced by the eccrine apparatus and secreted into human sweat (Schittek et al, 2001; Murakami
et al, 2002). After secretion onto the skin surface, LL-37 is processed by a serine protease-dependent mechanism into multiple distinct anti-microbial peptides, such as RK-31 or KS-30 (Murakami et al, 2004) that are more potent compared with the uncleaved mature peptide. The function of cathelicidins as a natural barrier to fungal infections of the skin has not been well characterized. This study characterized the anti-fungal activity of the cathelicidins against Candida albicans and studied the possible role of this family of peptides in skin fungal infection.
Results Anti-fungal activity of cathelicidins The anti-fungal activity of cathelicidins was measured against C. albicans in low salt 20% mDixon medium (pH 5.5). Two similar ahelical cathelicidin homologues, human (LL-37) and mouse (mCRAMP), showed similar activity with delay or complete inhibition of growth at an minimum inhibitory concentration (MIC) range of 15–20 mM, whereas the linear porcine cathelicidin PR-39 was inactive (Fig 1A). Activities of the human and mouse cathelicidins were pH dependent but not affected by change in temperature from 251C to 371C. Growth inhibition of C. albicans in the presence of 15 mM of LL-37 was observed at acidic and neutral pH values, with growth of 19%, 21%, and 60% at pH 4.5, 5.5, and 7.2, respectively (Fig 1B). Growth in the absence of peptide under identical conditions was considered 100% growth. Processed forms of LL-37 have been shown to kill C. albicans in vitro (Murakami et al, 2004). To compare the activity of these against PR-39, LL-37, and mCRAMP,
Abbreviations: CFU, colony-forming units; GAS, Group A Streptococcus; HPLC, high-pressure liquid chromatography; SG, Sytox green; SwB, sweat buffer
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
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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 2.55 104 colony-forming units (CFU) per mL. Data represent the mean (SD) of triplicate determinations from a single experiment representative of at least two experiments.
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. 2.55 104 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 mM 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.
fungicidal activity was measured in 20% mDixon, pH 5.5 (Fig 2). These results suggest that the shorter processed forms of LL-37 present at the skin surface (RK-31 and KS30) have enhanced anti-fungal effect against the yeast compared with forms of cathelicidins released by neutrophils such as mCRAMP and LL-37 (Fig 2). LL-37 and RK-31 permeabilize C. albicans To evaluate the kinetics and mechanism of killing of C. albicans by cathelicidins, the survival and permeability of the yeast were investigated in the presence of LL-37 or RK-31 (Fig 3). A higher initial concentration of yeast was used in these experiments compared with Fig 2. At this initial C. albicans load (106 colony-forming units (CFU) per mL), 25 mM LL-37 had less fungicidal activity than at (2.5–5 104 CFU per mL), whereas 8 mM RK-31 continued to kill approximately 90% of the initial inoculum within 15 min (Fig 3A). To determine fungal membrane integrity simultaneously, an assay based on the uptake of the fluorescent dye Sytox green (SG) was used (Fig 3B). SG does not penetrate live eukaryotic or prokaryotic cell membranes or walls but binding to nucleic acids results in a 4500-fold enhancement in its fluorescence emission, thus enabling sensitive detection of a disruption in the outer membrane (Roth et al, 1997; Thevissen et al, 1999). Incubation of yeast cells with 25 mM LL-37
Figure 3 Kinetics of cathelicidin activity against Candida albicans. Yeast (106 colony-forming units per mL, initial number) was incubated with 0 mM (black diamond), 25 mM LL-37 (black square), or 8 mM RK-31 (white triangle). (A) Percent of remaining cells was calculated as the colonyforming 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.
or 8 mM RK-31 allowed rapid uptake of SG in both cases (Fig 3B). After 5 min of incubation, LL-37 and RK-31 permeabilized more than 50% of the cells versus 12% permeable cells observed in samples without peptide. These results indicate that the kinetics of permeabilization correlated with the fungicidal effect of RK-31 and LL-37. Candida skin infection increases cathelicidin expression In humans and mice, cathelicidins are not easily
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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 ¼ 10 mm) of dermis from (B) shows evidence of multiple C. albicans organisms indicated by arrows and mCRAMP containing neutrophils (hematoxylin counterstain; scale bar ¼ 40 mm in (A, B, D)).
detectable in normal adult skin, but are usually abundantly expressed after inflammation or injury (Frohm et al, 1997; Dorschner et al, 2001). To determine the expression after fungal infection, mice were subcutaneously infected with C. albicans and tissues were sampled. Immunohistochemical analysis showed that mCRAMP expression was higher in infected tissue (Fig 4B and C) compared with expression in healthy tissue (Fig 4A). mCRAMP appeared to be induced specifically at areas of C. albicans involvement. This induction of mCRAMP protein expression was confirmed by western blot (data not shown). Control sections stained with rabbit pre-immune sera demonstrated that the staining pattern seen was not because of endogenous peroxidase activity (Fig 4D).
Role of cathelicidins in C. albicans infection in vivo To explore whether cathelicidins are responsible for controlling C. albicans infection in mice, two complementary experiments were carried out. Using blood from mice deficient for mCRAMP (Cnlp/), an in vitro killing assay was carried out to determine fungicidal activity against C. albicans. No statistical differences were seen between the growth of the yeast in blood from wild-type versus knockout Cnlp/ mice (Fig 5A). As reported previously, however, a difference was observed with GAS (Nizet et al, 2001). Next, subcutaneous skin infections of C. albicans were carried out in wildtype and Cnlp/ mice. Three days after inoculation of yeast, lesions were biopsied and colony counts were per-
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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-valuep0.001). (B) C. albicans cultured from tissue biopsies of wild-type and mCRAMP knockout mice 3 d after subcutaneous injection of 150 mL 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.
formed. Colony-forming units per milliliter were similar from knockout and wild-type mice (Fig 5B). Processing of LL-37 in sweat increases activity against C. albicans The apparent discordant results between killing in vitro and our inability to detect a contribution for cathelicidins in mouse blood or subcutaneous injections suggested that cathelicidins may only be active against C. albicans on the skin surface. Since the anti-microbial activity of cathelicidins is dependent on ionic and serum components, and can be neutralized in ionic environments containing high concentrations of NaCl (Bals et al, 1998; Turner et al, 1998; Zasloff, 2002), we next evaluated whether cathelicidins were active in the ionic environment of the human skin surface, that of sweat. First, the abundance of cathelicidin peptides in their processed form compared with LL-37 was measured. Human sweat was collected, analyzed by high-pressure liquid chromatography (HPLC), and the mass of fractions eluting at the expected site for KS-30 and LL-37 was confirmed by mass spectrometry. This analysis yielded an approximate concentration of 1.165 mM for KS-30/RK-31 and 0.013 mM of LL-37 in normal human sweat (Fig 6A). The human sweat was next analyzed for its anti-Candida activity before and after concentration by evaporation.
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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 mM (black bars), 2 mM (gray bars), or 10 mM (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.
Figure 6 Detection, dependence on proteolysis, and potency of cathelicidin peptides in sweat. (A) Analysis of normal human sweat by reversephase 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) 2.55 104 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 mM LL-37 or 8 mM 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-valueo0.05). (C) 2.55 104 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.
Following partial evaporation to reduce the volume 4-fold, C. albicans growth was inhibited by the sweat concentrate containing a complex mixture of cathelicidins and other potential anti-microbials (Murakami et al, 2002) (Fig 6B). This inhibitory effect of concentrated sweat was not because of concentrated salt content, as a salt buffer mimicking sweat (SwB) and concentrated 4 had no effect on growth (Fig 6B). Addition of 25 mM LL-37 to 1 sweat inhibited fungal growth, but this inhibition appeared to be the consequence of protease activity in the natural sweat
solution as the anti-fungal activity of LL-37 was blocked in the presence of protease inhibitors. Conversely, the antifungal activity of 8 mM RK-31 in sweat was not blocked by protease inhibitors. The anti-fungal activity of LL-37, KS-30, and RK-31 was further analyzed in sweat buffer (SwB) solution to directly determine the function of these peptides in the sweat ionic environment without complication of native proteases or as yet undefined inhibitory factors present in normal human sweat. In buffer containing 40 mM NaCl, 10 mM KCl, 1 mM CaCl2, and 1mM MgCl2, the derived peptides KS-30 and RK-31 were fungicidal against C. albicans, whereas LL-37 in the absence of proteolytic processing was not active (Fig 6C). Since the buffer conditions of C. albicans growth strongly influence the apparent activity of cathelicidin peptides, additional experiments were performed to mimic the effects of sweat evaporation at the skin surface. When added to the SwB solution, concentration by a factor of 5 or greater decreased the relative fungicidal activity (Fig 7A), thus suggesting that by itself the cathelicidin peptides may be inactivated in high ionic strength solutions. When cathelicidin peptides were added to normal sweat concentrated by 2 or 5 (Fig 7B), the anti-fungal activity of the concentrated sweat was greater than any additional activity detectable by supplementing with synthetic peptides.
Discussion Little is known about the contribution of naturally occurring anti-microbial peptides to defense against fungal infection.
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In this study, we characterized the anti-fungal activity of human and mice cathelicidin peptides. The form of these peptides found in human neutrophils (LL-37) or mouse granulocytes (mCRAMP) was effective against C. albicans in 20% mDixon medium with an MIC of 15–20 mM and minimal fungicidal concentration (MFC) of 25–30 mM. This activity was pH and growth media dependent. As previously published (Marr et al, 1999), pH is a critical factor in antifungal susceptibility testing. Using an acidic pH of 4–5 rather than neutral pH decreased the MIC of LL-37 against C. albicans, an effect seen with other anti-fungal agents (Rogers and Galgiani, 1986; McIntyre and Galgiani, 1989). Also, comparison of Figs 2 and 6B shows that the MFC of cathelicidin peptides is greater in salt-containing solutions than in low-salt Dixon media. Thus, conclusions regarding cathelicidin activity against C. albicans must be qualified by recognition of the assay conditions, a variable that is also true for other anti-microbial peptides and other target organisms including Gram and Gram þ bacteria. Importantly, the activity of cathelicidin against C. albicans could be demonstrated by the direct addition of the peptides in natural human sweat, and human sweat itself becomes anti-fungal once concentrated such as would normally occur during evaporation. Thus, the most superficial topical environment of human skin contains anti-fungal activity that may be partly because of the contribution of cathelicidins. Several studies have addressed the mode of action of anti-microbial peptides (Oren and Shai, 1998; Epand and Vogel, 1999; Shai et al, 2001; Zasloff, 2002; Lo´pez-Garcı´a et al, 2004). Membrane permeabilization drives the anti-microbial activity of most cationic and amphipathic peptides including LL-37 and mCRAMP. Our experiments using fluorescent SG indicate that the perturbation of the yeast membrane is a step in the anti-fungal activity of cathelicidins. Both eukaryotic and bacterial cells can be made transiently permeable with retention of viability; thus, permeability alone does not predict activity (Shapiro, 2001). Our data, however, support a good correlation between both permeability and anti-fungal activity for LL-37 and RK-31 on C. albicans (Fig 3). Other factors may also modulate the activity and specificity of peptide anti-microbial activity. For example, the cathelicidin PR-39 affects DNA and protein synthesis (Boman et al, 1993) and is a non-competitive and reversible inhibitor of the proteasome function (Gaczynska et al, 2003) but does not immediately disrupt bacterial membranes. Moreover, LL-37 can neutralize endotoxin, and has chemoattractive effects on leukocytes, which could provide additional mechanisms for protection independent of direct microbial killing (Larrick et al, 1995; Agerberth et al, 2000; De et al, 2000). Together, these observations support a role for membrane permeabilization in the activity of cathelicidins against C. albicans, but do not conclusively prove that this is the sole mechanism for cell killing. Injection of C. albicans into mouse skin indicated that cathelicidin is induced in the skin in response to C. albicans infection, but this induction did not confer subcutaneous resistance, as mCRAMP-deficient mice were not more susceptible to C. albicans in a subcutaneous model. Unlike Streptococcus pyogenes, blastospores of C. albicans grew equally well in blood from wild-type and Cnlp/ mice. This lack of response likely reflects several differences between
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S. pyogenes and C. albicans but may also several conditions in the subcutaneous environment that act to inhibit cathelicidin function. These include higher pH and concentrations of cations in vivo than in the fungal culture conditions and/or inactivation of peptide by interaction with high-molecularweight serum proteins (Wang et al, 1998). Susceptibility to C. albicans infection may also vary with the strain of mouse used (Hay et al, 1983). Given the results observed with mice and in mouse blood, these findings suggested that cathelicidins may not be involved in protection against C. albicans infection once it has penetrated the skin surface. An alternative role for cathelicidins in fungal infection may be to provide a superficial barrier against proliferation and potential fungal invasion. Our data show that cathelicidin peptides have activity against C. albicans when added to normal human sweat, or in ionic environments that mimic sweat. But LL-37 was poorly active in this environment without proteolytic processing. In this paper, we estimated that the concentration of these processed forms (KS-30 or RK31) and LL-37 is 1.165 and 0.013 mM, respectively (Fig 6A). These concentrations are likely a large underestimate of the actual abundance of cathelicidins at the skin surface. Cationic amphipathic peptides such as the human cathelicidins will partition into lipid components and bind anionic molecules and will not partition completely into the soluble sweat fraction recovered. But although the actual concentrations may be an underestimate, it appears that the proportion of LL-37 related to forms such as RK-31 is low. LL-37 is a minor form of human cathelicidin on the skin surface. The actual contribution of the human cathelicidins KS-30 and RK-31 to topical defense against C. albicans growth is difficult to determine. This data confirm that these peptides are present, and show activity against C. albicans under select growth conditions. Interestingly, RK-31 shows higher activity than KS-30 in higher ionic strength media (SwB, Fig 6C) but lesser activity in the low ionic strength medium (20% mDixon, Fig 2). This suggests that KS-30 activity may be more sensitive to inactivation by salt than RK-31. In SwB solutions alone, the amount of RK-31 required to completely kill C. albicans was greater than the amount of RK-31 estimated in normal sweat. Upon addition of 8 mM peptide to normal sweat, however, inhibitory activity was detectable. Activity at 8 mM in the complex mixture of normal sweat compared with 25 mM in SwB alone suggests that these peptides are most likely to be effective in combination with other factors, including other anti-microbial peptides. In summary, our findings show that naturally produced cathelicidins are present at sites of fungal infection and may form a barrier to C. albicans infection. Superficial infection with C. albicans is a common dermatological condition that is exacerbated by several factors including host immune status, location, and medications. In humans, abnormal production or an inability to appropriately increase cathelicidin expression following skin inflammation has been associated with disease (Frohm et al, 1997; Dorschner et al, 2001; Conner et al, 2002; Marchini et al, 2002; Ong et al, 2002; Heilborn et al, 2003). The observations made in this study suggest that unlike bacterial infections with S. pyogenes, the relevance of cathelicidins to C. albicans infection is more likely to be important at the skin surface. Understanding such interactions with innate immune effector
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molecules such as cathelicidins may explain the pathophysiology of common fungal skin diseases and offer alternative therapeutic approaches to conventional anti-fungal therapy. Materials and Methods Peptides All peptides were prepared by Synpep (Dublin, Oregon), purified by HPLC (95% purity), and identity confirmed by mass spectrometry. A 10 stock solution was prepared in H2O for each peptide. Media Primary Candida culture medium was a modified Dixon (mDixon) prepared in the laboratory from 4% malt extract (Fluka Biochemika, Steinheim, Germany), 0.6% Bacto Peptone (Becton, Dickinson and Company, Sparks, Maryland), 1% glucose (SigmaAldrich, St Louis, Missouri), and 1% Tween-80 (Sigma-Aldrich). The pH of this medium was 5.0 0.1. For anti-microbial assays, 20% mDixon medium in 1 mM sodium phosphate buffer pH 7 containing 16 mg per mL chloramphenicol was used. The final pH of the mixture was 5.5. Our 20% mDixon medium contained 5.0 mM Na þ , 1.6 mM K þ , 4 mg per dL phosphate, and undetectable amounts of Ca2 þ , Mg2 þ , and Cl. Microorganism C. albicans American Type Cultures Collection 14053 was cultured on mDixon agar medium (mDixon broth þ 1.5% agar). The day before use, colonies were transferred into mDixon broth medium and grown overnight at 371C with shaking. The turbidity of each suspension was adjusted to OD600 of 0.01 (corresponding to 2.5–5 105 CFU per mL).
In vitro anti-fungal activity assays The susceptibility of C. albicans was tested by broth microdilution assay. Yeast was grown on sterile 96-well microtiter plates (Corning, Corning, New York) in a final volume of 100 mL. The assay mixture contained 2.5–5 104 CFU per mL in 90 mL of 20% mDixon medium (final concentration), 10 mL of a 10 stock solutions of each synthetic peptide was added to yeast and incubated at 25 or 371C for initial determination of growth by absorbance at A600 with a Spectra max PLUS 384 (Molecular Devices, Sunnyvale, California) over 2 d. In all the experiments, the blank mean A600 value from one row of mock inoculations was subtracted from each well A600 measurement, and the mean and SD were calculated for each treatment. The MIC was defined as the lowest peptide concentration that showed no growth at the end of the experiment (usually, after 2 d of incubation). After 24 h of incubation, aliquots of each yeast–peptide combination were plated in mDixon agar plates in order to determine the MFC values. In the kinetic assays, the colony-forming units per milliliter was determined by plating aliquots of each yeast–peptide combination at different times. The plates were incubated at 371C for 1 d and the colonies were counted. Membrane permeabilization assay Fungal membrane permeabilization was determined using the fluorescent dye SG (Molecular Probes, Eugene, Oregon). The assay mixture contained 106 CFU per mL (final concentration) in 90 mL of 20% mDixon containing 0.2 mM of SG. A greater number of organisms was used in these experiments compared with anti-fungal assays in order to visualize cell permeation more easily. Ten microliters of a 10 stock solution of each peptide was added and the 96-well plates were incubated at 371C in the dark. Subsequently, stained blastospores were quantified at different times (5, 20, 40, 60, and 120 min) by microscopic observation with a fluorescence microscope Olympus BX41 (Olympus, Hamburg, Germany) with the filter set at an excitation of 460–490 nm and emission at 520 nm. The percentage of permeabilized cells was the ratio of the number of fluorescent green cells to the total number of cells counted. More than 100 cells were counted for each sample. In parallel, aliquots of each sample were plated to obtain the number of colony-forming units per milliliter.
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Mouse infection and tissue sampling Procedures were approved by the Veterans Affairs (VA) San Diego Healthcare System subcommittee on animal studies. The backs of BALB/c mice were shaved and hair removed by chemical depilation (Nair). Subcutaneous injections with 150 mL of 108 CFU per mL C. albicans complexed to Cytodex beads as a carrier were carried out. Three days after injection, the site was measured, then biopsied, and tissue weighed prior to homogenization in PBS for release of organisms and measurement of surviving C. albicans. Some skin specimens were immediately embedded in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) frozen into liquid nitrogen, and kept at 801C for immunohistochemistry. Also, skin tissues from areas of normal skin from the same mice were sampled. Sections from fresh-frozen tissues were cut at 10 mm. For protein extraction, skin from infected and non-infected areas was biopsied and homogenized immediately in 1% Triton X-100 in PBS buffer. Immunohistochemistry Fresh-frozen sections were cut at 10 mm and sections fixed with 10% buffered formalin for 5 min at room temperature and immersed in PBS for 10 min. Endogenous peroxidase activity was blocked with 30 min in 0.3% H2O2. Sections were blocked with 2% goat serum in PBS for 30 min, and incubated with primary antibody, rabbit anti-CRAMP polyclonal antibody (1:500 in PBS) (Dorschner et al, 2001), and 3% bovine serum albumin (BSA) overnight at 41C. As a negative control, the polyclonal antibody was replaced by normal rabbit pre-immune IgG diluted with PBS containing 3% BSA at the same protein concentration as the primary antibody. After washing the slides 3 with PBS for 15 min, the signal was detected with goat biotinylated antirabbit IgG and a Vectastain ABC Rabbit IgG Elite kit (Vector Laboratories, Burlingame, California) according to the manufacturer’s instructions. Finally, sections were incubated in 0.02% 3,30 -diaminobenzidine with 0.03% H2O2/urea in PBS for 3–5 min. Sections were counterstained with hematoxylin for 1 s. Protein extraction and western blotting Skin was dissolved in 1 mL of 1% Triton X-100 in PBS buffer containing protease inhibitor (Complete Mini, EDTA-free, Roche Diagnostics, Mannheim, Germany) and then vortexed, incubated 1–2 h on ice, homogenized with a Dounce homogenizer (Kente Glassio, Vineland, New Jersey) and centrifuged for 10 min at 16,000 g. Five microliters of 6 SDS loading buffer containing 10% 2-mercaptoethanol was added to 25 mL of the supernatants of each sample. Proteins were separated using 16% Tris/tricine peptide gels (GeneMate Express Gels, ISC BioExpress, Kaysville, Utah) and transferred to a PVDF membrane (0.45 mm) Immobilion-P (Millipore, Bedford, Massachusetts). For positive control, 80 ng mCRAMP synthetic peptide was applied. The membrane was treated with blocking solution (3% non-fat dry milk, 0.5% BSA, 0.87% NaCl, and 0.3% Tween-20 in TSB) overnight at 41C and then detected with rabbit anti-CRAMP polyclonal antibody (1:5000 in blocking solution) for 4 h at 41C. After washing 3 with 0.1% TTBS for 15 min, the signal was detected by using a goat anti-rabbit IgG conjugated with horseradish peroxidase (Dako A/S, Glostrup, Denmark) and developed by using Western Lightning Chemiluminescence Regent Plus (Perkin Elmer, Boston, Massachusetts). Whole-blood-killing assay Three hundred to 500 mL of mouse blood was collected into a tube with 2 mL of heparin (1000 U per mL). Ten microliters of mDixon containing C. albicans at 1.3 105 CFU per mL (final concentration 3 104 CFU per mL) was added to 35 mL of fresh blood. The mixture was incubated for 4 h at 371C and dilutions of the mixture plated on agar plates for enumeration of colony-forming units. As a control, the experiment was carried out with GAS as this organism has previously been shown to depend on mouse cathelicidin in the blood-killing assay. Ten microliters of TSB containing GAS 2.7 105 CFU per mL was added to 35 mL of blood and the enumeration of colony-forming units was carried out after 1 h of incubation at 371C. The growth index was
114 LO´PEZ-GARCI´A ET AL calculated as the ratio of colony-forming units recovered to the initial inoculum. Sweat collection and analysis Human sweat was collected on paper tissues (Kimwipes, Kimberly-Clark, Neenah, Wisconsin) from three healthy volunteers (two females and one males) after exercise as previously described (Murakami et al, 2002, 2004). The sweat was removed from paper tissue by centrifugation. For analysis of cathelicidin in human sweat, 1 mL was separated by reverse phase (RP) HPLC with a C18 column. The sweat was eluted at a flow rate of 2 mL per min using gradients of 10%–20% and 20%–70% acetonitrile containing 0.1% trifluoroacetic acid (solution B) for 2 and 32 min, respectively. Column effluent was monitored at 214, 230, and 280 nm. Identified peaks in sweat fractions were compared with the peaks of synthesized peptides (LL-37, KS-30, and RK-31) in an identical HPLC protocol, and the concentrations of peptides were estimated by the peak areas of chromatograms using UNICORN 3.00 (Amersham Pharmacia Biotech, Piscataway, New Jersey). The identity of samples was confirmed by Mass spectrometry (MALDI-MS) performed by Center for Mass Spectrometry, The Scripps Research Institute (La Jolla, California) as previously described (Murakami et al, 2004). Mass assignments were assigned with an accuracy of approximately 0.1% ( 1 Da/1000 Da). Anti-fungal activity in sweat To test the anti-fungal activity of human sweat, 4 mL of a 20 human sweat concentrate was added to 2.5–5 104 CFU per mL of C. albicans in 16 mL of 20% mDixon medium (final concentration). To study the importance of the proteolytic processing of LL-37 in more active peptides as KS30 and RK-31 for the anti-fungal activity, C. albicans at 2.5–5 104 CFU per mL (final concentration) was incubated in 1 sweat with the peptides LL-37 and RK-31 from 10 stock solutions. In some samples, 2 mL of a 10 protease inhibitor solution composed of a mixture of several protease inhibitors with broad inhibitory specificity against serine, cysteine, and metalloproteases, as well as calpains (Complete Mini, EDTA-free protease inhibitor, Roche Diagnostics) was added. To mimic the human sweat ionic environment, and evaluate individual synthetic cathelididin peptides, an SwB composed of 40 mM NaCl, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM NaH2PO4, pH 6.5 was also prepared (Murakami et al, 2002). This was sterile filtered using 0.2 mm filter and concentrated 20 to replicate evaporated conditions in some experiments. Two microliters of 10 synthetic cathelicidin peptides, LL37, KS-30, and RK-31 were added to 2.5–5 104 CFU per mL of C. albicans in 18 mL of 1 SwB. To test whether the peptides are active in sweat after the evaporation process, peptides RK-31, KS30, and KR-20 were mixed in sweat or SwB, at a final concentration of 2 or 10 mM of each peptide. Each sample was lyophilized and dissolved in H2O to final concentrations of 2 , 4 , and 10 . Ten microliters of 9 104 CFU per mL of C. albicans was incubated with 10 mL of each sample of mixed peptides in SwB or sweat. In all cases, each sample contained 16 mg per mL chloramphenicol to inhibit bacterial growth. Assay mixtures were incubated in sterile 96-well microtiter plates at 371C for 6 or 24 h and then aliquots of each sample were plated in mDixon agar plates to determine the colony-forming units per milliliter. These plates were then incubated at 371C for 1 d and colonies were counted.
This work was supported by VA Merit award and NIH grants AI48176 and AR45676 (R. G.). Bele´n Lo´pez-Garcı´a is a recipient of a postdoctoral fellowship from Fundacion Ramon Areces (Spain). Phillip H. Lee is supported by the Generalist Physician-Scientist Training Program NIH-NCI 1T32 CA81211. Kenshi Yamasaki is a recipient of a post-doctoral fellowship from the Association for Preventive Medicine of Japan. DOI: 10.1111/j.0022-202X.2005.23713.x Manuscript received April 12, 2004; revised December 28, 2004; accepted for publication January 1, 2005
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Address correspondence to: Dr Richard L. Gallo, Division of Dermatology, University of California, San Diego, and VA San Diego Healthcare System, San Diego, California 92161, USA Email: rgallo@vapop. ucsd.edu
References Agerberth B, Charo J, Werr J, et al: The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 96:3086–3093, 2000 Andreu D, Rivas L: Animal antimicrobial peptides: An overview. Biopolymers 47:415–433, 1998 Bals R, Wang X, Zasloff M, Wilson JM: The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA 95:9541–9546, 1998 Bjorck L, Akesson P, Bohus M, Trojnar J, Abrahamson M, Olafsson I, Grubb A: Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor. Nature 337:385–386, 1989 Boman HG, Agerberth B, Boman A: Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect Immun 61:2978–2984, 1993 Conner K, Nern K, Rudisill J, O’Grady T, Gallo RL: The antimicrobial peptide LL37 is expressed by keratinocytes in condyloma acuminatum and verruca vulgaris. J Am Acad Dermatol 47:347–350, 2002 De Y, Chen Q, Schmidt AP, et al: LL-37, the neutrophil granule-and epithelial cellderived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192:1069–1074, 2000 Dorschner RA, Pestonjamasp VK, Tamakuwala S, et al: Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus. J Invest Dermatol 117:91–97, 2001 Epand RM, Vogel HJ: Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28, 1999 Erdag G, Morgan JR: Interleukin-1alpha and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts. Ann Surg 235:113–124, 2002 Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S, Wigzell H, Gudmundsson GH: The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. J Biol Chem 272:15258–15263, 1997 Gaczynska M, Osmulski PA, Gao Y, Post MJ, Simons M: Proline- and argininerich peptides constitute a novel class of allosteric inhibitors of proteasome activity. Biochemistry 42:8663–8670, 2003 Gallo RL, Ono M, Povsic T, Page C, Eriksson E, Klagsbrun M, Bernfield M: Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc Natl Acad Sci USA 91:11035–11039, 1994 Hancock REW, Chapple DS: Peptide antibiotics. Antimicrob Agents Chemother 43:1317–1323, 1999 Harder J, Bartels J, Christophers E, Schroder JM: A peptide antibiotic from human skin. Nature 387:861, 1997 Hay RJ, Calderon RA, Collins MJ: Experimental dermatophytosis: The clinical and histopathologic features of a mouse model using Trichophyton quinckeanum (mouse favus). J Invest Dermatol 81:270–274, 1983 Heilborn JD, Nilsson MF, Kratz G, Weber G, Sorensen O, Borregaard N, StahleBackdahl M: The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium. J Invest Dermatol 120:379–389, 2003 Howell MD, Jones JF, Kisich KO, Streib JE, Gallo RL, Leung DY: Selective killing of vaccinia virus by LL-37: Implications for eczema vaccinatum. J Immunol 172:1763–1767, 2004 Larrick JW, Hirata M, Balint RF, Lee J, Zhong J, Wright SC: Human CAP18: A novel antimicrobial lipopolysaccharide-binding protein. Infect Immun 63:1291–1297, 1995 Lo´pez-Garcı´a B, Marcos JF, Pe´rez-Paya´ E: Stabilisation of mixed peptide/lipid complexes in selective antifungal hexapeptides. Biochim Biophys Acta— Biomembranes 1660:131–137, 2004 Marchini G, Lindow S, Brismar H, et al: The newborn infant is protected by an innate antimicrobial barrier: Peptide antibiotics are present in the skin and vernix caseosa. Br J Dermatol 147:1127–1134, 2002 Marr KA, Rustad TR, Rex JH, White TC: The trailing end point phenotype in antifungal susceptibility testing is pH dependent. Antimicrob Agents Chemother 43:1383–1386, 1999 McIntyre KA, Galgiani JN: pH and other effects on the antifungal activity of cilofungin (LY121019). Antimicrob Agents Chemother 33:731–735, 1989
125 : 1 JULY 2005 Murakami M, Lo´pez-Garcı´a B, Braff M, Dorschner RA, Gallo RL: Post-secretory processing generates multiple human cathelicidin peptides for enhanced topical antimicrobial defense. J Immunol 172:3070–3077, 2004 Murakami M, Ohtake T, Dorschner RA, Schittek B, Garbe C, Gallo RL: Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin. J Invest Dermatol 119:1090–1095, 2002 Nizet V, Ohtake T, Lauth X, et al: Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414:454–457, 2001 Ong PY, Ohtake T, Brandt C, et al: Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 347:1151–1160, 2002 Oren Z, Shai Y: Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47:451–463, 1998 Rao AG: Antimicrobial peptides. Mol Plant–Microbe Interact 8:6–13, 1995 Rogers TE, Galgiani JN: Activity of fluconazole (UK 49,858) and ketoconazole against Candida albicans in vitro and in vivo. Antimicrob Agents Chemother 30:418–422, 1986 Roth BL, Poot M, Yue ST, Millard PJ: Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 63:2421–2431, 1997 Schittek B, Hipfel R, Sauer B, et al: Dermcidin: A novel human antibiotic peptide secreted by sweat glands. Nat Immunol 2:1133–1137, 2001 Shafer WM, Pohl J, Onunka VC, Bangalore N, Travis J: Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide. J Biol Chem 266:112–116, 1991 Shai Y, Avrahami D, Gerber D, et al: Protein–membrane interaction: Role in cell permeation, virus-cell fusion, and organization of membrane proteins. In: Life Science Book 2000, www.weizmann.ac.il/Biology/open-day/images/ syehiel.pdf, 2001.
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Shapiro HM: Multiparameter flow cytometry of bacteria: Implications for diagnostics and therapeutics. Cytometry 43:223–226, 2001 Steiner H, Andreu D, Merrifield RB: Binding and action of cecropin and cecropin analogues: Antibacterial peptides from insects. Biochim Biophys Acta 939:260–266, 1988 Thevissen K, Terras FR, Broekaert WF: Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65: 5451–5458, 1999 Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI: Activities of LL-37, a cathelinassociated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42:2206–2214, 1998 Wang Y, Agerberth B, Lothgren A, Almstedt A, Johansson J: Apolipoprotein A-I binds and inhibits the human antibacterial/cytotoxic peptide LL-37. J Biol Chem 273:33115–33118, 1998 Wu H, Zhang G, Minton JE, Ross CR, Blecha F: Regulation of cathelicidin gene expression: Induction by lipopolysaccharide, interleukin-6, retinoic acid, and Salmonella enterica serovar typhimurium infection. Infect Immun 68:5552–5558, 2000 Wu M, Maier E, Benz R, Hancock RE: Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38:7235–7242, 1999 Zaiou M, Nizet V, Gallo RL: Antimicrobial and protease inhibitory functions of the human cathelicidin (hCAP18/LL-37) prosequence. J Invest Dermatol 120:810–816, 2003 Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 415:389–395, 2002
Anti-Fungal Activity of Cathelicidins and their Potential Role in Candida albicans Skin Infection Bele´n Lo´pez-Garcı´a, Phillip H. A. Lee, Kenshi Yamasaki, and Richard L. Gallo Division of Dermatology, University of California San Diego, and VA San Diego Healthcare Center, San Diego, California, USA
Cathelicidins have broad anti-microbial capacity and are important for host defense against skin infections by some bacterial and viral pathogens. This study investigated the activity of cathelicidins against Candida albicans. The human cathelicidin LL-37, and mouse cathelicidin mCRAMP, killed C. albicans, but this fungicidal activity was dependent on culture conditions. Evaluation of the fungal membrane by fluorescent dye penetration after incubation with cathelicidins correlated membrane permeabilization and inhibition of fungal growth. Anti-fungal assays carried out in an ionic environment that mimicked human sweat and with the processed forms of cathelicidin such as are present in sweat found that the cleavage of LL-37 to forms such as RK-31 conferred additional activity against C. albicans. C. albicans also induced an increase in the expression of cathelicidin in mouse skin, but this induction did not confer systemic or subcutaneous resistance as mCRAMP-deficient mice were not more susceptible to C. albicans in blood-killing assays or in an intradermal infection model. Therefore, cathelicidins appear active against C. albicans, but may be most effective as a superficial barrier to infection.
Key words: anti-microbial peptides/defensins/infection/innate immunity/LL-37/mCRAMP/sweat J Invest Dermatol 125:108 – 115, 2005
Cationic peptides play important roles in the defense systems of many organisms from insects to humans (Rao, 1995; Zasloff, 2002) and have been isolated from diverse organisms (Andreu and Rivas, 1998; Hancock and Chapple, 1999). A number of these have received attention because of their low toxicity against mammalian cells (Steiner et al, 1988). In particular, mammalian skin and other epithelial surfaces are a major producer of functional cathelicidins (Gallo et al, 1994; Frohm et al, 1997; Bals et al, 1998). LL-37 and mCRAMP belong to the cathelicidin family and are present in humans and mice, respectively. They are constitutively expressed, or can be induced, at the interface with the external environment where they act together with other anti-microbial molecules such as defensins (Harder et al, 1997), select proteases (Shafer et al, 1991), cytokines (Wu et al, 2000; Erdag and Morgan, 2002), and protease inhibitors (Bjorck et al, 1989; Zaiou et al, 2003). Cathelicidins have been shown to contribute to defense against invasive bacterial infection by Group A Streptococcus (GAS) (Dorschner et al, 2001; Nizet et al, 2001) and vaccinia virus (Howell et al, 2004). Whereas cathelicidins are typically expressed at low levels in normal keratinocytes, they are induced during inflammation and increase locally because of release from neutrophils and mast cells (Frohm et al, 1997). Recently, it has been demonstrated that cathelicidins are produced by the eccrine apparatus and secreted into human sweat (Schittek et al, 2001; Murakami
et al, 2002). After secretion onto the skin surface, LL-37 is processed by a serine protease-dependent mechanism into multiple distinct anti-microbial peptides, such as RK-31 or KS-30 (Murakami et al, 2004) that are more potent compared with the uncleaved mature peptide. The function of cathelicidins as a natural barrier to fungal infections of the skin has not been well characterized. This study characterized the anti-fungal activity of the cathelicidins against Candida albicans and studied the possible role of this family of peptides in skin fungal infection.
Results Anti-fungal activity of cathelicidins The anti-fungal activity of cathelicidins was measured against C. albicans in low salt 20% mDixon medium (pH 5.5). Two similar ahelical cathelicidin homologues, human (LL-37) and mouse (mCRAMP), showed similar activity with delay or complete inhibition of growth at an minimum inhibitory concentration (MIC) range of 15–20 mM, whereas the linear porcine cathelicidin PR-39 was inactive (Fig 1A). Activities of the human and mouse cathelicidins were pH dependent but not affected by change in temperature from 251C to 371C. Growth inhibition of C. albicans in the presence of 15 mM of LL-37 was observed at acidic and neutral pH values, with growth of 19%, 21%, and 60% at pH 4.5, 5.5, and 7.2, respectively (Fig 1B). Growth in the absence of peptide under identical conditions was considered 100% growth. Processed forms of LL-37 have been shown to kill C. albicans in vitro (Murakami et al, 2004). To compare the activity of these against PR-39, LL-37, and mCRAMP,
Abbreviations: CFU, colony-forming units; GAS, Group A Streptococcus; HPLC, high-pressure liquid chromatography; SG, Sytox green; SwB, sweat buffer
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
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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 2.55 104 colony-forming units (CFU) per mL. Data represent the mean (SD) of triplicate determinations from a single experiment representative of at least two experiments.
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. 2.55 104 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 mM 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.
fungicidal activity was measured in 20% mDixon, pH 5.5 (Fig 2). These results suggest that the shorter processed forms of LL-37 present at the skin surface (RK-31 and KS30) have enhanced anti-fungal effect against the yeast compared with forms of cathelicidins released by neutrophils such as mCRAMP and LL-37 (Fig 2). LL-37 and RK-31 permeabilize C. albicans To evaluate the kinetics and mechanism of killing of C. albicans by cathelicidins, the survival and permeability of the yeast were investigated in the presence of LL-37 or RK-31 (Fig 3). A higher initial concentration of yeast was used in these experiments compared with Fig 2. At this initial C. albicans load (106 colony-forming units (CFU) per mL), 25 mM LL-37 had less fungicidal activity than at (2.5–5 104 CFU per mL), whereas 8 mM RK-31 continued to kill approximately 90% of the initial inoculum within 15 min (Fig 3A). To determine fungal membrane integrity simultaneously, an assay based on the uptake of the fluorescent dye Sytox green (SG) was used (Fig 3B). SG does not penetrate live eukaryotic or prokaryotic cell membranes or walls but binding to nucleic acids results in a 4500-fold enhancement in its fluorescence emission, thus enabling sensitive detection of a disruption in the outer membrane (Roth et al, 1997; Thevissen et al, 1999). Incubation of yeast cells with 25 mM LL-37
Figure 3 Kinetics of cathelicidin activity against Candida albicans. Yeast (106 colony-forming units per mL, initial number) was incubated with 0 mM (black diamond), 25 mM LL-37 (black square), or 8 mM RK-31 (white triangle). (A) Percent of remaining cells was calculated as the colonyforming 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.
or 8 mM RK-31 allowed rapid uptake of SG in both cases (Fig 3B). After 5 min of incubation, LL-37 and RK-31 permeabilized more than 50% of the cells versus 12% permeable cells observed in samples without peptide. These results indicate that the kinetics of permeabilization correlated with the fungicidal effect of RK-31 and LL-37. Candida skin infection increases cathelicidin expression In humans and mice, cathelicidins are not easily
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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 ¼ 10 mm) of dermis from (B) shows evidence of multiple C. albicans organisms indicated by arrows and mCRAMP containing neutrophils (hematoxylin counterstain; scale bar ¼ 40 mm in (A, B, D)).
detectable in normal adult skin, but are usually abundantly expressed after inflammation or injury (Frohm et al, 1997; Dorschner et al, 2001). To determine the expression after fungal infection, mice were subcutaneously infected with C. albicans and tissues were sampled. Immunohistochemical analysis showed that mCRAMP expression was higher in infected tissue (Fig 4B and C) compared with expression in healthy tissue (Fig 4A). mCRAMP appeared to be induced specifically at areas of C. albicans involvement. This induction of mCRAMP protein expression was confirmed by western blot (data not shown). Control sections stained with rabbit pre-immune sera demonstrated that the staining pattern seen was not because of endogenous peroxidase activity (Fig 4D).
Role of cathelicidins in C. albicans infection in vivo To explore whether cathelicidins are responsible for controlling C. albicans infection in mice, two complementary experiments were carried out. Using blood from mice deficient for mCRAMP (Cnlp/), an in vitro killing assay was carried out to determine fungicidal activity against C. albicans. No statistical differences were seen between the growth of the yeast in blood from wild-type versus knockout Cnlp/ mice (Fig 5A). As reported previously, however, a difference was observed with GAS (Nizet et al, 2001). Next, subcutaneous skin infections of C. albicans were carried out in wildtype and Cnlp/ mice. Three days after inoculation of yeast, lesions were biopsied and colony counts were per-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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-valuep0.001). (B) C. albicans cultured from tissue biopsies of wild-type and mCRAMP knockout mice 3 d after subcutaneous injection of 150 mL 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.
formed. Colony-forming units per milliliter were similar from knockout and wild-type mice (Fig 5B). Processing of LL-37 in sweat increases activity against C. albicans The apparent discordant results between killing in vitro and our inability to detect a contribution for cathelicidins in mouse blood or subcutaneous injections suggested that cathelicidins may only be active against C. albicans on the skin surface. Since the anti-microbial activity of cathelicidins is dependent on ionic and serum components, and can be neutralized in ionic environments containing high concentrations of NaCl (Bals et al, 1998; Turner et al, 1998; Zasloff, 2002), we next evaluated whether cathelicidins were active in the ionic environment of the human skin surface, that of sweat. First, the abundance of cathelicidin peptides in their processed form compared with LL-37 was measured. Human sweat was collected, analyzed by high-pressure liquid chromatography (HPLC), and the mass of fractions eluting at the expected site for KS-30 and LL-37 was confirmed by mass spectrometry. This analysis yielded an approximate concentration of 1.165 mM for KS-30/RK-31 and 0.013 mM of LL-37 in normal human sweat (Fig 6A). The human sweat was next analyzed for its anti-Candida activity before and after concentration by evaporation.
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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 mM (black bars), 2 mM (gray bars), or 10 mM (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.
Figure 6 Detection, dependence on proteolysis, and potency of cathelicidin peptides in sweat. (A) Analysis of normal human sweat by reversephase 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) 2.55 104 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 mM LL-37 or 8 mM 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-valueo0.05). (C) 2.55 104 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.
Following partial evaporation to reduce the volume 4-fold, C. albicans growth was inhibited by the sweat concentrate containing a complex mixture of cathelicidins and other potential anti-microbials (Murakami et al, 2002) (Fig 6B). This inhibitory effect of concentrated sweat was not because of concentrated salt content, as a salt buffer mimicking sweat (SwB) and concentrated 4 had no effect on growth (Fig 6B). Addition of 25 mM LL-37 to 1 sweat inhibited fungal growth, but this inhibition appeared to be the consequence of protease activity in the natural sweat
solution as the anti-fungal activity of LL-37 was blocked in the presence of protease inhibitors. Conversely, the antifungal activity of 8 mM RK-31 in sweat was not blocked by protease inhibitors. The anti-fungal activity of LL-37, KS-30, and RK-31 was further analyzed in sweat buffer (SwB) solution to directly determine the function of these peptides in the sweat ionic environment without complication of native proteases or as yet undefined inhibitory factors present in normal human sweat. In buffer containing 40 mM NaCl, 10 mM KCl, 1 mM CaCl2, and 1mM MgCl2, the derived peptides KS-30 and RK-31 were fungicidal against C. albicans, whereas LL-37 in the absence of proteolytic processing was not active (Fig 6C). Since the buffer conditions of C. albicans growth strongly influence the apparent activity of cathelicidin peptides, additional experiments were performed to mimic the effects of sweat evaporation at the skin surface. When added to the SwB solution, concentration by a factor of 5 or greater decreased the relative fungicidal activity (Fig 7A), thus suggesting that by itself the cathelicidin peptides may be inactivated in high ionic strength solutions. When cathelicidin peptides were added to normal sweat concentrated by 2 or 5 (Fig 7B), the anti-fungal activity of the concentrated sweat was greater than any additional activity detectable by supplementing with synthetic peptides.
Discussion Little is known about the contribution of naturally occurring anti-microbial peptides to defense against fungal infection.
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In this study, we characterized the anti-fungal activity of human and mice cathelicidin peptides. The form of these peptides found in human neutrophils (LL-37) or mouse granulocytes (mCRAMP) was effective against C. albicans in 20% mDixon medium with an MIC of 15–20 mM and minimal fungicidal concentration (MFC) of 25–30 mM. This activity was pH and growth media dependent. As previously published (Marr et al, 1999), pH is a critical factor in antifungal susceptibility testing. Using an acidic pH of 4–5 rather than neutral pH decreased the MIC of LL-37 against C. albicans, an effect seen with other anti-fungal agents (Rogers and Galgiani, 1986; McIntyre and Galgiani, 1989). Also, comparison of Figs 2 and 6B shows that the MFC of cathelicidin peptides is greater in salt-containing solutions than in low-salt Dixon media. Thus, conclusions regarding cathelicidin activity against C. albicans must be qualified by recognition of the assay conditions, a variable that is also true for other anti-microbial peptides and other target organisms including Gram and Gram þ bacteria. Importantly, the activity of cathelicidin against C. albicans could be demonstrated by the direct addition of the peptides in natural human sweat, and human sweat itself becomes anti-fungal once concentrated such as would normally occur during evaporation. Thus, the most superficial topical environment of human skin contains anti-fungal activity that may be partly because of the contribution of cathelicidins. Several studies have addressed the mode of action of anti-microbial peptides (Oren and Shai, 1998; Epand and Vogel, 1999; Shai et al, 2001; Zasloff, 2002; Lo´pez-Garcı´a et al, 2004). Membrane permeabilization drives the anti-microbial activity of most cationic and amphipathic peptides including LL-37 and mCRAMP. Our experiments using fluorescent SG indicate that the perturbation of the yeast membrane is a step in the anti-fungal activity of cathelicidins. Both eukaryotic and bacterial cells can be made transiently permeable with retention of viability; thus, permeability alone does not predict activity (Shapiro, 2001). Our data, however, support a good correlation between both permeability and anti-fungal activity for LL-37 and RK-31 on C. albicans (Fig 3). Other factors may also modulate the activity and specificity of peptide anti-microbial activity. For example, the cathelicidin PR-39 affects DNA and protein synthesis (Boman et al, 1993) and is a non-competitive and reversible inhibitor of the proteasome function (Gaczynska et al, 2003) but does not immediately disrupt bacterial membranes. Moreover, LL-37 can neutralize endotoxin, and has chemoattractive effects on leukocytes, which could provide additional mechanisms for protection independent of direct microbial killing (Larrick et al, 1995; Agerberth et al, 2000; De et al, 2000). Together, these observations support a role for membrane permeabilization in the activity of cathelicidins against C. albicans, but do not conclusively prove that this is the sole mechanism for cell killing. Injection of C. albicans into mouse skin indicated that cathelicidin is induced in the skin in response to C. albicans infection, but this induction did not confer subcutaneous resistance, as mCRAMP-deficient mice were not more susceptible to C. albicans in a subcutaneous model. Unlike Streptococcus pyogenes, blastospores of C. albicans grew equally well in blood from wild-type and Cnlp/ mice. This lack of response likely reflects several differences between
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
S. pyogenes and C. albicans but may also several conditions in the subcutaneous environment that act to inhibit cathelicidin function. These include higher pH and concentrations of cations in vivo than in the fungal culture conditions and/or inactivation of peptide by interaction with high-molecularweight serum proteins (Wang et al, 1998). Susceptibility to C. albicans infection may also vary with the strain of mouse used (Hay et al, 1983). Given the results observed with mice and in mouse blood, these findings suggested that cathelicidins may not be involved in protection against C. albicans infection once it has penetrated the skin surface. An alternative role for cathelicidins in fungal infection may be to provide a superficial barrier against proliferation and potential fungal invasion. Our data show that cathelicidin peptides have activity against C. albicans when added to normal human sweat, or in ionic environments that mimic sweat. But LL-37 was poorly active in this environment without proteolytic processing. In this paper, we estimated that the concentration of these processed forms (KS-30 or RK31) and LL-37 is 1.165 and 0.013 mM, respectively (Fig 6A). These concentrations are likely a large underestimate of the actual abundance of cathelicidins at the skin surface. Cationic amphipathic peptides such as the human cathelicidins will partition into lipid components and bind anionic molecules and will not partition completely into the soluble sweat fraction recovered. But although the actual concentrations may be an underestimate, it appears that the proportion of LL-37 related to forms such as RK-31 is low. LL-37 is a minor form of human cathelicidin on the skin surface. The actual contribution of the human cathelicidins KS-30 and RK-31 to topical defense against C. albicans growth is difficult to determine. This data confirm that these peptides are present, and show activity against C. albicans under select growth conditions. Interestingly, RK-31 shows higher activity than KS-30 in higher ionic strength media (SwB, Fig 6C) but lesser activity in the low ionic strength medium (20% mDixon, Fig 2). This suggests that KS-30 activity may be more sensitive to inactivation by salt than RK-31. In SwB solutions alone, the amount of RK-31 required to completely kill C. albicans was greater than the amount of RK-31 estimated in normal sweat. Upon addition of 8 mM peptide to normal sweat, however, inhibitory activity was detectable. Activity at 8 mM in the complex mixture of normal sweat compared with 25 mM in SwB alone suggests that these peptides are most likely to be effective in combination with other factors, including other anti-microbial peptides. In summary, our findings show that naturally produced cathelicidins are present at sites of fungal infection and may form a barrier to C. albicans infection. Superficial infection with C. albicans is a common dermatological condition that is exacerbated by several factors including host immune status, location, and medications. In humans, abnormal production or an inability to appropriately increase cathelicidin expression following skin inflammation has been associated with disease (Frohm et al, 1997; Dorschner et al, 2001; Conner et al, 2002; Marchini et al, 2002; Ong et al, 2002; Heilborn et al, 2003). The observations made in this study suggest that unlike bacterial infections with S. pyogenes, the relevance of cathelicidins to C. albicans infection is more likely to be important at the skin surface. Understanding such interactions with innate immune effector
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molecules such as cathelicidins may explain the pathophysiology of common fungal skin diseases and offer alternative therapeutic approaches to conventional anti-fungal therapy. Materials and Methods Peptides All peptides were prepared by Synpep (Dublin, Oregon), purified by HPLC (95% purity), and identity confirmed by mass spectrometry. A 10 stock solution was prepared in H2O for each peptide. Media Primary Candida culture medium was a modified Dixon (mDixon) prepared in the laboratory from 4% malt extract (Fluka Biochemika, Steinheim, Germany), 0.6% Bacto Peptone (Becton, Dickinson and Company, Sparks, Maryland), 1% glucose (SigmaAldrich, St Louis, Missouri), and 1% Tween-80 (Sigma-Aldrich). The pH of this medium was 5.0 0.1. For anti-microbial assays, 20% mDixon medium in 1 mM sodium phosphate buffer pH 7 containing 16 mg per mL chloramphenicol was used. The final pH of the mixture was 5.5. Our 20% mDixon medium contained 5.0 mM Na þ , 1.6 mM K þ , 4 mg per dL phosphate, and undetectable amounts of Ca2 þ , Mg2 þ , and Cl. Microorganism C. albicans American Type Cultures Collection 14053 was cultured on mDixon agar medium (mDixon broth þ 1.5% agar). The day before use, colonies were transferred into mDixon broth medium and grown overnight at 371C with shaking. The turbidity of each suspension was adjusted to OD600 of 0.01 (corresponding to 2.5–5 105 CFU per mL).
In vitro anti-fungal activity assays The susceptibility of C. albicans was tested by broth microdilution assay. Yeast was grown on sterile 96-well microtiter plates (Corning, Corning, New York) in a final volume of 100 mL. The assay mixture contained 2.5–5 104 CFU per mL in 90 mL of 20% mDixon medium (final concentration), 10 mL of a 10 stock solutions of each synthetic peptide was added to yeast and incubated at 25 or 371C for initial determination of growth by absorbance at A600 with a Spectra max PLUS 384 (Molecular Devices, Sunnyvale, California) over 2 d. In all the experiments, the blank mean A600 value from one row of mock inoculations was subtracted from each well A600 measurement, and the mean and SD were calculated for each treatment. The MIC was defined as the lowest peptide concentration that showed no growth at the end of the experiment (usually, after 2 d of incubation). After 24 h of incubation, aliquots of each yeast–peptide combination were plated in mDixon agar plates in order to determine the MFC values. In the kinetic assays, the colony-forming units per milliliter was determined by plating aliquots of each yeast–peptide combination at different times. The plates were incubated at 371C for 1 d and the colonies were counted. Membrane permeabilization assay Fungal membrane permeabilization was determined using the fluorescent dye SG (Molecular Probes, Eugene, Oregon). The assay mixture contained 106 CFU per mL (final concentration) in 90 mL of 20% mDixon containing 0.2 mM of SG. A greater number of organisms was used in these experiments compared with anti-fungal assays in order to visualize cell permeation more easily. Ten microliters of a 10 stock solution of each peptide was added and the 96-well plates were incubated at 371C in the dark. Subsequently, stained blastospores were quantified at different times (5, 20, 40, 60, and 120 min) by microscopic observation with a fluorescence microscope Olympus BX41 (Olympus, Hamburg, Germany) with the filter set at an excitation of 460–490 nm and emission at 520 nm. The percentage of permeabilized cells was the ratio of the number of fluorescent green cells to the total number of cells counted. More than 100 cells were counted for each sample. In parallel, aliquots of each sample were plated to obtain the number of colony-forming units per milliliter.
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Mouse infection and tissue sampling Procedures were approved by the Veterans Affairs (VA) San Diego Healthcare System subcommittee on animal studies. The backs of BALB/c mice were shaved and hair removed by chemical depilation (Nair). Subcutaneous injections with 150 mL of 108 CFU per mL C. albicans complexed to Cytodex beads as a carrier were carried out. Three days after injection, the site was measured, then biopsied, and tissue weighed prior to homogenization in PBS for release of organisms and measurement of surviving C. albicans. Some skin specimens were immediately embedded in optimal cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) frozen into liquid nitrogen, and kept at 801C for immunohistochemistry. Also, skin tissues from areas of normal skin from the same mice were sampled. Sections from fresh-frozen tissues were cut at 10 mm. For protein extraction, skin from infected and non-infected areas was biopsied and homogenized immediately in 1% Triton X-100 in PBS buffer. Immunohistochemistry Fresh-frozen sections were cut at 10 mm and sections fixed with 10% buffered formalin for 5 min at room temperature and immersed in PBS for 10 min. Endogenous peroxidase activity was blocked with 30 min in 0.3% H2O2. Sections were blocked with 2% goat serum in PBS for 30 min, and incubated with primary antibody, rabbit anti-CRAMP polyclonal antibody (1:500 in PBS) (Dorschner et al, 2001), and 3% bovine serum albumin (BSA) overnight at 41C. As a negative control, the polyclonal antibody was replaced by normal rabbit pre-immune IgG diluted with PBS containing 3% BSA at the same protein concentration as the primary antibody. After washing the slides 3 with PBS for 15 min, the signal was detected with goat biotinylated antirabbit IgG and a Vectastain ABC Rabbit IgG Elite kit (Vector Laboratories, Burlingame, California) according to the manufacturer’s instructions. Finally, sections were incubated in 0.02% 3,30 -diaminobenzidine with 0.03% H2O2/urea in PBS for 3–5 min. Sections were counterstained with hematoxylin for 1 s. Protein extraction and western blotting Skin was dissolved in 1 mL of 1% Triton X-100 in PBS buffer containing protease inhibitor (Complete Mini, EDTA-free, Roche Diagnostics, Mannheim, Germany) and then vortexed, incubated 1–2 h on ice, homogenized with a Dounce homogenizer (Kente Glassio, Vineland, New Jersey) and centrifuged for 10 min at 16,000 g. Five microliters of 6 SDS loading buffer containing 10% 2-mercaptoethanol was added to 25 mL of the supernatants of each sample. Proteins were separated using 16% Tris/tricine peptide gels (GeneMate Express Gels, ISC BioExpress, Kaysville, Utah) and transferred to a PVDF membrane (0.45 mm) Immobilion-P (Millipore, Bedford, Massachusetts). For positive control, 80 ng mCRAMP synthetic peptide was applied. The membrane was treated with blocking solution (3% non-fat dry milk, 0.5% BSA, 0.87% NaCl, and 0.3% Tween-20 in TSB) overnight at 41C and then detected with rabbit anti-CRAMP polyclonal antibody (1:5000 in blocking solution) for 4 h at 41C. After washing 3 with 0.1% TTBS for 15 min, the signal was detected by using a goat anti-rabbit IgG conjugated with horseradish peroxidase (Dako A/S, Glostrup, Denmark) and developed by using Western Lightning Chemiluminescence Regent Plus (Perkin Elmer, Boston, Massachusetts). Whole-blood-killing assay Three hundred to 500 mL of mouse blood was collected into a tube with 2 mL of heparin (1000 U per mL). Ten microliters of mDixon containing C. albicans at 1.3 105 CFU per mL (final concentration 3 104 CFU per mL) was added to 35 mL of fresh blood. The mixture was incubated for 4 h at 371C and dilutions of the mixture plated on agar plates for enumeration of colony-forming units. As a control, the experiment was carried out with GAS as this organism has previously been shown to depend on mouse cathelicidin in the blood-killing assay. Ten microliters of TSB containing GAS 2.7 105 CFU per mL was added to 35 mL of blood and the enumeration of colony-forming units was carried out after 1 h of incubation at 371C. The growth index was
114 LO´PEZ-GARCI´A ET AL calculated as the ratio of colony-forming units recovered to the initial inoculum. Sweat collection and analysis Human sweat was collected on paper tissues (Kimwipes, Kimberly-Clark, Neenah, Wisconsin) from three healthy volunteers (two females and one males) after exercise as previously described (Murakami et al, 2002, 2004). The sweat was removed from paper tissue by centrifugation. For analysis of cathelicidin in human sweat, 1 mL was separated by reverse phase (RP) HPLC with a C18 column. The sweat was eluted at a flow rate of 2 mL per min using gradients of 10%–20% and 20%–70% acetonitrile containing 0.1% trifluoroacetic acid (solution B) for 2 and 32 min, respectively. Column effluent was monitored at 214, 230, and 280 nm. Identified peaks in sweat fractions were compared with the peaks of synthesized peptides (LL-37, KS-30, and RK-31) in an identical HPLC protocol, and the concentrations of peptides were estimated by the peak areas of chromatograms using UNICORN 3.00 (Amersham Pharmacia Biotech, Piscataway, New Jersey). The identity of samples was confirmed by Mass spectrometry (MALDI-MS) performed by Center for Mass Spectrometry, The Scripps Research Institute (La Jolla, California) as previously described (Murakami et al, 2004). Mass assignments were assigned with an accuracy of approximately 0.1% ( 1 Da/1000 Da). Anti-fungal activity in sweat To test the anti-fungal activity of human sweat, 4 mL of a 20 human sweat concentrate was added to 2.5–5 104 CFU per mL of C. albicans in 16 mL of 20% mDixon medium (final concentration). To study the importance of the proteolytic processing of LL-37 in more active peptides as KS30 and RK-31 for the anti-fungal activity, C. albicans at 2.5–5 104 CFU per mL (final concentration) was incubated in 1 sweat with the peptides LL-37 and RK-31 from 10 stock solutions. In some samples, 2 mL of a 10 protease inhibitor solution composed of a mixture of several protease inhibitors with broad inhibitory specificity against serine, cysteine, and metalloproteases, as well as calpains (Complete Mini, EDTA-free protease inhibitor, Roche Diagnostics) was added. To mimic the human sweat ionic environment, and evaluate individual synthetic cathelididin peptides, an SwB composed of 40 mM NaCl, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM NaH2PO4, pH 6.5 was also prepared (Murakami et al, 2002). This was sterile filtered using 0.2 mm filter and concentrated 20 to replicate evaporated conditions in some experiments. Two microliters of 10 synthetic cathelicidin peptides, LL37, KS-30, and RK-31 were added to 2.5–5 104 CFU per mL of C. albicans in 18 mL of 1 SwB. To test whether the peptides are active in sweat after the evaporation process, peptides RK-31, KS30, and KR-20 were mixed in sweat or SwB, at a final concentration of 2 or 10 mM of each peptide. Each sample was lyophilized and dissolved in H2O to final concentrations of 2 , 4 , and 10 . Ten microliters of 9 104 CFU per mL of C. albicans was incubated with 10 mL of each sample of mixed peptides in SwB or sweat. In all cases, each sample contained 16 mg per mL chloramphenicol to inhibit bacterial growth. Assay mixtures were incubated in sterile 96-well microtiter plates at 371C for 6 or 24 h and then aliquots of each sample were plated in mDixon agar plates to determine the colony-forming units per milliliter. These plates were then incubated at 371C for 1 d and colonies were counted.
This work was supported by VA Merit award and NIH grants AI48176 and AR45676 (R. G.). Bele´n Lo´pez-Garcı´a is a recipient of a postdoctoral fellowship from Fundacion Ramon Areces (Spain). Phillip H. Lee is supported by the Generalist Physician-Scientist Training Program NIH-NCI 1T32 CA81211. Kenshi Yamasaki is a recipient of a post-doctoral fellowship from the Association for Preventive Medicine of Japan. DOI: 10.1111/j.0022-202X.2005.23713.x Manuscript received April 12, 2004; revised December 28, 2004; accepted for publication January 1, 2005
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Address correspondence to: Dr Richard L. Gallo, Division of Dermatology, University of California, San Diego, and VA San Diego Healthcare System, San Diego, California 92161, USA Email: rgallo@vapop. ucsd.edu
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