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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: J Invest Dermatol. 2009 Jun 18;129(10):2489–2496. doi: 10.1038/jid.2009.106

Histone H4 Is a Major Component of the Antimicrobial Action of Human Sebocytes

Dong-Youn Lee 1,4, Chun-Ming Huang 1, Teruaki Nakatsuji 1, Diane Thiboutot 2, Sun-Ah Kang 3, Marc Monestier 3, Richard L Gallo 1
PMCID: PMC2765036  NIHMSID: NIHMS147619  PMID: 19536143

Abstract

Antimicrobial peptides, such as cathelicidin and β defensins, directly kill microbes and have been detected in human sebaceous glands and cell lines. Despite the presence of several such peptides, the apparent abundance of these is insufficient for direct killing of most skin pathogens. In this study, we sought to determine which molecules provide the majority of antimicrobial peptide activity in human sebocytes. Acid-soluble protein extracts of SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography and were assayed for their capacity to inhibit the growth of Staphylococcus aureus. Antimicrobial activity was isolated in a single major fraction and identified to be histone H4 by mass spectrometry and western blot analysis. The importance of histone H4 in the antimicrobial activity of sebocytes was confirmed by a specific neutralizing antibody and by direct demonstration that recombinant histone H4 had antimicrobial activity against S. aureus and Propionibacterium acnes. In addition, histone H4 enhanced the antimicrobial action of free fatty acids in human sebum. Taken together, these results indicate that the release of histone H4 by holocrine secretion from the sebaceous gland may play an important role in innate immunity.

INTRODUCTION

Antimicrobial peptides are crucial components of the innate immune system that protects against microbial infection (Zasloff, 2002). In part, acting as natural antibiotics, antimicrobial peptides have a distinct and overlapping antimicrobial activity against a variety of microbial pathogens (Bals, 2000; Nagaoka et al., 2000). They are produced constitutively or can be induced in a variety of types, including phagocytes and epithelial cells (Gallo et al., 1994; Ganz, 2002), and they can directly kill a broad spectrum of bacteria, fungi, and viruses. Their role is particularly important in the skin where they play an important role in first-line defense against infection (Izadpanah and Gallo, 2005). In the keratinocyte, cathelicidins and β defensins are major families of antimicrobial peptides (Frohm et al., 1997; Ali et al., 2001). In addition, however, many other peptides and proteins in the skin also show antimicrobial activity, including proteinase inhibitors, chemokines, and neuropeptides (Braff et al., 2005).

The sebaceous gland is a major appendage and exocrine gland in the skin consisting of specialized epithelial cells named sebocytes (Thody and Shuster, 1989; Thiboutot, 2004). Human sebaceous glands are distributed in all areas of the skin except in that of the palms and soles. Although the sebaceous gland produces and secretes lipid-rich sebum onto the external skin surface by the holocrine rupture of mature sebocytes, the fundamental functions of this gland have been only partly understood (Zouboulis, 2003). These functions include the hypothesis that the sebocyte provides an innate immune defensive function. This has been supported by observations that antimicrobial peptides are produced by these cells. For example, human β defensins (HBD)-1 and -2 were found in human pilosebaceous units by in situ hybridization and immunohistochemistry (Chronnell et al., 2001). An HBD-2 mRNA expression was induced in SZ95 sebocytes by Propionibacterium acnes (P. acnes), a predominant organism in pilosebaceous units (Nagy et al., 2006). Psoriasin (S100A7) has been detected in sebaceous glands by immunohistochemistry (Glaser et al., 2005). Recently, we reported that human sebocytes express cathelicidin antimicrobial peptides and can act to kill P. acnes (Lee et al., 2008). Moreover, sebaceous lipids exhibit antibacterial activity (Wille and Kydonieus, 2003; Georgel et al., 2005). These findings suggested that the human sebaceous gland may play an important role in the innate immunity of the skin through the expression of antimicrobial peptides and specific lipids. However, the measured concentration of the previously defined antimicrobial peptides in the extracts of sebocytes was lower than the concentration necessary for antimicrobial activity. Thus, although multiple antimicrobial peptides have been detected in sebocytes, it remains unclear which are functionally important to immune defense. Here, we carried out an unbiased functional screening of proteins derived from sebocytes for antimicrobial activity. On the basis of this screening, we identified histone H4 as the predominant peptide active against Staphylococcus aureus (S. aureus) and P. acnes.

RESULTS

Purification of antimicrobial activity from cultured human sebocytes

To identify antimicrobial molecules produced by the sebaceous gland, SEB-1 sebocytes were subjected to a variety of initial extraction methods including chloroform, detergent, and aqueous extraction techniques. Only acid extracts, predominantly representing the acid-soluble protein fraction of the cell, were antimicrobial in the solution-killing assay against S. aureus ΔmprF, a well-defined S. aureus strain selected for its sensitivity to cationic antimicrobial peptides. These extracts were subsequently separated by reverse-phase high-performance liquid chromatography (RP-HPLC) (Figure 1a) and by the bactericidal activity of the HPLC fractions examined against S. aureus by limiting dilution. In this screening, only a single fraction (fraction 13) was confirmed to have an antibacterial activity (Figure 1b).

Figure 1. Purification of the antimicrobial activity from extracts of cultured sebocytes.

Figure 1

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(a) RP-HPLC was carried out to separate antimicrobial peptides from acid-soluble extracts of SEB-1 sebocytes. A plot of absorbance at 214nm is indicated on the left y axis and that of the concentration of acetonitrile used for elution on the right y axis. (b) The antimicrobial activity of the HPLC fractions was evaluated against S. aureus ΔmprF by solution-killing assay. Growth of bacterial colonies on agar after incubation with fractions obtained by RP-HPLC separation is shown with limiting dilutions from top to bottom. A single fraction (fraction 13) showed maximal antimicrobial activity. (c) HPLC fractions were subjected to SDS-PAGE and stained with Coomassie’s blue. Arrow in the active fraction indicates a unique protein band that was further analyzed by mass spectrometry.

Identification of Histone H4 as an antimicrobial peptide

As predicted by the initial HPLC tracing, each of the fractions separated from sebocyte extracts contained multiple protein bands (Figure 1c). On the basis of the findings of the antimicrobial activity and the SDS-PAGE, one band migrating at approximately 11 kDa in the active fraction was found to be interesting. To identify this molecule, tryptic peptide digests were analyzed by Nano liquid chromatography-mass spectrometry (MS). Histones, H2A, H2B, and H4, were identified with H4 being most abundant (Table 1).

Table 1.

Proteins identified by mass spectrometry from sebocyte antimicrobial fraction

Identified
protein		 Accession
no./database		 Peptide sequence Sequence
coverage (%)
Histone H4 NP_778224.1/ ISGLIYEETR 63.1
NCBI TLYGFGG
DAVTYTEHAK
KTVTAMDVVYALK
TVTAMDVVYALK
VFLENVIR
RISGLIYEETR
DNIQGITKPAIR
DNIQGITK
DNIQGITKPAIRR
NP_003538.1/ ILGLIYEETRR 19.4
NCBI ILGLIYEETR
DNIQGITK
Histone H2B NP_778225.1/ AMGIMNSFVNDIFER 27
NCBI KESYSIYVYK
LLLPGELAK
NP_066407.1/ KESYSVYVYK 27
NCBI LLLPGELAK
AMGIMNSFVNDIFER
Histone H2A NP_002097.1/ ATIAGGGVIPHIHK 23.4
NCBI AGLQFPVGR
HLQLAIR
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To confirm these results, each of the HPLC fractions was evaluated by western blot with an anti-histone antibody (BWA-3). BWA-3 is a monoclonal antibody that reacts with a common sequence in the amino-terminus of histones H2A and H4 (Monestier et al., 1993). In fraction 13, one strong band was seen at 11 kDa, which corresponded to the expected size of histone H4 (Figure 2a) and to the unique protein predicted to have antimicrobial activity in Figure 1c. In fraction 12, one strong band was observed at 13 kDa, which corresponded to the expected size of histone H2A (Figure 2a). An additional western blot analysis with a different antibody that specifically reacts with acetyl histone H4 confirmed that the band in fraction 13 is H4 (Figure 2b). Evaluation of the total acid-soluble extracts of SEB-1 by western blot with the anti-histone antibody (BWA-3) further confirmed the presence of the two histone bands at 11 and 13 kDa, corresponding to histones H4 and H2A (Figure 2c). Histone H4 was much more abundant than histone H2A in the total extract. A similar result was obtained in the acid-soluble fraction of the SEB-1 debris released into the culture medium, also showing two bands, with the majority being histone H4 (Figure 2c). To further confirm histone H4 expression in human sebaceous glands, histological sections of normal human scalp tissue were examined for histone H4 expression by immunohistochemistry with BWA-3. As expected for this essential component of the chromosome, histone H4 was detected in the nuclei of the sebocytes and in those of all other cells within the biopsy (data not shown). Thus, on the basis of the results of functional HPLC analysis, Coomassie staining, mass spectrometry, and western blot analysis, histone H4 was identified as a top candidate responsible for the antimicrobial activity in fraction 13.

Figure 2. Identification of histone H4 in antimicrobial fractions purified from cultured sebocytes.

Figure 2

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(a) Western blot analysis with anti-histone antibody (BWA3, specific for histones H2A and H4) was carried out on HPLC fractions shown in Figure 1. In fraction 13, a single strong band was observed at 11 kDa, which corresponded to the expected mass of histone H4. In fraction 12, a single strong band was observed at 13 kDa, which corresponded to the expected mass of histone H2A. The lane marked as H4 was loaded with recombinant histone H4 used as a positive control. (b) Western blot analysis with the specific anti-acetyl histone H4 antibody showed a single band at 11 kDa only in fraction 13. (c) Acid-soluble extracts of SEB-1 sebocytes and the culture medium containing SEB-1 sebocyte debris was evaluated by western blot with an anti-histone H2A and H4 antibody (BWA-3). Two bands were observed at 11 and 13 kDa, which corresponded to histones H4 and H2A. H4, recombinant histone H4, was used as a positive control.

Histone H4 has direct antimicrobial activity against S. aureus and P. acnes

To confirm whether histone H4 can kill bacteria found on the skin, bactericidal assays were carried out using a purified recombinant histone H4 protein against relevant skin pathogens such as S. aureus or P. acnes. Bacteria were incubated for 3 hours at 37°C in an assay buffer consisting of a 10mm sodium phosphate buffer (pH 6.5) and 100mm NaCl. Histone H4 showed bactericidal activity against both S. aureus ΔmprF (25 µg ml−1) and P. acnes (12.5 µg ml−1) in a dose-dependent manner compared with the control (Figure 3). Furthermore, as histone H4 is released from sebocytes along with lipids, which are metabolized into free fatty acids with antimicrobial activity, the combined antimicrobial activity of histone H4 in the presence of free fatty acids was examined. Lauric acid and oleic acid at low concentrations (1.5 µg ml−1) enhanced the antimicrobial action of histone H4 (Figure 4). At higher concentrations of fatty acids (60 µg ml−1), lauric acid completely inhibited S. aureus survival at 12.5 µg ml−1 histone H4, but oleic acid at this concentration killed all of the bacteria. Thus, as expected, free fatty acids present on the skin are inherently antimicrobial, but will also enhance the antimicrobial potency of peptides such as histone H4 released by the sebocyte.

Figure 3. Recombinant histone H4 kills S. aureus and P. acnes.

Figure 3

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The antimicrobial activity of recombinant histone H4 was evaluated against S. aureus ΔmprF and P. acnes by the solution-killing assay. Bacteria were incubated for 3 hours at 37°C in a 10mm sodium phosphate buffer (pH 6.5) containing 100mM NaCl. Recombinant histone H4 showed significant bactericidal activity against S. aureus ΔmprF at 25 µg ml−1 and above and against P. acnes at 12.5 µg ml−1 and above. Data shown are from triplicate determinations (**P<0.01; Student’s t-test).

Figure 4. Recombinant histone H4 enhances the antimicrobial action of free fatty acids.

Figure 4

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The antimicrobial activity of histone H4 was evaluated in the presence of free fatty acids. Lauric and oleic acids (1.5 µg ml−1) synergistically exerted antimicrobial effects in combination with histone H4. Lauric acid (60 µg ml−1) exerted a stronger synergy with histone H4, but oleic acid at this concentration killed all bacteria. Data shown are from triplicate determinations (*P<0.05; Student’s t-test). DMSO, dimethyl sulfoxide: LA, lauric acid: OA, oleic acid.

Histone H4 is a major component of the antimicrobial activity of sebocyte extracts

Having found that histone H4 is a possible antimicrobial protein, we next sought to determine the relative contribution of histone H4 to the functional antimicrobial activity observed from sebocytes. The specific histone antibody (BWA-3) described earlier (Kim et al., 2002) was used in a functional assay in an attempt to block the activity of histones H4 and H2A in the entire SEB-1 extract. The addition of BWA-3 to the extracts of SEB-1 sebocytes significantly inhibited antimicrobial activity against S. aureus ΔmprF as seen by an approximately two logs better growth of bacteria in the presence of a sebocyte extract plus BWA-3 compared with that with a sebocyte extract plus IgG at a similar concentration, or with PBS as controls (Figure 5). Thus, the ability to neutralize the majority of the antimicrobial action of sebocyte extracts with a specific antibody to histone H4 suggests that histone H4 is the major acid-soluble antimicrobial protein released by the sebocyte.

Figure 5. Neutralization of histone H4 in sebocyte extracts removes antimicrobial activity.

Figure 5

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BWA-3 neutralizing antibody was used to treat extracts of SEB-1 sebocytes. Addition of BWA-3 (400 µg ml−1) to the extracts of SEB-1 sebocytes significantly decreased antimicrobial activity against S. aureus ΔmprF compared with an equal concentration of IgG added to separate aliquots, or aliquots to which an equal volume of PBS was added as controls. Data shown are from triplicate determinations (*P<0.05; Student’s t-test).

DISCUSSION

Many studies have shown that antimicrobial peptides are essential elements of epithelial defense, providing a barrier against infection of the skin and other epithelial organs (Zasloff, 2002; Braff et al., 2005). Human sebaceous glands may contribute to the defense of the skin through the release of antimicrobial peptides (Chronnell et al., 2001; Glaser et al., 2005, Lee et al., 2008). In this study, we examined human sebocytes to identify functionally important antimicrobials in human sebaceous glands. In a non-biased biochemical screening, histone H4 was found to be the major antimicrobial protein in the extracts of SEB-1 sebocytes. Recombinant histone H4 showed antimicrobial activity against S. aureus and P. acnes, both potential pathogens of the human pilosebaceous unit. Finally, by selective neutralization of histone, this protein was found to have a major role in the antimicrobial activity of sebocytes. These observations suggest that histone H4 in the human sebaceous gland can contribute to the innate immune defense of the skin, providing an additional source of antimicrobial activity that acts in the defense against infection.

To our knowledge, this is the first report showing that histones can act as antimicrobial peptides on human skin. Although histone proteins exist primarily in the nucleus and are known for their involvement in chromatin formation and in the regulation of transcription, there is precedence for the conclusion that histones or histone-derived peptides can have antimicrobial properties (Parseghian and Luhrs, 2006). Other activities such as hormone-like activity and endotoxin-neutralizing activity have also been reported for histones (Reichhart et al., 1985; Kim et al., 2002). In humans, histone H1, derived from epithelial cells in the gastrointestinal tract, is active against Salmonella typhimurium (Rose et al., 1998). The histones, H2A and H2B, are secreted into the amniotic fluid from epithelial cells on the surface of the human placenta and they participate in host defenses of the fetus (Kim et al., 2002). Recently, neutrophil extracellular traps containing histones in human neutrophils were identified as a novel antimicrobial mechanism (Brinkmann et al., 2004). In this study, the antimicrobial action of histone is particularly relevant in the setting of the sebaceous gland function, as these cells secrete their contents into the sebum by disintegration of the entire cell, a process known as holocrine secretion. Thus, it is particularly relevant for sebocytes to use histones as a part of their antimicrobial system. The current data detecting antimicrobial activity for histone H4 in the extracts of human sebocyte cultures, and in the culture medium from these cells, support the conclusion that histone H4 from human sebaceous glands in vivo is released with sebum onto the surface of the skin, and once released can act as an antimicrobial.

As major components of the nucleosome structure in eukaryotic cells, the histones are functionally classified into two groups: linker histones (H1), which seal loops of DNA and keep the nucleosome structures condensed in compact conformations, and core histones (H2A, H2B, H3, and H4), which form an octameric complex to produce the nucleosome. Most earlier studies have shown that the lysine-rich histones (H1, H2A, and H2B) and their fragments have antimicrobial activity (Hiemstra et al., 1993; Rose et al., 1998; Kim et al., 2002; Howell et al., 2003). There have been only a few reports on the antimicrobial activities of histone H4. A mixture of histones H2B and H4 obtained from hemocytes of Pacific shrimp showed an activity against Gram-positive bacteria (Patat et al., 2004). In human meconium, the histones H2A and H4 have been isolated and identified by their antimicrobial activity (Kai-Larsen et al., 2007). Recently, the post-translationally modified histone H4 was purified from the skin extract of a Japanese tree frog, but the protein did not show any antimicrobial activity (Kawasaki et al., 2008). Histone H4, as well as H2A, H2B, and H3, have shown bacterial LPS-binding abilities (Augusto et al., 2003). In addition, as mentioned earlier, the neutrophil extracellular traps have been observed to react with antibodies against the histones, H1, H2A, H2B, H3, and H4, and may be a part of their antimicrobial function (Brinkmann et al., 2004). In this study, we showed by solution-killing assay that histone H4 contributes to the antimicrobial activity of the extracts of SEB-1 sebocytes, and that this occurs in physiologically relevant ionic conditions and against relevant microbes such as S. aureus and P. acnes.

To detect histones in HPLC fractions and in the extracts of sebocytes, we used a monoclonal anti-histone antibody (BWA-3). BWA-3 reacts with peptides l–20 of histone H2A and l–29 of histone H4 (Monestier et al., 1993). This antibody was previously also used as a neutralizing antibody (Kim et al., 2002). As a result, it was relevant to test the addition of this antibody as a neutralizing antibody to the extracts of SEB-1 sebocytes, and results show that this significantly decreased the antimicrobial activity. Although the neutralizing antibody blocked both histones H2A and H4, the extracts of the sebocytes had a higher histone H4 content than a histone H2A content, and antimicrobial activity was not detected in HPLC fractions that contained abundant histone H2A but not histone H4. These results suggested that the antimicrobial activity in the extracts of sebocytes was likely because of histone H4 and not because of histone H2A.

Several antimicrobial peptides are found in sebocytes and may work with histone H4 to increase antimicrobial activity. Earlier, human β defensins-1 and -2 were described in human pilosebaceous units, including the sebaceous glands (Chronnell et al., 2001). Psoriasin (S100A7) was found in human sebaceous glands (Glaser et al., 2005) and our lab has recently shown that cathelicidin antimicrobial peptides are present in cultured human sebocytes and glands (Lee et al., 2008). On the basis of the results of the neutralization of antimicrobial activity with an anti-histone H4 antibody, our data suggest that histone H4 probably plays a larger role as a direct antimicrobial substance than do the other antimicrobial peptides previously identified in the sebocyte. This conclusion is consistent with the approximate concentration of these antimicrobial peptides compared with their potency to kill bacteria. However, the actual concentration of histone H4 and other antimicrobial peptides released by human sebaceous glands remains unknown. Nevertheless, on the basis of the known additive or synergistic activity of antimicrobial peptides (Nagaoka et al., 2000), it is likely that these factors work together to provide an immune defense.

In addition to antimicrobial peptides in the human sebaceous gland, sebaceous lipids have been shown to exhibit an innate antimicrobial activity (Georgel et al., 2005; Clarke et al., 2007). In the sebum, oleic acid predominates, and lauric acid is one of the strongest antimicrobial lipids against Gram-positive bacteria, although it is a minor free fatty acid (Wille and Kydonieus, 2003; Bodoprost and Rosemeyer, 2007). Thus, we chose these free fatty acids for combined effects with histone H4. Histone H4 showed synergistic antimicrobial effects with free fatty acids against S. aureus. These findings suggest that the production of lipids by the sebaceous gland, and the metabolism to antimicrobial free fatty acids by resident microbes, may further contribute to epithelial defense by combining activity with histone H4 and the antimicrobial peptides. However, an alternative explanation for the role of histone H4 in the sebaceous gland is that it may serve a more specialized and focused role in the protection of the gland itself against infection. In this study, histone H4 was shown to kill P. acnes, a normal microflora in the pilosebaceous unit. Therefore, sebaceous antimicrobial peptides and lipids may act only under special circumstances, such as microbial proliferation in the follicle.

Taken together, our data suggest that histone H4 is a functionally important antimicrobial peptide released by the holocrine secretion mechanism of human sebocytes. Further, in vivo studies of the surface expression of histones on human skin, and mechanistic studies to determine how histone H4 acts as an antimicrobial peptide, are required to fully elucidate their role in skin defense.

MATERIALS AND METHODS

Culture of SEB-1 human sebocytes

The immortalized human sebaceous gland cell (sebocyte) line, SEB-1, was grown in a culture medium consisting of DMEM with 5.5mm glucose/Ham’s F-12 3:1, fetal bovine serum 2.5%, adenine 1.8 × 10−4 M, hydrocortisone 0.4mg ml−1, insulin 10 ng ml−1, epidermal growth factor 3 ngml−1, and cholera toxin 1.2 × 10−10 M at 37°C in a humidified atmosphere containing 5% CO2 in air (Thiboutot et al., 2003).

Extraction of acid-soluble proteins from SEB-1 sebocytes

SEB-1 sebocytes were cultured to confluence in 75 cm2 culture flasks. After washing with PBS, SEB-1 sebocytes were extracted in 1M HCl-1% trifluoroacetic acid. The extracts of the SEB-1 sebocytes in the acid were centrifuged at 12,000 r.p.m., and the supernatant was collected. The supernatant was then frozen and lyophilized. In addition, the debris of the SEB-1 sebocytes in the culture medium was centrifuged and extracted in the same way as the extraction of the SEB-1 sebocytes.

Separation of antimicrobial peptides

Antimicrobial peptides were separated from the acid extracts of the SEB-1 sebocytes by a previously described method with slight modifications (Murakami et al., 2004). The lyophilized material of the acid-soluble protein extracts of the SEB-1 sebocytes was dissolved in 10% acetonitrile-0.1% trifluoroacetic acid. Peptide separation was carried out using an AKTA purification system (Amersham Pharmacia Biotech, Piscataway, NJ) on a Sephasil peptide C18 column (12 µm, ST 4.6/250; Amersham Pharmacia Biotech). The acid-soluble protein extracts of the SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) after column equilibration in 0.1% trifluoroacetic acid at a flow rate of 2ml min−1. Elution was carried out using gradients of 0–25% and 25–60% acetonitrile for 5 or 25 minutes. The column effluent was monitored at 214, 230, and 280 nm. All collected fractions (1.5 ml) were frozen and lyophilized, and then resuspended in distilled water for antimicrobial assay and immunoassay. The protein concentration was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL), with BSA as a standard.

Antimicrobial activity assay of HPLC fractions

For screening the antimicrobial activity of the HPLC fractions, a solution-killing assay was carried out as described earlier (Lee et al., 2008). Lyophilized HPLC fractions were resuspended in 30 µl of distilled water and were then tested against S. aureus ΔmprF (a gift from A. Peschel, Microbial Genetics, University of Tubingen, Tubingen, Germany). This strain of S. aureus was selected for screening because of its increased sensitivity to cationic peptides. S. aureus ΔmprF was grown in a tryptic soy broth and collected in the log phase. The number of S. aureus ΔmprF was enumerated by applying a conversion factor (2.0 × 108 bacteria per ml=0.6 OD unit at 600 nm). The bacteria (CFU) were suspended in a 10mm sodium phosphate buffer (pH 6.5) supplemented with 100mm NaCl, and 5 µl of HPLC fractions were added and incubated at 37°C for 3 h. After incubation, 10-fold dilutions were prepared and plated on tryptic soy broth agar and the plates were incubated for 1 day at 37°C.

SDS-PAGE

The HPLC fractions were analyzed for the content of proteins/peptides by SDS-PAGE. An equal volume of HPLC fractions was prepared by mixing with a sample buffer, and was then denatured at 95°C for 5 minutes. Electrophoresis was carried out on 16% Tris-tricine gels (NuSep, Austell, GA) with running buffer for 2 hours at 100 V. To visualize the protein/peptide bands, the gel was stained with Coomassie solution according to the manufacturer’s instructions.

Mass spectrometry analysis

For protein identification, one band of interest in the active fraction was cut out of the Coomassie stained gel and enzymatically digested with trypsin (0.2 µg, 25mm Tris, 4M urea, pH 7.8) at 37°C overnight. Tryptic peptide digests were analyzed by Nano liquid chromatography-mass spectrometry as described earlier (Nakatsuji et al., 2008). The automated Nano liquid chromatography-mass spectrometry setup consisted of an Agilent 1200 Nano 2D LC system, a switch valve, a C18 trap column (Agilent, Santa Clara, CA), and a capillary reverse-phased column (10 cm in length, 75 µm i.d.) packed with 5 µm C18 AQUASIL resin with an integral spray tip (Picofrit, 15 µm tip, New Objective, Woburn, MA). A reverse-phase LC, directly linked to a Bruker Daltonics HCTultra PTM system mass spectrometer (Bruker Daltoniks, Bremen, Germany), was carried out using a linear gradient elution from buffer A (H2O plus 0.1% formic acid) to 50% buffer A plus 50% buffer B (acetonitrile plus 0.1% formic acid) in 100 minutes. The setup was operated in the data-dependent mode. Data on the four strongest ions above an intensity of 2 × 105 were collected with dynamic exclusion enabled, and the collision energy was set at 35%. The MS/MS spectra were extracted using a default value by BioTools 3.1 (Bruker Daltoniks). All MS/MS spectra were analyzed using in-house Mascot 2.1 server (Matrix Science, London, UK) for protein identification. Mascot was established to search the human database (downloaded from the NCBI website http://www.ncbi.nlm.nih.gov/) containing protein sequences in both forward and reverse orientations using trypsin as a digestion enzyme.

Western blot analysis

The fractions separated by HPLC were evaluated by western blot analysis. First, SDS-PAGE was carried out as described above and then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Danvers, MA). For a positive control, the recombinant histone H4 peptide (Biolabs, New England) was used. The membrane was treated with a blocking solution for 30minutes at room temperature, and then a mouse monoclonal anti-histone antibody (BWA-3) (10 µg ml−1 in blocking solution) or a rabbit polyclonal antiacetyl histone H4 antibody (Cell Signaling, Danvers, MA) was incubated with the membrane overnight at 4°C. BWA-3 reacts with histones, H2A and H4 (Monestier et al., 1993). After washing the membrane three times with 0.1% TTBS, a horseradish peroxidase-labeled goat anti-mouse polyclonal antibody (1:5,000 in the blocking solution; DAKO, Carpinteria, CA) or a horseradish peroxidase-labeled goat anti-rabbit polyclonal antibody was incubated with the membrane for 60 minutes at room temperature. After washing the membrane with 0.1% TTBS, it was immersed in an ECL solution (Western Lightning Chemiluminescence Reagents Plus; Perkin-Elmer Life Sciences, Boston, MA) for 60 seconds, and was then exposed to an X-ray film (X-Omat; Eastman Kodak, Rochester, NY). In addition, acid-soluble extracts of the SEB-1 sebocytes, and of the debris of the SEB-1 sebocytes in the culture medium, were evaluated by western blot analysis with an anti-histone antibody (BWA-3).

Antimicrobial activity assays

To evaluate the antimicrobial activity of the recombinant histone, H4, solution-killing assay was performed. Recombinant histone H4 peptide (Biolabs), which was dissolved in the PBS buffer, was tested against S. aureus ΔmprF and P. acnes. The bacteria were exposed to a range of recombinant histone H4 peptide concentrations (100 µg ml−1, 50µg ml−1, 25µg ml−1, and 12.5 µg ml−1) at 37°C for 3 hours. The same PBS buffer was used as a negative control. S. aureus ΔmprF was grown as described above. The P. acnes strain, ATCC6919 (American Type Cell Culture, Manassas, Virginia), was grown under anaerobic conditions in Reinforced Clostridial Medium (Oxoid, Basingstroke, England) for 2 days and collected in the log phase. The number of P. acnes was enumerated by applying a conversion factor (P. acnes 5 × 108 bacteria per ml=1.0 OD unit at 600 nm). After incubation, 10-fold dilutions were prepared and plated on solid media comprised of Brucella broth (BD Biosciences, San Diego, California) with 5% sheep red blood cells (Remel, Lenexa, Kansas), 0.5mgl−1 vitamin K, and 5.0 mgl−1 hemin (Remel). The plates were incubated for 4 days at 37°C under anaerobic conditions, and then individual colonies were counted. The killing efficiency of the recombinant histone H4 peptide was plotted using CFU per ml−1 after peptide incubation versus peptide concentrations, representing the survivor cells and the toxic potency of the histone H4 peptide against S. aureus ΔmprF and P. acnes.

To explore the contribution of the histone H4 to the antimicrobial activity of the acid-soluble protein extracts of the SEB-1 sebocytes, the lyophilized extracts of the SEB-1 sebocytes were resuspended in PBS. The extracts of the SEB-1 sebocytes were combined in triplicate with a monoclonal mouse anti-histone antibody (BWA-3) (400 µg ml−1) or with the same concentration of control IgG or PBS for 2 hours at room temperature and analyzed for antimicrobial activity against S. aureus ΔmprF using the solution-killing assay.

To evaluate the combined antimicrobial activity of histone H4 with free fatty acids, a solution-killing assay was carried out. Two free fatty acids (Sigma, St. Louis, MO), which were dissolved in dimethyl sulfoxide (DMSO), were tested against S. aureus ΔmprF. The bacteria were exposed to a range of recombinant histone H4 peptide concentrations (50 µg ml−1, 25µg ml−1, and 12.5 µg ml−1) in the PBS buffer in the presence of lauric or oleic acid (1.5 µg ml−1 and 60 µg ml−1) at 37°C for 3 hours. DMSO [5% (v v−1)] was used as a negative control. After incubation, 10-fold dilutions were prepared and plated on tryptic soy broth agar, and the plates were incubated for 1 day at 37°C.

Abbreviations

HPLC

high-performance liquid chromatography

MS

mass spectrometry

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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