Acta Dermato-Venereologica 98-7CompleteContent | Page 21

708 SHORT COMMUNICATION Identification of Antibacterial Components in Human Hair Shafts* Roopa S. SUBBAIAH 1 , Swat Kim KERK 1 , Yilong LIAN 1 , Declan LUNNY 2 , Siu Kwan SZE 3 , Kee Woei NG 4–6 , Artur SCHMIDTCHEN 1,7,8 and Sunil S. ADAV 1# 1 Lee Kong Chian School of Medicine, 3 School of Biological Sciences, 4 School of Materials Science and Engineering, 5 Nanyang Environment and Water Research Institute, (Environmental Chemistry and Materials Centre), Interdisciplinary Graduate School, Nanyang Technological University, Singapore, 2 Epithelial Biology, Institute of Medical Biology, Immunos, 6 Skin Research Institute of Singapore, Singapore, 7 Wound Healing Center, Bispebjerg University Hospital, Copenhagen, Denmark, and 8 Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden. # E-mail: [email protected] Accepted Apr 24, 2018; Epub ahead of print Apr 24, 2018 Antimicrobial peptides (AMPs) are evolutionarily old components of innate immunity. AMPs identified in human skin include defensins, cathelicidin, dermcidin, psoriasin, and RNase7 (1, 2). Some AMPs, such as the human cathelicidin peptide LL-37, are upregulated during inflammation in skin (1). Various microbes co- lonize the hair follicular canal, and various AMPs may modulate their population and composition (3, 4). It is not known whether the hair shaft per se contains functionally active antimicrobials. In a recent study, we explored different extraction procedures to determine the complete proteome of human hair shafts. That work focused on establishing potential biomarkers for hair with importance for eva- luating factors, such as ageing, infection, and potentially underlying follicular disorders. A multitude of potential antimicrobial peptides and proteins were observed, in- cluding lysozyme, various S100 proteins, and histones (5). This report focuses on the identification of hair- derived molecules with potential antimicrobial activity. Hair shafts were subjected to an acid-based extraction method (Appendix S1 1 ). The extracted material was fractionated using reverse-phase high-pressure liquid chromatography (RP-HPLC), and each fraction was analysed for antimicrobial activity by radial diffusion assay. Potential AMPs were identified by Western blot- ting combined with high-throughput mass spectrometry coupled with liquid chromatography. RESULTS AND DISCUSSION Hair shaft extraction using a buffer containing 8 M. urea (see Appendix S1 1 ), followed by mass spectrometri c analysis of this extract revealed the presence of multi- ple proteins (Table SI 1 ). Next, we attempted to detect possible antibacterial activity derived from hair shafts. Using RP-HPLC, a major protein peak was observed (Fig. 1A, peak at 18.44, fractions 17–23). The eluent was characterized using SDS-PAGE analysis (Fig. 1A insert) and antibacterial activity was analysed using a radial diffusion assay (RDA) by collecting and analysing *The Editor-in-Chief (AS) has not had responsibility for this article; it has been handled fully by the former Editor-in Chief, who made the decision to accept it. https://www.medicaljournals.se/acta/content/abstract/10.2340/00015555-2952 1 doi: 10.2340/00015555-2952 Acta Derm Venereol 2018; 98: 708–710 fractions eluted between 17 and 23 min (corresponding to 23–40% acetonitrile) (Fig. 1B). The results indicated that the antibacterial activity was related to proteins with molecular masses of 4–15 kDa. The RDA analysis demonstrated that fractions 19 and 20 showed zones of inhibition against Escherichia coli, indicating the presence of antimicrobials (Fig. 1B). The fractions showing antimicrobial activity were selected for further analysis by mass spectrometry. The fractions were found to contain multiple protein sequences (Table SII 1 ), in particular those linked to host defence, including multiple histone sequences. The gene ontology analysis is shown in Fig. 1C. One example of an identified histone sequence, H2A, is shown in Fig. 2A. To confirm the mass spectrometry results, RP-HPLC fractions 18–23 were further analysed by Western blot using specific rabbit polyclonal antibodies against histo- nes. The results are presented in Fig. 2B and demonstrate proteins of 24, 17 and 14 kDa, which correspond to histones H1, H2B and H3 and H2A, respectively. To illustrate the presence of one such histone variant, im- munohistochemistry analysis using an antibody against H3 was performed. The results indicated that this histone is localized in the cortex of hair shafts (Fig. 2C). Antimicrobial activity of histones was first reported in 1958 by Hirsch (6), who demonstrated that arginine- rich preparations (later denoted as histones) isolated from calf thymus exerted potent bactericidal activity. Subsequent reports showed that histones H1, H2A and H2B isolated from different species can act as potent antimicrobial agents (7–9). Recombinant human histone H1.2 shows activity against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, such as multidrug-resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) (10). Histone H2B is found in human placenta and co- lonic epithelial cells, but also in the skin of tree frogs (11). Chicken H2B (12) displays antimicrobial activity against both Gram-positive and Gram-negative bacte- ria. Histones H4 and H3 obtained from calf thymus are active against E. coli, and histone H4 also exerts anti- microbial activity (13). Recombinant histone H4 from human sebocytes inhibited the growth of S. aureus and Propionibacterium acnes. Antimicrobial histones are also major components of neutrophil extracellular traps This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2018 Acta Dermato-Venereologica.