Peptides 25 (2004) 1075–1077 In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis Lanfranco Fattorini a , Renato Gennaro b , Margherita Zanetti c,d , Dejiang Tan a , Lara Brunori a , Federico Giannoni a , Manuela Pardini a , Graziella Orefici a,∗ a Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Rome, Italy b Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università di Trieste, Trieste, Italy c Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Udine, Italy d Laboratorio Nazionale CIB, AREA Scienze Park, Padriciano, Italy Received 29 January 2004; received in revised form 31 March 2004; accepted 1 April 2004 Available online 10 May 2004 Abstract The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone. © 2004 Elsevier Inc. All rights reserved. Keywords: Mycobacterium tuberculosis; Protegrin-1; Human beta-defensin-1; Isoniazid; Activity in vitro 1. Introduction Cationic antimicrobial peptides (AMPs) from eukary- otes are components of the innate immune defense and display potent antimicrobial activity against a variety of microorganisms [13]. AMPs appear to interact and cross cell envelope membranes of microbes and kill cells by a multihit mechanism that involves action on more than one anionic target. Protegrin-1 (PG-1), human alpha-defensin-1, and PR-39 are known to be active against Mycobacterium tuberculosis (MTB) [7–9], the organism responsible for tuberculosis, but no studies on the combination between AMPs and anti-MTB drugs have been performed. We there- fore tested the activity of two cysteine-rich AMPs, namely the 18-residue beta-sheet PG-1 from porcine leukocytes [3] and the 36-residue beta-sheet epithelial beta-defensin-1 (HBD-1) from humans [4], both alone and in combi- nation with the anti-MTB drug isoniazid (INH), against drug-susceptible or multidrug-resistant (MDR) MTB strains. ∗ Corresponding author. Tel.: +39-06-499-02-333; fax: +39-06-493-871-12. E-mail address: gorefici@iss.it (G. Orefici). 2. Materials and methods The drug-susceptible strain M. tuberculosis H37Rv (ATCC 27294) and the MDR RM22 strain resistant to iso- niazid, rifampin, streptomycin, ethambutol, pyrazinamide, rifabutin, rifapentine were used [2]. Strains were grown on Middlebrook 7H11 agar (Difco Laboratories, Detroit, MI) at 37 ◦ C under a humidified 5% CO 2 atmosphere for 3 and 4 weeks, respectively. Bacterial suspensions were prepared by dispersing colonies with glass beads in Middlebrook 7H9 broth (Difco). The tubes were vortexed for 1 min and allowed to stand for 30 min to allow larger particles to settle; the upper supernatant was stored at -80 ◦ C until use. PG-1 and HBD-1 were synthesized by the solid phase method, using an automated Milligen 9050 peptide synthesizer [1]. Carboxyl activation was carried out with TBTU/HOBt or with HATU for difficult couplings. After cleavage and de- protection, the peptides were purified by RP-HPLC and their molecular mass was determined with an API I ion spray mass spectrometer. Both PG-1 and HBD-1 were air-oxidized in diluted solution under continuous stirring for 36–48 h. Disulfide bond formation was verified by mass spectrom- etry. The AMPs were dissolved in 0.01% acetic acid and diluted with Middlebrook 7H9 broth for drug susceptibility testing. The anti-MTB activity was determined by a 96-well microplate assay. One hundred and sixty microliters of 0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.04.003