Abstract The biochemical mechanism of action of hu- man neutrophil peptide-1 (HNP-1) against Mycobac- terium tuberculosis H 37 Ra was studied. Mycobacteria grown in the presence of a subinhibitory concentration (IC 50 ) of HNP-1 showed a significant decrease in the biosynthesis of vital macromolecules, as shown by the in- corporation of various radiolabeled precursors. Mycobac- terial cells grown in the presence of HNP-1 exhibited sur- face changes, as was evident from the increased number of binding sites for L-anilinonaphthalene 8-sulfonate. Per- meability studies carried out with spheroplasts showed a significantly high permeability to a fluorescent probe, N-phenyl naphthylamine, in the presence of HNP-1. Sig- nificant changes in the cell wall and cell membrane were observed when HNP-1-grown cells were analysed by transmission electron microscopy. Our results suggest the mycobacterial cell wall/membrane to be the major tar- get(s) of HNP-1. Key words Defensins · HNP-1 · Mycobacterium tuberculosis · Anti-mycobacterial activity · Membrane permeability Introduction Although the incidence of tuberculosis had been declining in the past few decades, the trend has now reversed, and tuberculosis is now the most prevalent infectious disease in the world (Bloom and Murray 1992). This resurgence has been attributed to the AIDS epidemic and to the emer- gence of multidrug resistance, and it is further aggravated by operational difficulties in the tuberculosis control pro- gramme. Although antitubercular therapy constitutes the most important component of the tuberculosis control pro- gramme, the mortality from multi-drug-resistant strains of Mycobacterium tuberculosis is still very high. This has fo- cused attention on alternative therapeutic regimens with an effective bactericidal mechanism of action different from that of the presently available chemotherapeutic agents. Defensins are a family of small (3–4 kDa) antimicro- bial cationic peptides consisting of 29–35 amino acid residues. These peptides have been recognised as lysoso- mal cationic proteins from rabbit and guinea pig neu- trophils and have been shown to possess antibacterial ac- tivity (Zeya and Spitznagel 1963). They are present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) of human (Ganz 1987), rabbit (Selsted et al. 1984), rat (Eisenhauer et al. 1989) and guinea pig (Sel- sted and Harwig 1987), and in rabbit lung macrophages (Selsted et al. 1983). They have also been reported in hu- man and mouse small intestinal paneth cells (Jones and Bevins 1992). In humans, four endogenous antimicrobial peptides exist in neutrophils, viz. human neutrophil pep- tides 1–4. Human and animal defensins exhibit potent an- timicrobial activity against a wide variety of microorgan- isms such as gram-positive bacteria (Shimoda et al. 1995), gram-negative bacteria (Lehrer et al. 1989), fungi (Selsted et al. 1985), enveloped viruses (Lehrer et al. 1985) and spirochaetes (Borenstein et al. 1991), and they have been shown to kill Mycobacterium avium, Myco- bacterium intracellulare (Ogata et al. 1992) and M. tuber- culosis (Miyakawa et al. 1996). The mode of antimicro- bial action of defensins is not well-understood. However, the available evidence suggests that they act by disrupting cell membranes. It has been reported that they permeabi- lize the outer and inner membranes of Escherichia coli (Lehrer et al. 1989). Since no information is available on the biochemical mechanism of action of defensins against mycobacteria, the present study was designed to deter- mine the effect of human neutrophil peptide-1 (HNP-1) on M. tuberculosis H 37 Ra. Sudhir Sharma · Indu Verma · G. K. Khuller Biochemical interaction of human neutrophil peptide-1 with Mycobacterium tuberculosis H 37 Ra Arch Microbiol (1999) 171 : 338–342 © Springer-Verlag 1999 Received: 7 January 1999 / Accepted: 15 February 1999 ORIGINAL PAPER Sudhir Sharma, Indu Verma, G. K. Khuller () Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh – 160012, India Tel. +91-0172-541031-39, Ext. 274 and 282; Fax +91-0172-540401