In vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against Mycobacterium tuberculosis

Abstract

Three independent assay methods were used to investigate the activities of antimicrobial peptides (human and rabbit defensins and protegrin from porcine leukocytes) against Mycobacterium tuberculosis in vitro. M. tuberculosis H37Ra was cultured in the presence of human neutrophil peptide 1, synthetic rabbit neutrophil peptide 1, or porcine protegrin 1 at 37؇C for 6 to 48 h, and antimycobacterial activity was measured by CFU assay. These peptides at a concentration of 50 g/ml showed significant antibacterial effects on M. tuberculosis after 24 and 48 h of incubation (85.9 to 97.5% at 24 h and 91.6 to 99.4% at 48 h). A radiometric method and a radial diffusion assay confirmed these observations. Antibacterial activity against M. tuberculosis was independent of calcium (1.0 mM) or magnesium (1.0 mM) and not inhibited by sodium chloride (100 mM). The optimal pH for antibacterial activity against M. tuberculosis was greater than 4.0. Three clinical isolates of M. tuberculosis were also studied, and these peptides showed 86.3 to 99.0% reduction in CFU of these organisms. Morphological studies using scanning electron microscopy showed that defensins caused lesions on the surface of H37Ra. These observations suggest that antimicrobial peptides such as defensins and protegrins may represent an important component of the host defense mechanism against M. tuberculosis and offer a potential new approach to therapy. Mycobacterium tuberculosis is still a leading cause of worldwide disease morbidity and is responsible for more deaths each year than any other single pathogen (32). In addition, the AIDS epidemic has exacerbated the problem. Interest in tuberculosis has been rekindled by the recent resurgence of cases both in the United States and worldwide. The increasing number of multidrug-resistant M. tuberculosis isolates that can be exceedingly difficult and expensive to treat is of particular concern (17). The combination of the increased frequency of infection due to M. tuberculosis and the increase in multidrugresistant M. tuberculosis isolates in patients with AIDS has raised a great deal of concern across the country. Defensins are endogenous antimicrobial peptides (AMPs) that contain 29 to 35 amino acid residues (27). These peptides were first recognized in rabbit and guinea pig neutrophils and in rabbit alveolar macrophages as ''lysosomal cationic proteins'' with antimicrobial properties (42, 43). Much earlier, calf thymus peptide (7) and lysozyme (34) were shown to inhibit the growth of pathogenic mycobacteria. More than 15 mammalian defensins derived from five species have been purified and sequenced, and human neutrophils were found to contain four defensin peptides (15). Defensins have been shown to possess antifungal (10), antibacterial (13, 16, 37), and antiviral (4) activities in vitro. We have previously demonstrated that the defensins human neutrophil peptide 1 (HNP-1), HNP-2, and HNP-3 have activity against Mycobacterium avium-Mycobacterium intracellulare (33). More recently, we have synthesized rabbit neutr...

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis

“…Perhaps is at this time-point when CRAMP exerts its maximal antimicrobial activity, as suggested by the electronmicroscopy study that revealed strong CRAMP immunolabelling in the cell wall and cytoplasm of phagocytosed mycobacteria. Our in vitro study showed that CRAMP has modest anti-mycobacterial activity with similar minimum inhibitory concentration to that of LL-37, which is not better than that reported elsewhere for other anti-microbial peptides, such as human neutrophil peptides (HNPs) [21][22][23][24] and b-defensins [23,25]. The ultrastructural study showed cell membrane dissolution in the areas where CRAMP labelling was strong, indicating structural damage.…”

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

“…91 The activity of NP-1 was followed by HNP-1, PG-1, Np-2, and HNP-2. 91 Similarly, Sharma and colleagues reported the half maximal inhibitory concentration (IC 50 ) and minimal inhibitory concentration (MIC) values for HNP-1 against the virulent M tuberculosis strain H37Rv as 0.8 μg/mL and 2.5 μg/mL, respectively. 90 They also demonstrated that HNP-1 inhibited 98% of H37Rv in 48 hours when used to treat infected murine J744A macrophages at a concentration of 40 μg/mL.…”

“…[74][75][76][77][78][79][80][81][82]86,87 In the search for smaller molecules with possible anti-mycobacterial activity, studies have identified several antimicrobial peptides (AMPs), including fragments of antimicrobial proteins and their analogues. 7,[88][89][90][91][92][93][94][95][96][97][98][99][100][101] AMPs are typically short (less than 50 amino acid residues), mostly cationic and amphipathic. 99,102,103 They are promising as novel anti-mycobacterial agents, especially against strains of MDR M tuberculosis, due to their direct and swift bactericidal activity against a wide range of microbes and due to the low probability of resistance acquisition by pathogens.…”

“…Miyakawa and colleagues identified NP-1 as the lead active peptide, which inhibited 98.9% of the avirulent M tuberculosis strain H37Ra at a concentration of 50 μg/mL. 91 The activity of NP-1 was followed by HNP-1, PG-1, Np-2, and HNP-2. 91 Similarly, Sharma and colleagues reported the half maximal inhibitory concentration (IC 50 ) and minimal inhibitory concentration (MIC) values for HNP-1 against the virulent M tuberculosis strain H37Rv as 0.8 μg/mL and 2.5 μg/mL, respectively.…”

Defensins and Other Antimicrobial Peptides and Proteins

“…Both human and rabbit defensins show activity against mycobacteria, including M. tuberculosis (Miyakawa et al 1996;Sharma et al 2000) and M. avium-intracellulare (Ogata et al 1992), even in the presence of divalent cations or physiologic salt concentrations. Although the plasma membrane of M. tuberculosis is the principal target for HNP-1 action, its DNA may be a secondary target (Sharma and Khuller 2001).…”