Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a major worldwide health problem in part due to the lack of development of new treatments and the emergence of new strains such as multidrug-resistant (MDR) and extensively drug-resistant strains that are threatening and impairing the control of this disease. In this study, the efficacy of natural and synthetic cationic antimicrobial (host defence) peptides that have been shown often to possess broad-spectrum antimicrobial activity was tested. The natural antimicrobial peptides human LL-37 and mouse CRAMP as well as synthetic peptides E2, E6 and CP26 were tested for their activity against Mycobacterium tuberculosis both in in vitro and in vivo models. The peptides had moderate antimicrobial activities, with minimum inhibitory concentrations ranging from 2 μg/mL to 10 μg/mL. In a virulent model of M. tuberculosis lung infection, intratracheal therapeutic application of these peptides three times a week at doses of ca. 1 mg/kg led to significant 3–10-fold reductions in lung bacilli after 28–30 days of treatment. The treatments worked both against the drug-sensitive H37Rv strain and a MDR strain. These results indicate that antimicrobial peptides might constitute a novel therapy against TB.

Introduction

Tuberculosis (TB) [1], caused by the bacterium Mycobacterium tuberculosis, remains one of the leading causes of disease and mortality due to an infectious agent. According to recent data from the World Health Organization (WHO), in 2010 there were 8.8 million active TB cases worldwide and nearly 1.5 million deaths. It has been estimated that one-third of the human population carries M. tuberculosis and 10% of these people will develop active disease at some time in their lives, creating an enormous reservoir [2].

Treatment of pulmonary TB has become increasingly challenging due in part to the required long duration of therapy and the advent of multiple drug resistance. One of the most important factors is the emergence of multidrug-resistant (MDR) bacilli that has been associated with inadequate use of antibiotics and poor adherence to recommended treatment regimens [3]. In recent years, new strains have emerged, termed extensively drug-resistant (XDR), that are also resistant to second-line antibiotics such as fluoroquinolones and either kanamycin, amikacin or capreomycin. These strains lead to poor treatment outcomes and a considerably increased rate of mortality [4]. Recent reports suggest the possible existence of cases of completely resistant TB in the Middle East, raising concerns regarding how to treat these TB cases effectively [5].

In the past 40 years, no broadly successful new TB drug has been developed. Therefore, there is a strong drive to develop new treatments for TB and/or to improve those currently in use. Important advances have been made and there are several clinical trials underway that have utilised fluoroquinolones in place of ethambutol, leading to preliminary indications of a significant reduction in the duration of therapy and encouraging the possibility of an improvement in patient survival [6].

Antimicrobial peptides (AMPs) are gene-encoded, amphipathic, cationic peptides that are produced by several species of mammals, birds, reptiles and amphibians. These peptides can inhibit microbial growth through a variety of often complex mechanisms, including membrane interactions that lead to permeabilisation of cells, inhibition of cell wall synthesis, and entry into cells leading to inhibition of macromolecular synthesis [7], [8], [9]. In addition, these peptides, also termed host defence peptides, can profoundly and favourably modulate innate immunity, upregulating protective immunity such as increasing the production of chemokines to recruit immune cells whilst dampening potentially harmful inflammation [9], [10]. The major groups of AMPs in humans are the defensins and a single cathelicidin, LL-37. It has been reported that alterations in the production of these molecules increase susceptibility to infectious diseases, including TB [11]. Conversely, upregulation of cathelicidin LL-37 through use of vitamin D supplementation has been considered to be a potential strategy to improve TB infection outcomes, although current data do not necessarily favour this possibility [12].

Previous studies by our group have reported that during M. tuberculosis infection of lung epithelial cells, there was a high production of β-defensins-3 and -4, and both were associated with mycobacteria in the lung, suggesting their possible participation in clearance of M. tuberculosis [13], [14]. Subsequently, it was reported that in murine TB models, BALB/c mice produced low quantities of murine β-defensins-3 and -4 during late progressive TB, and when both defensins were overproduced by intratracheal administration of isoleucine (a defensin inducer) these animals efficiently controlled infection both by drug-sensitive and drug-resistant bacilli [14], [15]. In addition, it has been shown that the interaction of a 19-kDa lipopeptide of M. tuberculosis with Toll-like receptor-2 on the macrophage surface upregulated the expression of vitamin D receptor leading to the induction of cathelicidin LL-37, promoting the killing of intracellular M. tuberculosis [16], [17].

Recently, methodologies have been developed to enable the enhanced design of AMPs (e.g. [18]). Rational substitution studies led to an enhanced 26-amino-acid β-helical peptide CP26 derived from a hybrid peptide comprising the amphipathic α-helical N-terminal region of cecropin A and the hydrophobic N-terminal α-helix of the bee venom peptide melittin [17]. Peptide array methods and substitution studies, starting from the smallest known broad-spectrum natural AMP bactenecin (also known as bovine dodecapeptide), led to peptides E2 (also known as Bac8c), an 8-amino-acid peptide, as well as E6 (also called Sub3), a 12-amino-acid peptide, both of which demonstrated enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria and the yeast Candida albicans [18], [19].

In this study, the antimicrobial activity of five natural and synthetic peptides against M. tuberculosis was evaluated in an in vitro setting.