Drug discovery and resistanceCationic amphipathic D-enantiomeric antimicrobial peptides with in vitro and ex vivo activity against drug-resistant Mycobacterium tuberculosis
Introduction
Tuberculosis (TB) is a severe infectious disease of which there were 8.6 million new cases and caused 1.3 million deaths globally in 2012 [1]. Although the absolute number and the incidence rate of TB has been falling in the last few years, cases of multi-drug resistant TB (MDR-TB) are rising, reaching almost 450,000 worldwide in 2012 [1]. The situation has been worsened by the emergence of extensively drug-resistant TB (XDR-TB). The average proportion of MDR-TB cases with XDR-TB is 9.6%. According to the Global Tuberculosis Report 2013 [1], the progress towards targets for diagnosis and treatment of MDR-TB is far off-track. The target treatment success rate of 75% or higher for patients with MDR-TB was reached by only 34 of 107 countries that reported treatment outcomes [1]. Within the UK, of the 8751 cases in 2012, 73% were non-UK born and the majority of cases were likely due to reactivation of latent TB infections, with 6.8% of cases in the UK resistant to isoniazid [2]. There is an urgent need to develop safe and effective TB therapies that can combat latent TB infections and/or MDR-TB.
TB is caused by Mycobacterium tuberculosis (Mtb), an intracellular pathogen which can survive inside human alveolar macrophages. The exact mechanisms underlying the virulence of Mtb have not been fully elucidated. Extensive studies have indicated that despite the antimicrobial activity of macrophages, Mtb has developed multiple strategies to interfere with the phagosomal maturation, manipulate the host machinery, neutralize toxic factors and eventually survive the harsh intracellular environment within the host macrophages [3], [4], [5], [6]. Antimicrobial peptides (AMPs) are produced by a wide variety of living organisms, serving an essential role in the host innate immune system. Various studies have demonstrated that both naturally occurring AMPs and their synthetic analogues have a wide spectrum activity against pathogenic organisms, including Gram-positive and Gram-negative bacteria, as well as intracellular pathogens like Plasmodium falciparum and M. tuberculosis [7], [8], [9], [10], [11]. AMPs are usually positively charged amphipathic small peptides consisting around 12 to 50 amino acid residues [11], [12], [13]. Although their mechanisms of action have not been fully understood, it is generally believed that cationic AMPs can selectively bind to the negatively charged membrane components of the pathogens and destabilize and/or penetrate their plasma membrane. Subsequently, this membrane activity causes either leakage of the cellular fluid or facilitates additional intracellular nucleic acid or protein inhibition activity and eventually the death of the pathogenic microorganism [13], [14], [15].
AMPs that assume an α-helical structure are widespread and abundant in nature. The selectivity, potency and safety of α-helical AMPs heavily depend on their physicochemical properties including their amphipathic nature, net charge, charge angle, overall hydrophobicity and conformational flexibility [8]. In addition, the use of D-conformation peptides is preferred because it has been shown that the L to d-amino acid substitution of AMPs confers resistance to the activity of proteolytic enzymes and therefore improves AMP biostability, without impairing antimicrobial activity [16], [17]. In our pervious study, we have already identified that a charge angle of 120° conferred the highest potency against Mtb using the attenuated laboratory strain H37Ra as a model [7]. When proline is incorporated to the hydrophilic face of the amphipathic α-helical AMPs, an improvement of antibacterial activity and reduction of hemolytic effect were observed, as well as increasing the conformational flexibility [18], [19]. In addition, increasing the conformational flexibility within the peptide backbone, through incorporation of a proline residue at position 13 has been shown to enable effective killing of P. falciparum within an erythrocyte host while minimizing collateral toxicity as determined by hemolysis [7]. We therefore tested the hypothesis that the same modifications to the peptides would promote activity against Mtb within a macrophage host while mitigating toxicity to macrophages.
In this study, the anti-TB activity of six structurally similar AMPs of the D-LAK family, with different physicochemical properties, was evaluated both in vitro and ex vivo (Table 1). These six D-AMPs consist of 25 d-enantiomer amino acid residues in a primary sequence designed to adopt a left-handed α-helix conformation with charge angle of 120°. Each of them contains eight lysine residues with a nominal charge of +9 at neutral pH. The major difference between these six D-AMPs is the complement of histidine, alanine and presence/absence of proline residues, which in turn affects their overall hydrophobicity and conformational flexibility. We tested the D-AMPs against clinical strains of Mtb, including MDR-TB and XDR-TB. In addition, since AMPs possess membrane active properties, they may facilitate the access of anti-TB drugs such as isoniazid (INH) into the Mtb, potentiating the efficacy of the anti-TB drugs [20], [21]. Therefore D-LAK peptides were also used in combination with INH to investigate whether these peptides can enhance the efficacy of INH against drug-resistant TB in vitro. Although other mycobacteria such as Mycobacterium smegmatis or Mycobacterium marinum are commonly used as a surrogate model as they grow faster than tuberculosis and require a lower biosafety level for experiment, they are also less relevant [22], [23]. Therefore clinical isolates of drug-resistant Mtb strains were employed in our study, allowing us to give a more representative examination of the potential of the D-AMPs for clinical development.
Section snippets
Antimicrobial peptides
Six structurally similar D-AMPs (Table 1) were synthesized using standard manual Fmoc (N-(9-fluorenyl)methyoxycarbonyl) solid state chemistry as previously described [7]. High performance liquid chromatography (HPLC) purification was performed using acetonitrile/water gradients on an Agilent 1100 system using a SymmetryPrep™ C8 7 μm, 19 × 300 mm column (Waters, Milford, MA) and the identity of the product was confirmed by matrix assisted laser desorption ionization mass spectrometry. Peptides
In vitro anti-TB assay
The in vitro anti-TB activities of the D-AMPs were screened by visually comparing the growth and the morphology of the mycobacterial colonies with the control samples by light microscopy (Figure 1). All six D-AMPs tested in this study showed concentration-dependent antibacterial activity against the three clinical strains of Mtb. At low concentrations of D-AMPs, the mycobacteria appeared to be dispersed and scattered inside the culture well instead of remaining as colonies of aggregated
Discussion
The extensive use and, particularly, mismanagement of TB antibiotics in the clinic has favored the emergence of drug-resistant strains of mycobacteria. There is an urgent need for the development of new classes of antimicrobial agents particularly for the treatment of drug-resistant TB which is very challenging. Our group is currently investigating a series of linear cationic amphipathic α-helical AMPs, LAK peptides, which have the potential to be developed into therapeutic agents for clinical
Acknowledgments
The authors thank Dr Vincenzo Abbate and Dr Sukhi Bansal (both KCL) for peptide synthesis.
References (42)
Mycobacterium tuberculosis and the macrophage: maintaining a balance
Cell Host Microbe
(2008)- et al.
Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic alpha-helical peptides
J Biol Chem
(2012) Role of antimicrobial peptides in host defense against mycobacterial infections
Peptides
(2008)- et al.
Human macrophage host defense against Mycobacterium tuberculosis
Curr Opin Immunol
(2008) - et al.
Effect of D-amino acid substitution on the stability, the secondary structure, and the activity of membrane-active peptide
Biochem Pharmacol
(1999) - et al.
The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: identification of a potent anti-Candida peptide
Biochim Biophys Acta
(2013) - et al.
Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity
Biochem Biophys Res Commun
(2004) - et al.
In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis
Peptides
(2004) - et al.
Mycobacterial cell wall: structure and role in natural resistance to antibiotics
FEMS Microbiol Lett
(1994) - et al.
Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update
Tuber Lung Dis Off J Int Union Against Tuberc Lung Dis
(1998)
Chapter 2: Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis
Adv Appl Microbiol
Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis
Int J Antimicrob Agents
Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis
Lancet Infect Dis
Global tuberculosis report 2013
Tuberculosis in the UK: 2013 report
Immunopathology of tuberculosis: roles of macrophages and monocytes
Infect Immun
Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages
Infect Immun
Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages
J Innate Immun
Anti-tuberculosis activity of alpha-helical antimicrobial peptides: de novo designed L- and D-enantiomers versus L- and D-LL-37
Protein Peptide Lett
Antimicrobial peptides: primeval molecules or future drugs?
PLoS Pathog
Antimicrobial peptides of multicellular organisms
Nature
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