Identification of Peptides Derived from the Human Antimicrobial Peptide LL-37 Active against Biofilms Formed by Pseudomonas aeruginosa Using a Library of Truncated Fragments

Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients and are linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 is the only human antimicrobial peptide derived from cathelicidin. The effects on the P. aeruginosa PAO1 strain of synthetic truncated fragments of this peptide were compared with the effects of the original peptide. Fragments of LL-37 composed of 19 residues (LL-19, LL13-31, and LL7-25) inhibited biofilm formation. The strongest antibiofilm activity was observed with the peptides LL7-37 and LL-31, which decreased the percentage of biomass formation at a very low concentration. Some peptides were also active on the bacteria within an established biofilm. LL7-31, LL-31, and LL7-37 increased the uptake of propidium iodide (PI) by sessile bacteria. The peptide LL7-37 decreased the height of the biofilm and partly disrupted it. The peptides active within the biofilm had an infrared spectrum compatible with an α-helix. LL-37, but not the peptides LL7-31 and LL7-37, showed cellular toxicity by permeabilizing the eukaryotic plasma membrane (uptake of ethidium bromide and release of lactate dehydrogenase [LDH]). None of the tested peptides affected mitochondrial activity in eukaryotic cells. In conclusion, a 25-amino-acid peptide (LL7-31) displayed both strong antimicrobial and antibiofilm activities. The peptide was even active on cells within a preformed biofilm and had reduced toxicity toward eukaryotic cells. Our results also suggest the contribution of secondary structures (α-helix) to the activity of the peptides on biofilms.     

Antimicrobial Peptide GL13K Is Effective in Reducing Biofilms of Pseudomonas aeruginosa

Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH₂ (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa, 8 μg/ml [5.6 μM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 10², and 100 μg/ml (70 μM) GL13K reduced bacteria by 10³. Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 μM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.     

Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide

Biofilms cause up to 80% of infections and are difficult to treat due to their substantial multidrug resistance compared to their planktonic counterparts. Based on the observation that human peptide LL-37 is able to block biofilm formation at concentrations below its MIC, we screened for small peptides with antibiofilm activity and identified novel synthetic cationic peptide 1037 of only 9 amino acids in length. Peptide 1037 had very weak antimicrobial activity, but at 1/30th the MIC the peptide was able to effectively prevent biofilm formation (>50% reduction in cell biomass) by the Gram-negative pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia and Gram-positive Listeria monocytogenes. Using a flow cell system and a widefield fluorescence microscope, 1037 was shown to significantly reduce biofilm formation and lead to cell death in biofilms. Microarray and follow-up studies showed that, in P. aeruginosa, 1037 directly inhibited biofilms by reducing swimming and swarming motilities, stimulating twitching motility, and suppressing the expression of a variety of genes involved in biofilm formation (e.g., PA2204). Comparison of microarray data from cells treated with peptides LL-37 and 1037 enabled the identification of 11 common P. aeruginosa genes that have a role in biofilm formation and are proposed to represent functional targets of these peptides. Peptide 1037 shows promise as a potential therapeutic agent against chronic, recurrent biofilm infections caused by a variety of bacteria.     

Activities of LL-37, a Cathelin-Associated Antimicrobial Peptide of Human Neutrophils

Human neutrophils contain two structurally distinct types of antimicrobial peptides, β-sheet defensins (HNP-1 to HNP-4) and the α-helical peptide LL-37. We used radial diffusion assays and an improved National Committee for Clinical Laboratory Standards-type broth microdilution assay to compare the antimicrobial properties of LL-37, HNP-1, and protegrin (PG-1). Although generally less potent than PG-1, LL-37 showed considerable activity (MIC, <10 μg/ml) against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, and vancomycin-resistant enterococci, even in media that contained 100 mM NaCl. Certain organisms (methicillin-resistant S. aureus, Proteus mirabilis, and Candida albicans) were resistant to LL-37 in media that contained 100 mM NaCl but were susceptible in low-salt media. Burkholderia cepacia was resistant to LL-37, PG-1, and HNP-1 in low- or high-salt media. LL-37 caused outer and inner membrane permeabilization of E. coli ML-35p. Chromogenic Limulus assays revealed that LL-37 bound to E. coli O111:B4 lipopolysaccharide (LPS) with a high affinity and that this binding showed positive cooperativity (Hill coefficient = 2.02). Circular dichroism spectrometry disclosed that LL-37 underwent conformational change in the presence of lipid A, transitioning from a random coil to an α-helical structure. The broad-spectrum antimicrobial properties of LL-37, its presence in neutrophils, and its inducibility in keratinocytes all suggest that this peptide and its precursor (hCAP-18) may protect skin and other tissues from bacterial intrusions and LPS-induced toxicity. The potent activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it or related molecules might have utility as topical bronchopulmonary microbicides in cystic fibrosis.     

Antibiofilm Peptides Increase the Susceptibility of Carbapenemase-Producing Klebsiella pneumoniae Clinical Isolates to β-Lactam Antibiotics

Multidrug-resistant carbapenemase-producing Klebsiella pneumoniae (KpC) strains are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and β-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of β-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates.     

Sodium Nitrite Blocks the Activity of Aminoglycosides against Pseudomonas aeruginosa Biofilms

Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm.     

A Broad-Spectrum Antibiofilm Peptide Enhances Antibiotic Action against Bacterial Biofilms

Biofilm-related infections account for at least 65% of all human infections, but there are no available antimicrobials that specifically target biofilms. Their elimination by available treatments is inefficient since biofilm cells are between 10- and 1,000-fold more resistant to conventional antibiotics than planktonic cells. Here we describe the synergistic interactions, with different classes of antibiotics, of a recently characterized antibiofilm peptide, 1018, to potently prevent and eradicate bacterial biofilms formed by multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Combinations of peptide 1018 and the antibiotic ceftazidime, ciprofloxacin, imipenem, or tobramycin were synergistic in 50% of assessments and decreased by 2- to 64-fold the concentration of antibiotic required to treat biofilms formed by Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus. Furthermore, in flow cell biofilm studies, combinations of low, subinhibitory levels of the peptide (0.8 μg/ml) and ciprofloxacin (40 ng/ml) decreased dispersal and triggered cell death in mature P. aeruginosa biofilms. In addition, short-term treatments with the peptide in combination with ciprofloxacin prevented biofilm formation and reduced P. aeruginosa PA14 preexisting biofilms. PCR studies indicated that the peptide suppressed the expression of various antibiotic targets in biofilm cells. Thus, treatment with the peptide represents a novel strategy to potentiate antibiotic activity against biofilms formed by multidrug-resistant pathogens.     

Enhancement of Antimicrobial Activity of Neuropeptide Y by N-Terminal Truncation

The activity of neuropeptide Y (NPY) against Candida albicans, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 μM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 μM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the α-amino group. These results suggest that only the α-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.     

Synergistic Effects and Antibiofilm Properties of Chimeric Peptides against Multidrug-Resistant Acinetobacter baumannii Strains

The increasing prevalence of drug-resistant pathogens highlights the need to identify novel antibiotics. Here we investigated the efficacies of four new antimicrobial peptides (AMPs) for potential drug development. The antibacterial activities, synergistic effects, and antibiofilm properties of the four chimeric AMPs were tested against Acinetobacter baumannii, an emerging Gram-negative, nosocomial, drug-resistant pathogen. Nineteen A. baumannii strains resistant to ampicillin, cefotaxime, ciprofloxacin, tobramycin, and erythromycin were isolated at a hospital from patients with cholelithiasis. All four peptides exhibited significant antibacterial effects (MIC = 3.12 to 12.5 μM) against all 19 strains, whereas five commercial antibiotics showed little or no activity against the same pathogens. An exception was polymyxin, which was effective against all of the strains tested. Each of the peptides showed synergy against one or more strains when administered in combination with cefotaxime, ciprofloxacin, or erythromycin. The peptides also exhibited an ability to prevent biofilm formation, which was not seen with cefotaxime, ciprofloxacin, or erythromycin, though polymyxin also inhibited biofilm formation. Indeed, when administered in combination with ciprofloxacin, the AMP HPMA exerted a potent synergistic effect against A. baumannii biofilm formation. Collectively, our findings indicate that the AMPs tested have no cytotoxicity but possess potent antibacterial and antibiofilm activities and may act synergistically with commercial antibiotics.     

LL-37-Derived Peptides Eradicate Multidrug-Resistant Staphylococcus aureus from Thermally Wounded Human Skin Equivalents

Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria.     

Inhibition of Nucleic Acid Biosynthesis Makes Little Difference to Formation of Amphotericin B-Tolerant Persisters in Candida albicans Biofilm

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml⁻¹; biofilm cells, 1 μg ml⁻¹) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml⁻¹ for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.     

Sustained Release of a Novel Anti-Quorum-Sensing Agent against Oral Fungal Biofilms

Thiazolidinedione-8 (S-8) has recently been identified as a potential anti-quorum-sensing/antibiofilm agent against bacteria and fungi. Based on these results, we investigated the possibility of incorporating S-8 in a sustained-release membrane (SRM) to increase its pharmaceutical potential against Candida albicans biofilm. We demonstrated that SRM containing S-8 inhibits fungal biofilm formation in a time-dependent manner for 72 h, due to prolonged release of S-8. Moreover, the SRM effectively delivered the agent in its active form to locations outside the membrane reservoir. In addition, eradication of mature biofilm by the SRM containing S-8 was also significant. Of note, S-8-containing SRM affected the characteristics of mature C. albicans biofilm, such as thickness, exopolysaccharide (EPS) production, and morphogenesis of fungal cells. The concept of using an antibiofilm agent with no antifungal activity incorporated into a sustained-release delivery system is new in medicine and dentistry. This concept of an SRM containing a quorum-sensing quencher with an antibiofilm effect could pave the way for combating oral fungal infectious diseases.     

Antibiofilm and Membrane-Damaging Potential of Cuprous Oxide Nanoparticles against Staphylococcus aureus with Reduced Susceptibility to Vancomycin

The antimicrobial effects of copper ions and salts are well known, but the effects of cuprous oxide nanoparticles (Cu₂O-NPs) on staphylococcal biofilms have not yet been clearly revealed. The present study evaluated Cu₂O-NPs for their antibacterial and antibiofilm activities against heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA). Nanoscaled Cu₂O, generated by solution phase technology, contained Cu₂O octahedral nanoparticles. Field emission electron microscopy demonstrated particles with sizes ranging from 100 to 150 nm. Cu₂O-NPs inhibited the growth of S. aureus and showed antibiofilm activity. The MICs and minimum biofilm inhibitory concentrations ranged from 625 μg/ml to 5,000 μg/ml and from 2,500 μg/ml to 10,000 μg/ml, respectively. Exposure of S. aureus to Cu₂O-NPs caused leakage of the cellular constituents and increased uptake of ethidium bromide and propidium iodide. Exposure also caused a significant reduction in the overall vancomycin-BODIPY (dipyrromethene boron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding and a decrease in the viable cell count in the presence of 7.5% sodium chloride. Cu₂O-NP toxicity assessment by hemolysis assay showed no cytotoxicity at 625 to 10,000 μg/ml concentrations. The results suggest that Cu₂O-NPs exert their action by disruption of the bacterial cell membrane and can be used as effective antistaphylococcal and antibiofilm agents in diverse medical devices.     

Oral Administration of the Broad-Spectrum Antibiofilm Compound Toremifene Inhibits Candida albicans and Staphylococcus aureus Biofilm Formation In Vivo

We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.     

High Activity of Fosfomycin and Rifampin against Methicillin-Resistant Staphylococcus aureus Biofilm In Vitro and in an Experimental Foreign-Body Infection Model

Increasing antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the activity of fosfomycin alone and in combination with vancomycin, daptomycin, rifampin, and tigecycline against MRSA (ATCC 43300) in a foreign-body (implantable cage) infection model. The MICs of the individual agents were as follows: fosfomycin, 1 μg/ml; daptomycin, 0.125 μg/ml; vancomycin, 1 μg/ml; rifampin, 0.04 μg/ml; and tigecycline, 0.125 μg/ml. Microcalorimetry showed synergistic activity of fosfomycin and rifampin at subinhibitory concentrations against planktonic and biofilm MRSA. In time-kill curves, fosfomycin exhibited time-dependent activity against MRSA with a reduction of 2.5 log₁₀ CFU/ml at 128 × the MIC. In the animal model, planktonic bacteria in cage fluid were reduced by <1 log₁₀ CFU/ml with fosfomycin and tigecycline, 1.7 log₁₀ with daptomycin, 2.2 log₁₀ with fosfomycin-tigecycline and fosfomycin-vancomycin, 3.8 log₁₀ with fosfomycin-daptomycin, and >6.0 log₁₀ with daptomycin-rifampin and fosfomycin-rifampin. Daptomycin-rifampin cured 67% of cage-associated infections and fosfomycin-rifampin cured 83%, whereas all single drugs (fosfomycin, daptomycin, and tigecycline) and rifampin-free fosfomycin combinations showed no cure of MRSA cage-associated infections. No emergence of fosfomycin resistance was observed in animals; however, a 4-fold increase in fosfomycin MIC (from 2 to 16 μg/ml) occurred in the fosfomycin-vancomycin group. In summary, the highest eradication of MRSA cage-associated infections was achieved with fosfomycin in combination with rifampin (83%). Fosfomycin may be used in combination with rifampin against MRSA implant-associated infections, but it cannot replace rifampin as an antibiofilm agent.     

Fungicidal Mechanisms of Cathelicidins LL-37 and CATH-2 Revealed by Live-Cell Imaging

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.     

Effect of monovalent salt concentration and peptide secondary structure in peptide-micelle binding

Recently, we reported a cationic 14 residue peptide LL-14 (LKWLKKLLKWLKKL) with salt-sensitive broad-spectrum antimicrobial potency. However, the mechanism of its salt (NaCl) sensitivity remained unclear. In this study, we have reported computational (∼14.2 μs of MD) and experimental (CD, fluorescence) investigations to examine the salt-sensitivity and the role of peptide secondary structure on LL-14 binding to simple membrane mimetic (SDS, DPC) systems. LL-14 was shown to adopt a random coil (P^c) conformation in water and α-helical conformation (P^h) in the peptide:SDS micelle complex, accompanied by tryptophan burial, using both simulations and experiments. Simulations successfully deconvoluted the LL-14:micelle binding event in terms of secondary structure (random coil P^cversus helix P^h) and gave atomic insight into the initial and final LL-14:SDS complexes. Electrostatics drove the N-terminus (L1 and K2) of LL-14 (P^c or P^h) to bind the SDS micellar surface, initiating complex formation. LL-14 in amphipathic P^h conformation bound faster and buried deeper into the SDS micelle relative to P^c. Increasing NaCl concentration incrementally delayed LL-14:micelle binding by shielding the overall charges of the interacting partners. LL-14 binding to the SDS micelle was significantly faster relative to that of the zwitterionic DPC micelle due to electrostatic reasons. Cationic α-helical amphipathic peptides (with positively charged N-terminus) with low salt-ion concentration seemed to be ideal for faster SDS binding.

We report computational (∼14.2 μs of MD) and experimental (CD, fluorescence) investigations to examine the salt-sensitivity and the role of the peptide secondary structure on LL-14 binding to simple membrane mimetic systems.     

Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.