The rapid increase in the emergence of antifungal-resistant Candida albicans strains is becoming a serious health concern. Because antimicrobial peptides (AMPs) may provide a potential alternative to conventional antifungal agents, we have synthesized a series of peptides with a varying number of lysine and tryptophan repeats (KW_n-NH₂). The antifungal activity of these peptides increased with peptide length, but only the longest KW₅ peptide displayed cytotoxicity towards a human keratinocyte cell line. The KW₄ and KW₅ peptides exhibited strong antifungal activity against C. albicans, even under conditions of high-salt and acidic pH, or the addition of fungal cell wall components. Moreover, KW₄ inhibited biofilm formation by a fluconazole-resistant C. albicans strain. Circular dichroism and fluorescence spectroscopy indicated that fungal liposomes could interact with the longer peptides but that they did not release the fluorescent dye calcein. Subsequently, fluorescence assays with different dyes revealed that KW₄ did not disrupt the membrane integrity of intact fungal cells. Scanning electron microscopy showed no changes in fungal morphology, while laser-scanning confocal microscopy indicated that KW₄ can localize into the cytosol of C. albicans. Gel retardation assays revealed that KW₄ can bind to fungal RNA as a potential intracellular target. Taken together, our data indicate that KW₄ can inhibit cellular functions by binding to RNA and DNA after it has been translocated into the cell, resulting in the eradication of C. albicans.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.

In our previous work, we showed that short antimicrobial hexapeptides (AMPs) containing three Trp and three Arg residues had a potent antibiofilm activity against a pathogenic Gram-positive Staphylococcus aureus MRSA strain. However, the activity of these hexapeptides against a Gram-negative Pseudomonas aeruginosa PA01 strain was relatively poor. Herein, we tested the longer 13-residue synthetic AMP tritrpticin-NH₂ (Tritrp) and several of its analogs as potential antibiofilm agents that can prevent biofilm formation (MBIC) and/or cause biofilm dissolution (MBEC) for two P. aeruginosa PA01 strains, one of which expressed the GFP protein. Tritrp, a porcine cathelicidin, is currently the only known naturally occurring cationic AMP that has three Trp in sequence (WWW), a feature that was found to be important in our previous study. Our results show that several Tritrp analogs were effective. In particular, analogs with Pro substitutions that had altered peptide backbone structures compared to the naturally occurring amphipathic two-turn structure showed more potent MBIC and MBEC antibiofilm activities. Selectivity of the peptides towards P. aeruginosa could be improved by introducing the non-proteinogenic amino acid 2,3-diaminopropionic acid, rather than Arg or Lys, as the positively charged residues. Using ¹H NMR spectroscopy, we also reinvestigated the role of the two Pro residues in cis–trans isomerism of the peptide in aqueous solution. Overall, our results show that the WWW motif embedded in longer cationic AMPs has considerable potential to combat biofilm formation in pathogenic Gram-negative strains.

Recently, several antimicrobial peptides (AMPs), varying in length from 12 to 37 residues, have been shown to act as antibiofilm agents. Here, we report a study of 23 hexapeptides modeled after four different Trp- and Arg-rich AMPs, including the RRWQWR-NH₂ peptide, derived from bovine lactoferrin. They were tested against the pathogenic Gram-negative Pseudomonas aeruginosa PAO1 strain and a Gram-positive Staphylococcus aureus MRSA strain. Both strains were engineered to express the green fluorescent protein (GFP) protein, and fluorescence detection was used to measure the ability of the peptides to prevent biofilm formation (minimum biofilm inhibitory concentration (MBIC)) or to cause the breakdown of established biofilms (minimum biofilm eradication concentration (MBEC)). Similar antibiofilm activities were obtained with the standard crystal violet dye assay. Most Trp- and Arg-rich hexapeptides displayed a potent antibiofilm activity against the Gram-positive S. aureus MRSA strain. In particular, hexapeptides with 3 Arg and 3 Trp were very effective, especially when they contained the three Trp in sequence. Somewhat unexpectedly, the antimicrobial (MIC) values correlated with the MBIC and MBEC values, which has not been seen for several other AMP/antibiofilm peptides. Our results demonstrate that short Trp- and Arg-rich peptides merit further studies as antibiofilm agents that could be deployed to address part of the antimicrobial resistance problem.

Cationic antimicrobial peptides (AMPs) that can target multidrug-resistant pathogenic bacteria via multiple mechanisms are considered promising alternatives to antibiotics. Small (~9 kDa) highly acidic acyl carrier proteins (ACPs), which are a well-known cofactor protein in bacterial fatty acid synthesis (FAS), are a potential intracellular target for AMPs. A previous study has demonstrated that the human AMP LL-37 can bind to ACP and thereby affect FAS and the bacterial membrane integrity. In this work, we have investigated the interactions of different classes of AMPs and antibiofilm peptides (ABPs) with the ACPs of two pathogens. We first studied the folding characteristics of the two ACPs and found that Pseudomonas aeruginosa ACP (PaACP) is fully folded at neutral pH in the absence of divalent cations. On the other hand, the homologous Francisella novicida ACP (FnACP) is unfolded at low ionic strength, but it adopts a fully folded conformation after the addition of divalent cations such as Ca²⁺ or Mg²⁺. These distinct characteristics were shown to be related to a unique His residue that is involved in a stabilizing cation–π interaction. Subsequent biophysical SPR and NMR interaction studies reveal that cationic AMPs and ABPs such as LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B and IDR-1018, but not F5W-magainin 2, can bind to both apo- and holo-ACPs. Binding of Arg-rich peptides is preferred over their Lys-rich analogs. Interestingly, all the peptides bind to holo-ACP with higher affinity than to apo-ACP, which lacks the functionally important phosphopantothenate group. NMR peak intensity perturbation data reveal that helix II of ACP, which is known to be directly involved in complex formation with bacterial FAS enzymes, acts as a common and main recognition site for the peptides. We propose that binding of AMPs and ABPs to this region of bacterial ACPs can directly block fatty acid synthesis and interfere in other ACP-dependent biosynthetic and regulatory events, which in turn could contribute to killing the bacteria and could also intervene in biofilm formation.