Tamara Lushnikova

Antimicrobial Peptides in 2014

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

Decoding the Functional Roles of Cationic Side Chains of the Major Antimicrobial Region of Human Cathelicidin LL-37

ABSTRACT Human cathelicidin LL-37 is a critical cationic antimicrobial peptide for host defense against infection, immune modulation, and wound healing. This article elucidates the functional roles of the cationic side chains of the major antimicrobial region of LL-37, corresponding to residues 17 to 32 (designated GF-17). Antimicrobial assays, killing kinetics studies, and vesicle leakage experiments all indicate that a conversion of lysines to arginines affected the ability of the peptide to kill the Gram-positive Staphylococcus aureus strain USA300. Alanine scanning experiments show that S. aureus is less sensitive than Escherichia coli to a single cationic residue mutation of GF-17. Among the five cationic residues, R23 appears to be somewhat important in killing S. aureus. However, R23 and K25 of GF-17 are of prime importance in killing the Gram-negative organism E. coli. In particular, R23 is essential for (i) rapid recognition, (ii) permeation of the E. coli outer membrane, (iii) clustering of anionic lipids in a membrane system mimicking the E. coli inner membrane, and (iv) membrane disruption. Bacterial aggregation (i.e., rapid recognition via charge neutralization) is the first step of the peptide action. Structurally, R23 is located in the interface (i.e., the first action layer), a situation ideal for the interactions listed above. In contrast, residues K18, R19, and R29 are on the hydrophilic surface of the amphipathic helix and play only a secondary role. Mapping of the functional spectrum of cationic residues of GF-17 provides a solid basis for engineering bacterium-specific antimicrobials using this highly potent template.

Transformation of Human Cathelicidin LL-37 into Selective,Stable, and Potent Antimicrobial Compounds

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens.

Design and surface immobilization of short anti-biofilm peptides

Short antimicrobial peptides are essential to keep us healthy and their lasting potency can inspire the design of new types of antibiotics. This study reports the design of a family of eight-residue tryptophan-rich peptides (TetraF2W) obtained by converting the four phenylalanines in temporin-SHf to tryptophans. The temporin-SHf template was identified from the antimicrobial peptide database (http://aps.unmc.edu/AP). Remarkably, the double arginine variant (TetraF2W-RR) was more effective in killing methicillin-resistant Staphylococcus aureus (MRSA) USA300, but less cytotoxic to human skin HaCat and kidney HEK293 cells, than the lysine-containing dibasic combinations (KR, RK and KK). Killing kinetics and fluorescence spectroscopy suggest membrane targeting of TetraF2W-RR, making it more difficult for bacteria to develop resistance. Because established biofilms on medical devices are difficult to remove, we chose to covalently immobilize TetraF2W-RR onto the polyethylene terephthalate (PET) surface to prevent biofilm formation. The successful surface coating of the peptide is supported by FT-IR and XPS spectroscopies, chemical quantification, and antibacterial assays. This peptide-coated surface indeed prevented S. aureus biofilm formation with no cytotoxicity to human cells. In conclusion, TetraF2W-RR is a short Trp-rich peptide with demonstrated antimicrobial and anti-biofilm potency against MRSA in both the free and immobilized forms. Because these short peptides can be synthesized cost effectively, they may be developed into new antimicrobial agents or used as surface coating compounds. STATEMENT OF SIGNIFICANCE It is stunning that the total deaths due to methicillin-resistant Staphylococcus aureus (MRSA) infection are comparable to AIDS/HIV-1, making it urgent to explore new possibilities. This study deals with this problem by two strategies. First, we have designed a family of novel antimicrobial peptides with merely eight amino acids, making it cost effective for chemical synthesis. These peptides are potent against MRSA USA300. Our study uncovers that the high potency of the tryptophan-rich short peptide is coupled with arginines, whereas these Trp- and Arg-rich peptides are less toxic to select human cells than the lysine-containing analogs. Such a combination generates a more selective peptide. As a second strategy, we also demonstrate successful covalent immobilization of this short peptide to the polyethylene terephthalate (PET) surface by first using a chitosan linker, which is easy to obtain. Because biofilms on medical devices are difficult to remove by traditional antibiotics, we also show that the peptide coated surface can prevent biofilm formation. Although rarely demonstrated, we provide evidence that both the free and immobilized peptides target bacterial membranes, rendering it difficult for bacteria to develop resistance. Collectively, the significance of our study is the design of novel antimicrobial peptides provides a useful template for developing novel antimicrobials against MRSA. In addition, orientation-specific immobilization of the same short peptide can prevent biofilm formation on the PET surface, which is widely used in making prosthetic heart valves cuffs and other bio devices.

Anti-Staphylococcal Biofilm Effects of Human Cathelicidin Peptides

Staphylococcus aureus can live together in the form of biofilms to avoid elimination by the host. Thus, a useful strategy to counteract bacterial biofilms is to re-engineer human antimicrobial peptide LL-37 so that it can be used as a remedy for preventing and removing biofilms. This study reports antibiofilm effects of four human cathelicidin LL-37 peptides against community-associated and hospital isolated methicillin-resistant Staphylococcus aureus (MRSA) strains. Although the intact molecule LL-37 inhibited biofilm formation at low concentrations, it did not inhibit bacterial attachment nor disrupt preformed biofilms. However, two 17-residue peptides, GF-17 and 17BIPHE2, inhibited bacterial attachment, biofilm growth, and disrupted established biofilms. An inactive peptide RI-10 was used as a negative control. Our results obtained using the S. aureus mutants in a static biofilm model are consistent with the literature obtained in a flow cell biofilm model. Because 17BIPHE2 is the most effective biofilm disruptor with desired stability to proteases, it is a promising lead for developing new anti-MRSA biofilm agents.

Small lipopeptides possess anti-biofilm capability comparable to daptomycin and vancomycin

Antibiotic resistance, to a large extent, is related to the formation of bacterial biofilms. Thus, compounds with anti-biofilm capability are of practical importance. Inspired by the recent discovery of two amino acid lipopeptides from marine bacteria, we constructed a family of small lipopeptides with 2-3 amino acids. While no antimicrobial activity was found for anionic lipopeptides, cationic candidates are potent against Staphylococcus strains, such as methicillin-resistant Staphylococcus aureus (MRSA) USA200, USA300, USA400, UAMS-1, Newman, and Mu50. In the simplest design, two lysines (C14-KK) or three arginines (C14-RRR) attached to an acyl chain of 14 carbons were sufficient to make the compounds antimicrobial. These simple lipopeptides are inherently stable towards S. aureus V8 proteinase and fungal proteinase K, more soluble in water, and more selective than other lipopeptides containing a mixture of hydrophobic and cationic amino acids. Furthermore, the activity of C14-RRR was not compromised by salts, serum, or a change in pH. Live cell experiments revealed that these lipopeptides, with a detergent-like structure, killed bacteria rapidly by targeting cell membranes. Importantly, these compounds were also able to inhibit biofilm formation and could even disrupt preformed biofilms of clinically relevant MRSA strains with an in vitro efficacy comparable to daptomycin and vancomycin. These results indicate that small lipopeptides are potentially useful candidates for preventing or eliminating bacterial biofilms alone or in combination with daptomycin or vancomycin.

Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes

Antimicrobial peptides are essential components of the innate immune system of multicellular organisms. Although cationic and hydrophobic amino acids are known determinants of these amphipathic molecules for bacterial killing, it is not clear how lysine-arginine (K-R) positional swaps influence peptide structure and activity. This study addresses this question by investigating two groups of peptides (GF-17 and 17BIPHE2) derived from human cathelicidin LL-37. K-R positional swap showed little effect on minimal inhibitory concentrations of the peptides. However, there are clear differences in bacterial killing kinetics. The membrane permeation patterns vary with peptide and bacterial types, but not changes in fluorescent dyes, salts or pH. In general, the original peptide is more efficient in bacterial killing, but less toxic to human cells, than the K-R swapped peptides, revealing the evolutionary significance of the native sequence for host defense. The characteristic membrane permeation patterns for different bacteria suggest a possible application of these K-R positional-swapped peptides as molecular probes for the type of bacteria. Such differences are related to bacterial membrane compositions: minimal for Gram-positive Staphylococcus aureus with essentially all anionic lipids (cardiolipin and phosphatidylglycerol), but evident for Gram-negative Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli with a mixture of phosphatidylethanolamine and phosphatidylglycerol. Biophysical characterization found similar structures and binding affinities for these peptides in vesicle systems mimicking E. coli and S. aureus. It seems that interfacial arginines of GF-17 are preferred over lysines in bacterial membrane permeation. Our study sheds new light on the design of cationic amphipathic peptides.

The π Configuration of the WWW Motif of a Short Trp-Rich Peptide Is Critical for Targeting Bacterial Membranes, Disrupting Preformed Biofilms, and Killing Methicillin-Resistant Staphylococcus aureus

Tryptophan-rich peptides, being short and suitable for large-scale chemical synthesis, are attractive candidates for developing a new generation of antimicrobials to combat antibiotic-resistant bacteria (superbugs). Although there are numerous pictures of the membrane-bound structure of a single tryptophan (W), how multiple Trp amino acids assemble themselves and interact with bacterial membranes is poorly understood. This communication presents the three-dimensional structure of an eight-residue Trp-rich peptide (WWWLRKIW-NH2 with 50% W) determined by the improved two-dimensional nuclear magnetic resonance method, which includes the measurements of 13C and 15N chemical shifts at natural abundance. This peptide forms the shortest two-turn helix with a distinct amphipathic feature. A unique structural arrangement is identified for the Trp triplet, WWW, that forms a π configuration with W2 as the horizontal bar and W1/W3 forming the two legs. An arginine scan reveals that the WWW motif is essential for killing methicillin-resistant Staphylococcus aureus USA300 and disrupting preformed bacterial biofilms. This unique π configuration for the WWW motif is stabilized by aromatic-aromatic interactions as evidenced by ring current shifts as well as nuclear Overhauser effects. Because the WWW motif is maintained, a change of I7 to R led to a potent antimicrobial and antibiofilm peptide with 4-fold improvement in cell selectivity. Collectively, this study elucidated the structural basis of antibiofilm activity of the peptide, identified a better peptide candidate via structure-activity relationship studies, and laid the foundation for engineering future antibiotics based on the WWW motif.

Amino Acid Composition Determines Peptide Activity Spectrum and Hot‐Spot‐Based Design of Merecidin

There is a great interest in developing the only human cathelicidin into therapeutic molecules. The major antimicrobial region of human LL‐37 corresponds to residues 17–32. The resultant peptide GF‐17 shows a broad spectrum of antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. By reducing the hydrophobic content, converting the broad‐spectrum GF‐17 to two narrow‐spectrum peptides (GF‐17d3 and KR‐12) with activity against Gram‐negative bacteria is successful. This study demonstrates that substitution of multiple basic amino acids by hydrophobic alanines makes a broad‐spectrum peptide 17BIPHE2 (designed based on GF‐17d3) active against Staphylococcal pathogens but not other bacteria tested. Taken together, the results reveal distinct charge and hydrophobic requirements for peptides to kill Gram‐positive or Gram‐negative bacteria. This finding is in line with the bioinformatics analysis of the peptides in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP). In addition, a hot‐spot arginine is identified and used to design merecidin with reduced toxicity to human cells. Merecidin protects wax moth larvae (Galleria mellonella) from the infection of methicillin‐resistant Staphylococcus aureus USA300. These new selective peptides constitute interesting candidates for future development.

De novo design of antiviral and antibacterial peptides with varying loop structures.

Although the rate of new HIV infections has been declining, AIDS continues to be one of the leading causes of death worldwide. The lack of an effective HIV vaccine makes it necessary to develop alternative strategies, such as the development of topical microbicides, to prevent transmission. Antimicrobial peptides represent promising microbicide candidates. Previously, we succeeded in enhancing the anti-HIV activities of several peptides that form helical structures based on the bioinformatic results learned from the antimicrobial peptide database. This study showed that Lys-to-Arg alterations also improved the HIV inhibitory activity of thanatin which is known to form a β-hairpin structure. Using a previously reported de novo designed HIV inhibitory peptide GLR-19 as the starting template, loop structures of varying sizes were generated by restraining a disulfide bond at different positions. The thanatin-mimicking constructs are referred to as GLRC peptides since they are composed of only four amino acid residues G, L, R, and C. While GLRC-2, the peptide with a medium-sized loop structure, was most potent against HIV-1 and HSV-2, GLRC-3, with the small loop structure, was most potent against Escherichia coli K12. Thus, the efficacy of the GLRC peptides is microbe dependent. Further terminal sequence truncation of GLRC-2 reduced antimicrobial activity against both viruses and bacteria. It appears that the high antiviral potency of GLRC-2 is related to high hydrophobicity, although a wide-range correlation is lacking. In addition, GLRC-2, which is more active against viruses, is also more resistant to the action of chymotrypsin. Therefore, GLRC-2, a novel peptide that acquired not only higher stability but also higher anti-HIV activity than the GLR-19 template, serves as the starting point for additional rounds of peptide engineering.