Histone-derived Antimicrobial Peptides Research Articles

The Importance of the Proline Hinge in the Action of Histone-Derived Antimicrobial Peptides

Antimicrobial peptides (AMPs) are short, polycationic proteins capable of killing a wide variety of bacterial species through a number of different mechanisms. The ability to effectively engineer potent, cell-penetrating AMPs would maximize the full potential of these molecules. Buforin II (BF2), a cell-penetrating histone-derived antimicrobial peptide (HDAP), served as a model for the design of three novel histone-derived peptides, DesHDAPs1-3. BF2 is has a C-terminal α-helix that is broken by a proline hinge. Because this proline hinge determines, in part, BF2's ability to translocate into and kill bacterial cells, DesHDAPs1-3 were designed to also contain a helix-breaking proline residue. To determine whether this structural feature plays the same role in the designed peptides, the activity of proline to alanine mutants of each designed peptide were tested against a variety of bacterial species. As expected, circular dichroism measurements indicate that the proline to alanine mutation increases the α-helical character of BF2 and all designed peptides. For both BF2 and DesHDAP1, proline to alanine mutants show decreased antimicrobial activity against all species tested. In contrast, proline to alanine mutations in both DesHDAP2 and DesHDAP3 either do not affect or slightly increase the observed antimicrobial activity. This suggests that α-helicity is a poor predictor of antimicrobial activity for this family of HDAPs, and that the proline residue may play a different role in DesHDAP2 and DesHDAP3 than it does in BF2 and DesHDAP1. In order to explain these trends, we have further characterized the translocation and membrane permeabilization properties of the proline to alanine mutations. As well, we have used molecular dynamics simulations to explore the structure of the membrane bound peptides.

Design of novel histone-derived antimicrobial peptides

Previous studies have identified several naturally occurring antimicrobial peptides derived from histone proteins. This research aimed to design novel histone-derived antimicrobial peptides (HDAPs). To this end, three novel peptides (DesHDAP1, DesHDAP2, and DesHDAP3) were designed based on a histone–DNA crystal structure and structural properties of buforin II, the best characterized naturally occurring HDAP. Molecular dynamics simulations and circular dichroism spectroscopy were used to further support the predicted structure and potential nucleic acid interactions of these three designed peptides. The antibacterial activity of the three peptides was then verified experimentally against a series of bacterial strains using a radial diffusion assay. One of these peptides is the first known fragment of histone H3 with antibacterial properties. Optical density measurements of bacterial cells exposed to the designed peptides implied that at least two of the novel peptides can induce cell death without causing significant membrane permeabilization, as observed for buforin II. The antibacterial potency of these designed HDAPs does not appear to correlate with their overall α-helical content, unlike previous observations for analogs of buforin II. However, the most potent designed peptide, DesHDAP1, shares a markedly similar circular dichroism spectrum with buforin II. These results demonstrate the potential of using histone structures as a framework for designing novel antimicrobial peptides. As well, these studies represent an important starting point for a broader characterization of properties shared by HDAPs.

Investigating the Bactericidal Mechanism of Three Novel Histone-Derived Antimicrobial Peptides

Many antimicrobial peptides elicit bacterial death via cell membrane lysis. However, buforin II (BF2), a 21-amino acid peptide derived from histone H2A, is unique due to its hypothesized ability to translocate across cell membranes and interact with bacterial nucleic acids to cause cell death. Since cell-entry peptides, such as BF2, often are effective at lower concentrations than peptides that target membranes, increasing their potential as therapeutics in vivo. To this end, we developed three novel histone-derived antimicrobial peptides based on fragments of histones H2A (Des1), H3 (Des2) and H4 (Des3). These histones were previously found to exhibit translocation behavior. The designed peptides' antimicrobial properties were verified using a radial diffusion assay. In this assay, BF2 exhibited the greatest antimicrobial activity, followed by Des1, Des3 and Des2, respectively. We also measured the absorbance of bacterial cells incubated with these peptides. Based on the similarity between the absorbance versus time trend for the designed peptides with other DNA-binding antimicrobial peptides, such as BF2 and indolicidin, molecular dynamics simulations were used to model the peptides' interactions with nucleic acids. MM-GBSA analyses of the simulations were used to calculate DNA binding energies of individual peptide residues. We used these analyses to create mutant versions of the designed peptides that were predicted to have altered DNA binding. Experimental measurements of the DNA binding and antimicrobial properties of these variants will help us determine whether nucleic acid interactions are important in the bactericidal mechanism of the designed peptides. Ongoing work on the designed peptides is aimed at investigating their translocation behavior in vitro with lipid vesicles and in vivo with bacterial cells using confocal microscopy and fluorescently tagged peptides.

Antimicrobial effects of H4‐(86–100), histogranin and related compounds – possible involvement of DNA gyrase

Histone-derived antimicrobial peptides have been identified in various organisms from plants to humans. The rat histone H4 mRNA variants, H4-v.1 and rat histogranin (HNr) mRNAs, were recently reported to be involved in the synthesis of H4-(86-100) and its related peptide HNr, respectively. Herein, the two peptides were investigated for putative antimicrobial activity and found to inhibit growth of gram-negative (Escherichia coli, Pseudomonas aeruginosa) and gram-positive (Bacillus subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in E. coli (LD(50): 3.48 and 4.34 microg x mL(-1)) are comparable to that of the antimicrobial peptide LL-37 (LD(50): 4.10 microg x mL(-1)). The antimicrobial activities of H4-(86-100) and HNr depend upon the integrity of the molecules, as precursors [H4-(84-102), pro-HNr] and fragments [bovine histogranin (HNb)-(1-13), HNb-(3-13), H4-(89-102) or OGP] are at least five times less potent than the parent peptides. Among various HN-like compounds, cyclo-(-Gly-pCl-Phe-Tyr-D-Arg) (compound 3) and N-5-guanidino pentanamide-(2R)-yl-2-N-(p-hydroxyphenylacetyl)-4-(p-chlorobenzoyl)-phenylene diamine (compound 8) display antimicrobial activities comparable to that of HNr. Interestingly, the antimicrobial activities of H4-(86-100), HNr and compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mM) and coumermycin A1 (a DNA gyrase-linked ATPase inhibitor) and blocked by 2,4-dinitrophenol (DNP, an uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic poison). Finally, in vitro experiments indicate that H4-(86-100), HNr, compound 3 and compound 8, but not HNb-(1-13) or HNb-(3-13), inhibit DNA gyrase-mediated supercoiling of pBR322 DNA. These data indicate that the naturally occurring H4-(86-100) and HNr display antimicrobial effects that involve a modulation of ATP-dependent DNA gyrase.