Bioinformatics

The native, plasma-derived peptide sequences from the de novo sequencing [8] were used to predict the secondary structure and placement within the parent proteins. Apo5 and Apo6 are both part of the C-terminus of an apolipoprotein, apolipoprotein C-1. Apo5 comprises amino acids (aa) 64-86, while Apo6 is the smaller fragment (aa 67-86, shown in Fig. 1a). Both peptides have a +4 charge and a hydrophobic ratio of just over 30 % as determined using APD2 [16]. Apo5 is cleaved at a Glu-Phe site and Apo6 at a Thr-Lys site. In the full apolipoprotein, both cleavages sites are located in a disordered hinge preceding a C-terminal alpha helix as seen in Fig. 1c.

Fig. 1 Primary and tertiary structure of parental proteins and peptide placement within. a Amino acid sequence of A. mississippiensis apolipoprotein C-1 and the Apo5 and Apo6 fragments. b Amino acid sequence of A. mississippiensis alpha-1-proteinase 2-like protein and the A1P fragment. c Ribbon model of apolipoprotein C-1 showing cleavages points of Apo5 (long dashes) and Apo6 (short dashes). d Ribbon model of alpha-1-proteinase showing cleavage point of A1P Full size image

A1P is also the cleaved C-terminus of its parent protein, a serpin proteinase inhibitor (aa 394-428, shown in Fig. 1b). It carries a +4 charge and a 45 % hydrophobicity ratio. The peptide is cleaved from the parent protein at Asp-Pro site in a disordered region on the exterior of the folded protein, as shown in Fig. 1d. The cleaved peptide itself consists of the two β-sheet regions that run through the interior of the proteinase.

Secondary structure determination

To determine general secondary structure of Apo5, Apo6, and A1P, CD spectroscopy was used. CD was performed in 10 mM phosphate buffer, 60 mM SDS, and 50 % TFE in phosphate buffer. Many CAMPs, such as LL-37, maintain a random or disordered structure until associated with a membrane or micelle. SDS forms micelles with a negatively charged surface [32], mimicking the bacterial membrane and forcing the CAMP into a more ordered conformation [33, 34].

TFE is used in CD to promote a helical structure [35] and stabilize secondary structure [36].

As expected, Apo5 (Fig. 2a) and Apo6 (Fig. 2b) had nearly identical CD spectra. Both peptides have random coil and β-sheet characteristics in 10 mM phosphate buffer, and are primarily α-helical when CD is measured in buffers with SDS and TFE. Interestingly, Apo5, the longer of the two peptides, is calculated to have more α-helical character than Apo6 in both SDS (63.3 % vs. 57.0 %) and TFE (51.0 % vs. 50.3 %), shown in Table 3. When evaluating the α-helical properties of these two peptides by simple intensity at 208 nm and 222 nm, it is notable that the peaks at these wavelengths are more intense for Apo5 than Apo6, though the two Apo peptides maintain 12.5 % turn no matter the buffer used. Apo5 and Apo6 likely have a primarily α-helical structure with some random coil portions. Based on helical wheel projections, it appears that both Apo5 and Apo6 have significant amphipathic character, with several hydrophobic residues on one face and several basic amino acids on the other. The structure of Apo5 and Apo6 were predicted using I-TASSER and the resulting.pdb file was visualized using Chimera, shown in Fig. 3a and b. Consistent with the CD spectra, both are predicted to be α-helical structures. Apo5, the longer peptide, has a longer random coil portion at the N-terminus. This is consistent with the predicted structure of the C-terminal portion of the parental protein, apolipoprotein C-1 (Fig. 1c). Because Apo5 and Apo6 are predicted to helical, helical wheels were produced using Heliquest and modified (Fig. 4). Apo5 and Apo6 have a similar hydrophobic moment (0.436 vs 0.484); Apo6 likely has a slightly stronger hydrophobic moment due to the loss of polar Ser in the hydrophobic face of the helix. In 10 mM phosphate buffer, TFE, and SDS, A1P has significantly different spectra (Fig. 2c).

In 10 mM phosphate buffer, A1P is calculated to have primarily β-sheet contributions (57.9 %), as well as some random coil characteristics (34.7 %), shown in Table 3. By our calculations, A1P also has a negative percentage of contribution from the α-helix, which may be an artifact of the equations used. In TFE, A1P maintains its random coil nature (31.2 %), but also becomes strongly α-helical (38.2 %), with only 16.3 % contribution from the β-sheet. In SDS, A1P is calculated to be nearly equal parts random coil (36.9 %), turn (21.5 %), β-sheet (24.2 %), and α-helix (21.6 %). It has been shown by several groups that TFE can induce and support α-helical structures in peptides [37, 38]. By qualitatively evaluating the spectra, we conclude that only in TFE does A1P have a notable α-helical structure, though this conformation may be interrupted by several proline residues found along the polypeptide chain. It is likely that A1P has a mixed structure that may change dramatically based on environmental factors. The secondary structure of A1P was predicted by I-TASSER and visualized using Chimera. I-TASSER predicts that A1P is primarily random coil with two anti-parallel β-sheet in a hairpin formation (Fig. 3c), which corresponds with the structure of the full alpha-1-proteinase structure.

Fig. 2 CD spectra of peptides to determine secondary structure. a Apo5, b Apo6, c A1P. All spectra were taken with peptide concentrations of 100 μg/ml in a 1 mm pathway cuvette. Spectra were read in 10 mM phosphate buffer (long dashes), 50 % TFE in 10 mM phosphate buffer (dash dot dash), and 60 mM SDS in 10 mM phosphate buffer (solid line) Full size image

Table 3 Percent secondary structure contribution as calculated by a method described by Raussens et al. [27] Full size table

Fig. 3 Secondary structure prediction by I-TASSER [24], visualized by Chimera [25]. a Apo5, b Apo6, c A1P Full size image

Fig. 4 Helical wheels of a Apo5 and b Apo6, modified from Heliquest Full size image

Antimicrobial activity

Previously, we reported that Apo5 and Apo6 had strong broad-spectrum antimicrobial activity against E. coli and B. cereus, as well as S. aureus and P. aeruginosa [8]. It was also found that A1P had greater activity against S. aureus than P. aeruginosa [8], indicating it may have stronger activity against Gram-positive organisms.

Antibiotic resistance has been increasing steadily for the past several decades, and CAMPs are considered a possible basis for novel antimicrobials. Because of this, these new peptides were tested against clinically isolated and multi-drug resistant strains of S. aureus, E. coli, P. aeruginosa and A. baumannii. Numerical and statistical data can be found in Table 4. Sharing a salt-sensitive phenotype with LL-37 [34, 39, 40], these peptides had no effect in MIC experiments as high as 250 μg/ml, so experiments were performed to determine the EC 50 in 10 mM phosphate buffer. We performed these low-salt experiments using vancomycin as a control for Gram-positive bacteria and polymyxin B as a control for Gram-negative bacteria. Polymyxin B was found to be very effective against all strains tested, with all EC50 values being under 1 μg/ml, except against E. coli ATCC 4157 (2.50 μg/ml). In the case of vancomycin, we found that this antibiotic did not kill either S. aureus strain under 100 μg/ml, though the MIC for both strains was 1 μg/ml, indicating that vancomycin is bacteriostatic but not reliably bactericidal.

Table 4 Antimicrobial activity and statistical data Full size table

In general, it was found that the EC 50 values of Apo5 and Apo6 were statistically similar and showed broad-spectrum activity. We found that both apolipoprotein-derived peptides had strong activity against a clinical isolate of S. aureus ATCC BAA-1718 (EC 50 < 5 μg/ml). Both Apo5 and Apo6 were found to have very strong activity (EC 50 < 1 μg/ml) against drug sensitive and MDR A. baumannii (ATCC BAA-1794), MRSA (ATCC 33592), and MDR P. aeruginosa (ATCC BAA-2110). It was also found that Apo5 and Apo6 were somewhat less active against both strains of E. coli tested, with EC 50 values ranging from 4 to 20 μg/ml, though our previous work demonstrated that these peptides were extremely effective against E. coli ATCC 25922 [8]. Apo5 and Apo6 had differing activities against P. aeruginosa PAO1; Apo5 was found to have stronger activity against this strain than Apo6 (0.0878 μg/ml vs 1.17 μg/ml).

A1P was found to have stronger broad-spectrum activity than anticipated from our previous study [8]. Although A1P had weak activity against P. aeruginosa PAO1 (EC 50 = 38.6 μg/ml), it was not effective at concentrations tested against MDR P. aeruginosa (ATCC BAA-2110) (Table 4). A1P had stronger antimicrobial activity against MDR strains of E. coli (ATCC 51659), S. aureus (ATCC 33592), and A. baumannii (ATCC BAA-1794), with EC 50 values between 2 and 3 μg/ml, than against the antibiotic sensitive strains tested, which had EC 50 values ranging from 9 to 36 μg/ml.

Membrane permeabilization and depolarization by peptides

To determine whether Apo5, Apo6, and A1P interacted with the bacterial membrane, each peptide’s ability to disrupt or permeabilize the membrane was measured by the ethidium bromide uptake assay, while membrane depolarization was measured with the fluorescent chemical DiSC 3 (5), which is sensitive to the polarization of membranes.

When the ethidium bromide uptake assay was performed (Fig. 5a), it was found that Apo5 and Apo6 permeabilized the E. coli membrane at concentrations at 50 μg/ml quickly, comparable to control peptide LL-37, a known pore-forming peptide. Neither Apo peptide permeabilized membranes at lower concentrations. Like LL-37, these peptides permeabilized the membrane in a significant manner (p < 0.001), with peak fluorescence occurring within 3 min. A1P also permeabilized the membrane at 50 μg/ml significantly higher than the untreated control (p < 0.001), but displayed very different and slower kinetics. With this peptide, fluorescence gradually increased over the 20 min experimental time frame, until reaching nearly equivalent maximum fluorescence as Apo5 and Apo6 by the end of the experiment (Fig. 5b).

Fig. 5 Pore-forming activity by Apo5, Apo6, and A1P. An increase in fluorescence demonstrates greater binding of DNA by ethidium bromide, which indicates the formation of pores in the bacterial membrane. a. Kinetics of permeabilization and binding of ethidium bromide to DNA after treatment with Apo5 (dashes), Apo6 (solid line), A1P (dots), and LL-37 (dash dot) b. Permeabilization of membrane after 20 min Full size image

Depolarization of a bacterial membrane indicates transient membrane disruption that allows for ion leakage, which damages the proton motive force and other gradients that store chemical energy. As shown in Fig. 6, within 1 min it was found that Apo5 and Apo6 depolarized bacterial membranes at concentrations as low as 0.5 μg/ml (p < 0.001), with depolarization showing a clear dose-dependent response to peptide concentration. A1P did not depolarize membranes except at the highest concentration tested, 50 μg/ml (p < 0.05). Depolarization signals at this concentration were well below those achieved at the lowest concentrations used for Apo5, Apo6, and LL-37 (p < 0.001).

Fig. 6 Membrane depolarization activity by Apo5, Apo6, and A1P. Depolarization determined using DiSC 3 (5) for each peptide at 50 μg/ml (■), 5 μg/ml ( ), 0.5 μg/ml ( ), as well as no treatment ( ) Full size image

These results indicate that Apo5 and Apo6 depolarize the bacterial membrane quickly, suggesting membrane disruption is the mechanism by which these peptides kill bacteria. A1P does not depolarize the membrane, nor does it form pores quickly except at high concentrations. This implies that the primary mechanism of A1P is not related to membrane disruption.

DNA binding

Some peptides, such as LL-37 or histone-derived Buforin II, bind nucleic acids [4, 41], inhibiting translation and transcription or promoting mutagenesis. In general, this binding mechanism has been shown to be non-specific [41, 42]. To determine if any of the CAMPs were able to bind DNA, a gel shift assay was performed, shown in Fig. 7. A1P bound DNA only at very high ratios, needing at least 20 times more mass of peptide than DNA to inhibit DNA movement. Apo6 did not bind DNA at any concentration, while Apo5 bound DNA only at the highest concentration tested. It is unlikely that the primary mechanism of action of these three peptides is related to DNA binding.

Fig. 7 DNA binding by a Apo5, b Apo6, c A1P, and d LL-37 as measured using a gel shift assay. Ratio of DNA:CAMP is shown Full size image

Cytotoxicity

The physiochemical properties of CAMPs preclude significant host-cell cytotoxicity; however, some CAMPs, such as SMAP-29, have been found to cause damage to eukaryotic cells at similar concentrations. Cytotoxicity against red blood cells and A549 lung epithelial cells was measured. For red blood cells, a spectrophotometric assay that measures free heme was used, while an MTT assay was used for A549 cells. As shown in Table 4, these peptides have EC50’s between 0.07–39 μg/ml. In Fig. 8a, hemolytic activity is shown as percent hemolysis. All four peptides showed hemolysis of RBCs of less than 1 % at 300 μg/ml, a, comparable to the control peptide LL-37. No statistically significant difference was found between the untreated control and all peptides, indicating that Apo5, Apo6, and A1P are not hemolytic. The MTT assay was used to measure cytotoxicity of other cells lines. After 24 h exposure, concentrations of peptide up to 100 μg/ml were not significantly cytotoxic against A549 cells, as shown in Fig. 8b, while the EC50 against A. baumannii was less than 1 μg/ml of Apo peptide.