Hybridization and antibiotic synergism as a tool for reducing the cytotoxicity of antimicrobial peptides

Figures & data

Figure 1 Analytical RP-HPLC chromatogram of the synthetic hybrid peptide H4 displaying a purity of >95%.

Abbreviation: RP-HPLC, reverse-phase high-performance liquid chromatography.

Figure 2 Positive ESI-MS analysis of the synthetic peptide H4 showing major peaks in the +4, +5, +6, and +7 charge states of 910.27, 728.38, 607.16, and 520.68 Da, respectively.

Abbreviation: ESI-MS, electrospray ionization-mass spectrometry.

Table 1 NPS HNN secondary structure analysis and percentage helicity generated for BMAP-27, OP-145, and the hybrid peptide H4 in addition to the α-helical fragments identified from each peptide

Peptide	Natural sequence	Helicity %	a-helical fragments
BMAP-27	GRFKRFRKKFKKLFKKLSPVIPLLHL-NH2	73.08	KFKKLFKKLSPV RFKRFRK RFKRF
OP-145	IGKEFKRIVERIKRFLRELVRPLR	91.8	GKEFKR IGKEFKRI IGKEFKRIVERIKRFL
H4	KFKKLFKKLSPVIGKEFKRIVERIKRFLR	90	KFKKLFKKLSPV IGKEFKRIVERIKRFL

Abbreviation: NPS HNN, Hierarchical Neural Network in Network Protein Sequence.

Table 2 The calculated mean hydrophobicity <H>, hydrophobic moment <µH>, and net charge of BMAP-27, OP-145, and the hybrid peptide H4

Peptide name	Hybrid sequence	Hydrophobicity <H>	Hydrophobic moment <μΗ>	Net charge z
BMAP-27	GRFKRFRKKFKKLFKKLSPVIPLLHL-NH2	0.394	0.474	10
OP-145	GIGAVLKVLTTGLPALISWIKRKRQQ	0.165	0.805	6
H4	KFKKLFKKLSPVIGKEFKRIVERIKRFLR	0.272	0.638	6

Figure 3 Three-dimensional structural modeling of H4. Red regions correspond to helical structures within the peptide, while the green regions represent hinged regions and unordered conformations, respectively. The structure was visualized using Accelrys Discovery Studio software.

Table 3 MICs of H4 against all the microbial strains employed in this study

Bacterial strain	ATCC	MIC (μΜ)
Standard strains (Gram-positive)
Staphylococcus aureus	29213	20
Enterococcus faecalis	19433	15
Staphylococcus epidermidis	12228	10
Resistant strains (Gram-positive)
S. aureus	33591	10
S. aureus	43300	10
S. aureus	BAA41	10
E. faecalis	BAA2365	5
Enterococcus faecium	BAA2316	5
Standard strain (Gram-negative)
Escherichia coli	25922	10
Pseudomonas aeruginosa	27853	20
Acinetobacter baumannii	19606	10
Klebsiella pneumoniae	13883	2.5
Resistant strains (Gram-negative)
P. aeruginosa	BAA2114	25
E. coli	BAA2452	10

Abbreviations: ATCC, American Type Culture Collection; MIC, minimum inhibitory concentration.

Table 4 MICs of individual antibiotics and those in combination with H4 including the percentage reduction of antibiotic MIC when in combination; MICs of individual H4 and those in antibiotic combination; and the FIC indices for the antimicrobial combinations against the tested bacterial species

Bacterial species	Antibiotics				H4		FIC index
		Antibiotic individual MIC μM)	Antibiotic MIC in combination (μM)	Reduction in antibiotic MIC (%)	H4 individual MIC μM)	H4 MIC in combination μM)
Enterococcus faecalis	Levofloxacin	12.5	5	60	5	0.25	0.45
ATCC BAA2316	Chloramphenicol	20	15	25	5	0.25	0.8
	Rifampicin	7.5	1.8	75	5	0.25	0.29
	Erythromycin	-	-	-	-	-	Antagonism
Staphylococcus aureus	Levofloxacin	0.75	0.25	67	10	7.5	1.08
ATCC 33591	Chloramphenicol	130	30	77	7.5	0.25	0.26
	Rifampicin	0.4	0.1	75	7.5	0.2	0.27
	Erythromycin	-	-	-	-	-	Antagonism
S. aureus ATCC	Levofloxacin	0.5	0.125	75	20	2.5	0.37
29213	Chloramphenicol	20	10	50	20	5	0.75
	Rifampicin	0.025	0.005	80	20	2.5	0.32
	Erythromycin	0.5	0.125	75	20	2.5	0.37
Pseudomonas	Levofloxacin	12	8	33	25	0.75	0.69
aeruginosa ATCC	Chloramphenicol	200	25	87	25	2.5	0.22
BAA2114	Rifampicin	50	20	60	25	2.5	0.5
	Erythromycin	-	-	-	-	-	Antagonism
P. aeruginosa ATCC	Levofloxacin	7.5	2.5	67	20	0.75	0.37
27853	Chloramphenicol	350	150	57	20	10	0.93
	Rifampicin	45	15	67	20	0.25	0.34
	Erythromycin	150	2.5	98	20	10	0.5

Abbreviations: ATCC, American Type Culture Collection; FIC, fractional inhibitory concentration; MIC, minimum inhibitory concentration.

Table 5 Antibiofilm activity of H4 against standard resistant strains of Staphylococcus aureus (BAA41) and Pseudomonas aeruginosa (BAA2114)

Antibiofilm activity	S.aureus(ATCC BAA41)	P. aeruginosa(BAA2114)
MBEC value	20 μM	25 μM

Abbreviations: ATCC, American Type Culture Collection; MBEC, minimum biofilm eradication concentration.

Table 6 Hemolytic activity of H4 against human erythrocytes after 1 hour of exposure to the peptide

Peptide concentration (μM)	Hemolysis (%) H4
5	0
10	0
25	0
40	0
55	0
70	0
85	0
100	2.1

Figure 4 Cell survival curves as measured by MTT assay for H4 against two normal mammalian cell lines, Vero and HEK293. Cells were incubated with various concentrations of the peptides, for 24 hours, at 37°C. Control cells represent 100% proliferation, and the mean absorbance of treated cells was related to control values to determine sensitivity. Error bars represent standard error from mean cell proliferation as determined by repeated experiments.

Abbreviation: MTT, methylthiazolyldiphenyl-tetrazolium bromide.

Table 7 Cytotoxicity effects (IC₅₀ value) using MTT assay of H4 on two eukaryotic mammalian cell lines (HEK293 and Vero)

Peptide	HEK293 IC50(μM)	Vero IC50(μM)
H4	61.9±0.18	64.9± 1.01

Note: Data represent the mean ± SD of three independent experiments.

Abbreviations: IC₅₀, half maximal inhibitory concentration; MTT, methylthiazolyldiphenyl-tetrazolium bromide.