A Temporin Derived Peptide Showing Antibacterial and Antibiofilm Activities against Staphylococcus aureus

Abstract

Background

Temporin is one family of the shortest antimicrobial peptides found in Ranidae frogs. Staphylococcus aureus is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections in humans. Temporin-GHa (GHa) was previously obtained from Hylarana guentheri, showing weak antibacterial activity against S. aureus. Most temporin peptides are positively charged by arginine and lysine; however, GHa contains histidine.

Objective

In order to investigate the impact of positively charged amino acid on its antibacterial and antibiofilm activity, GHa4R was designed and synthesized by replacing histidine with arginine in GHa.

Methods

The antibacterial activity and efficacy against S. aureus were detected by minimum inhibitory concentration, minimum bactericidal concentration, and time-killing kinetics assays. The action mechanism was determined by propidium iodide uptake and scanning electron microscopy assays. The antibiofilm activity was measured by the MTT method. Eradication of biofilm was observed by fluorescence microscope.

Results

Compared to GHa, GHa4R had stronger antibacterial activity and bactericidal efficacy against S. aureus. Impressively, GHa4R presented antibacterial activity against methicillin-resistant S. aureus (MRSA). It was barely affected by temperature, pH, and storage period, showing high stability. Furthermore, it increased the permeability of the cell membrane and damaged the membrane integrity, leading to cell death. In addition, GHa4R did not induce antibiotic resistance in S. aureus in 30 days, but the MIC of vancomycin was doubled. It not only inhibited S. aureus biofilm formation but also eradicated 24 h-biofilms.

Conclusion

The above-mentioned characteristics make GHa4R a promising candidate for the treatment of S. aureus infections.

1. INTRODUCTION

Due to the abuse of antibiotics, the drug resistance of pathogenic bacteria has become a serious problem threatening human health worldwide, which has been widely concerned. In recent decades, antibiotic-resistant infections have been increasing rapidly; however, the discovery and commercialization of new antibiotics is a difficult and time-consuming process [1]. Therefore, the innovation of novel antibiotics has become increasingly important and urgent.

Antimicrobial peptides (AMPs) are peptides that commonly consist of less than 50 amino acid residues and exhibit antibacterial, antifungal, antiviral, anticancer, or immunoregulatory activities [2-4]. They also share the outstanding advantages of thermal stability, high solubility, diverse structure, and low cytotoxicity. AMPs have been considered one of the promising candidates for novel antimicrobial agents [5, 6].

Staphylococcus aureus, a Gram-positive bacterium, is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections [7]. S. aureus can form biofilms on medical devices and patient tissues. Biofilms are highly organized bacteria communities attaching to different surfaces, in which bacteria secrete water-based polymer matrix to protect themselves from antimicrobial agents [8]. The biofilm is not only difficult to be removed but also one of the main reasons for bacteria to develop drug resistance [7].

In our previous study, an AMP was obtained from the frog Hylarana guentheri and characterized as a temporin peptide, namely temporin-GHa (GHa) [9]. Compared to most temporin peptides, GHa has a positively charged amino acid residue histidine at the fourth position from N-terminus rather than arginine or lysine [10]. In order to demonstrate the effect of different positively charged amino acids on the antibacterial activity of temporin peptides, the histidine at position four in GHa was replaced with arginine to obtain a derived peptide GHa4R. In this study, antibacterial and antibiofilm activities against S. aureus of GHa4R were investigated. In comparison with the wild peptide, GHa4R showed stronger efficacy than the parent peptide.

2. MATERIALS AND METHODS

2.1. Microorganisms and Growth Conditions

Bacterial strains were used to determine the antimicrobial activity of temporin peptides, including the Gram-positive bacteria S. aureus (ATCC 25923), Streptococcus mutans (ATCC 25175), Bacillus subtilis (ATCC 6633), methicillin-resistant S. aureus (ATCC 43300, three clinical isolates MRSA-1-3), and the Gram-negative bacteria Escherichia coli (ATCC 25922), E. coli (D31), Pseudomonas aeruginosa (ATCC 15442), P. aeruginosa PAO1 (wild type), as well as fungi Candida albicans (ATCC 10231). The laboratory standard strains were purchased from China General Microbiological Culture Collection Center (CGMCC, China). The clinical isolates were identified by bacterial microbiochemical tube (HuanKai Microbial, China). The bacterial strains were grown in Tryptic Soy Broth (TSB). For the colony-forming units (CFU) count, serially diluted bacterial suspensions were plated on Tryptic Soy Agar (TSA) and incubated for 24 h at 37°C. The fungi were grown in Sabouraud Dextrose Broth (SDB). All experiments were conducted using bacterial cells and C. albicans at the logarithmic phase of growth (OD600 = 0.4-0.6).

2.2. Antimicrobial Peptides

GHa and GHa4R were synthesized by GL Biochem (Shanghai, China) using the solid phase synthesis with the N-9-fluorenylmethyloxycarbonyl (Fmoc) strategy. The peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The purity of peptides was more than 95%. The peptides were stored at -80°C before use.

2.3. Structure Prediction

The antibacterial activities of AMPs were predicted by the online analysis software CAMPR3 (http://www.camp. bicnirrh.res.in/prediction.php). The analysis tools of Heliquest (https://heliquest.ipmc.cnrs.fr/), ExPasy (https:// web.expasy.org/compute_pi/), and the database of APD3 (http://aps.unmc.edu/AP/main.php) were used to calculate hydrophobic moment (μH), charge number, isoelectric point (PI), Boman index (BI) and grand average hydropathy (GRAVY) of the AMPs to assist in the design of peptides. HeliQuest was used to analyse the physicochemical properties and structure of AMPs, and PEP-FOLD3 (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) was used to predict the 3D model of AMPs.

2.4. Determination of MIC/MBC

The antimicrobial activities of the peptides were evaluated by the broth microdilution method with a slight modification [11]. S. aureus was cultured in TSB until the logarithmic growth phase was reached. The bacteria were diluted with TSB to a cell density of 2 × 10⁶ CFU/mL. A series of diluted peptides (the final concentration of 1.6-50 μM, 50 μL per well) was prepared in a 96-well plate, and the same volume of bacterial suspension was added to each well. Vancomycin and Kanamycin were used as the positive controls, and the bacterial suspension without GHa4R was used as the negative control. After incubating at 37ºC for 18-24 h, the absorbance at 600 nm was measured by Multiskan Spectrum (BioTek, USA). Minimum inhibitory concen-tration (MIC) is defined as the minimum peptide concentration that can completely inhibit bacterial growth. A total of 50 μL of the mixture in MIC assay (the concentration was equal to or higher than MIC) was plated on TSA and incubated at 37°C for 24 h. The peptide concentration without bacterial growth is defined as the minimum bactericidal concentration (MBC).

2.5. Growth Inhibition Kinetics

The growth inhibition kinetics of peptides against S. aureus was performed as described previously [10]. The peptide was serially double diluted in a 96-well plate with 100 μL solution in each well (the concentrations of 0.8-12.5 μM). S. aureus at logarithmic growth phase was diluted into 2 × 10⁶ CFU/mL with TSB, and 100 μL bacterial suspension was added into each well and incubated at 37°C for 24 h. TSB was used as a blank control. The absorbance at 600 nm was measured per hour by using Multiskan Spectrum (BioTek, USA).

2.6. Time-kill Kinetics

The experiment was performed as described by Grassi et al. [12]. The sample was serially double diluted with TSB in a centrifuge tube with a volume of 1 mL (the concentration ranging from 1 to 8 × MIC). S. aureus was diluted to 2 × 10⁶ CFU/mL with TSB, and the same volume of bacterial suspension was added to each centrifuge tube. After incubating at 37 °C for 0, 15, 30, 60, 90, 120, and 180 min, respectively, the mixture was diluted, and 50 μL of the bacterial suspension was coated on TSA. The plates were incubated at 37 °C for 18 h, and the number of colonies was counted. TSB without the peptides was used as a negative control. Killing kinetics was constructed by plotting the log10 CFU/mL against time.

2.7. Stability Analysis of GHa4R

To evaluate the stability of GHa4R, the MIC assay was conducted under different conditions, including temperature, metal cations, pH, storage cycle, and temperature, respectively. In the assay of thermal stability, GHa4R was treated at 40°C for 30 min. For cationic stability, GHa4R was incubated with Na⁺ (0.15 M), Ca²⁺ (2.5 mM), Mg²⁺ (1.5 mM), or K⁺ (4.5 mM). To determine the sensitivity of GHa4R to pH, the peptide was incubated in a solution of pH 5 or 7.4. For the storage cycle and temperature assay, aliquots of GHa4R were stored at 25°C, 4°C, and -20°C for 30 and 45 days individually.

2.8. Propidium Iodide (PI) Uptake Assay

The experiment was conducted as described previously [13]. The bacteria (2 × 10⁸ CFU/mL) were inoculated in a 96-well plate. The peptides (the final concentration of 3.1-25 μM) and PI (the final concentration of 20 μM) were added to each well. The suspension was mixed, and the fluorescence was detected every 5 min at the excitation of 584 nm and the emission of 620 nm by the microplate reader for 2 h. Meanwhile, bacterial growth was detected at 600 nm.

2.9. Scanning Electron Microscopy (SEM)

The assay was performed according to Zhong et al. [14]. S. aureus (1 × 10⁹ CFU/mL) was incubated with the same volume of 25 μM GHa4R at 37°C for 1 h and centrifuged to collect the bacteria. The bacteria were fixed with 2.5% glutaraldehyde and washed three times with PBS. After dehydration with 30, 50, 70, 90, and 100% ethanol, the bacteria were resuspended in absolute ethanol and dripped on the tin foil. The bacteria were freeze-dried overnight, sprayed with gold, and observed using a scanning electron microscope (Verios G4 UC, Thermo Scientific, USA). The bacteria untreated with GHa4R served as a negative control.

2.10. Drug Resistance Assays

S. aureus was inoculated in 3 mL TSB containing GHa4R (the final concentration of 3.1 μM). After incubation at 37°C for 24 h, 50 μL bacterial suspension was removed into fresh TSB containing GHa4R (the final concentration of 3.1 μM) and cultured. The subculture was performed daily and lasted for 30 days. MICs of GHa4R against S. aureus were determined on days 5, 10, 15, 20, 25, and 30. Vancomycin was used as a positive control and TSB as a negative control.

2.11. Biofilm Metabolic Activity Assay by MTT Method

The effect of GHa4R on biofilm formation was evaluated according to Yuan et al. [11]. S. aureus at the logarithmic growth phase was diluted to 2 × 10⁶ CFU/mL with TSB (containing 1% glucose). GHa4R (the final concentrations of 0.8-6.2 μM) was mixed with an equal volume of S. aureus suspension in each well on a 96-well plate and incubated at 37 °C for 24 h. The planktonic bacteria were carefully washed out with PBS. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, the final concentration of 5 mg/mL) was added to each well and incubated at 37°C for 3 h. The insoluble purple formazan was dissolved in DMSO. The absorbance at 560 nm was measured by using a microplate reader (Multiskan Spectrum, BioTek, USA).

To determine the eradication effect of GHa4R on biofilms, 200 μL of diluted bacterial suspension was inoculated into each well on a 96-well plate and incubated at 37°C for 24 h to establish 24 h-biofilms. After the planktonic bacteria were washed out with PBS, GHa4R was added (the final concentrations of 6.2-50 μM) to each well and incubated at 37°C for 24 h. The MTT method was used to detect the ability of GHa4R to eradicate 24 h-biofilms according to the above method. The biofilm formation of S. aureus was calculated as follows: (OD570 experimental group/OD570 blank group) × 100%.

2.12. Investigation of Biofilm Inhibition and Eradication by Fluorescence Microscope Analysis

For inhibition of biofilm formation assay, S. aureus at logarithmic phase was diluted with TSB (containing 1% glucose) to 2 × 10⁶ CFU/mL, and the same volume of samples (the final concentration of 1/4 ×, 1/2 ×, and 1 × MIC) was added to each well containing a cover slip on a 24-well plate. The wells in the absence of GHa4R were used as a positive control. The plate was cultured at 37°C for 24 h.

For eradication of biofilm test, 24 h-biofilm was obtained by inoculating bacterial suspension (1 × 10⁶ CFU/mL) into each well containing a cover slip on a 24-well plate for 24 h incubation. Planktonic bacteria were washed away with PBS, and established biofilms were treated with 200 μL GHa4R (the final concentration of 4 ×, 8 ×, and 16 × MIC) and incubated at 37°C for 24 h. The wells without GHa4R were used as blank control.

To observe the effect of GHa4R on the biofilm, 10 μM SYTO was added to each well in the 24-well plates and incubated for 15 min in the dark after the treatment described above. The biofilms on the coverslips were observed by using a fluorescence microscope (Leica, DM6000, Germany).

2.13. Statistical Analysis

The Graphpad Prism 6 (Software GraphPad, USA) was used for data analysis, and statistical significance was calculated with a t-test by comparison with the untreated control (*P < 0.05; **P < 0.01; ***P < 0.001). All experiments were conducted in triplicate independently.

3. RESULTS

3.1. Antibacterial Activity Prediction and Physico-chemi-cal Properties Analysis

The prediction of the antibacterial activity of AMPs determined by using CAMPR3 is illustrated in Table S1 ^{(130.9KB, pdf)} . CAMPR3 combines amino acid composition, physicochemical properties, and structural characteristics to construct four prediction models, namely support vector machine (SVM), random forest (RF), artificial neural network (ANN), and discriminant analysis (DA) [15]. Among them, SVM, RF, and ANN models give probability scores (between 1 and 0). The higher value of a peptide means it is more likely to exhibit antimicrobial activity. The predicted results of the ANN model are as follows: AMP is indicated to be an antibacterial sequence, and NAMP means no antibacterial effect. The values of SVM, RF, and DA of GHa4R were slightly higher than that of GHa, which means that GHa4R was more likely to exert antimicrobial activity. Moreover, GHa4R was predicted to be an antimicrobial peptide sequence by the ANN model. The sequences and physicochemical properties of the peptides are shown in Table 1. Compared to GHa, the grand average hydropathy (GRAVY) value of GHa4R was decreased, but the charge number and Boman index (BI) were increased, which might be beneficial to enhance the antibacterial activity. In the helical wheel projection, the hydrophobic and hydrophilic amino acids are distributed on the opposite side of the derived peptide to individually form a hydrophobic and hydrophilic surface. The PEP-FOLD predicted that GHa4R has an α-helix structure with a hydrophilic surface and a hydrophobic surface along the long axis, which was consistent with the spiral wheel (Figure 1).

Table 1.

The sequences and physicochemical properties of GHa and GHa4R.

Peptides	Sequence	MWa	μHb	Chargea	pIc	BIa	GRAVYa
GHa	FLQHIIGALGHLF	1464.76	1.71	0	7.67	-1.49	1.315
GHa4R	FLQRIIGALGHLF	1483.87	1.67	1	10.5	-0.7	1.215

Note: ^aThe data were determined at APD3. The charge was calculated under physiological conditions. MW is molecular weight; BI is the Boman index (kcal/mol); GRAVY is grand average hydropathy. ^bDetermined by using Heliquest. μH is a hydrophobic moment. ^c Determined by using ExPasy. PI is an isoelectric point.

Figure 1.

Helical wheel projection and the predicted structure of temporin peptides. The structure prediction of (A) GHa and (B) GHa4R. The left column shows the helical wheel illustration of the peptide generated by using Heliquest, and the arrow indicates the direction of the hydrophobic torque. The middle column demonstrates the 3D structure predicted by PEP-FOLD. The hydrophilic surface is expressed in blue, and the hydrophobic surface is in yellow. The right column depicts the secondary structure of the peptides predicted by PEP-FOLD. The helical, coil, and extended conformation are colored red, blue, and green.

3.2. MIC and MBC Measurement

GHa showed weak antimicrobial activity against S. aureus, S. mutans, and E. coli and exhibited no antifungal activity (Table 2). The derived peptide GHa4R exhibited more efficient antibacterial activity with a broad antimicrobial spectrum than GHa, and the MIC was reduced by 2-16 times. GHa4R showed not only stronger antibacterial activity against both tested Gram-positive and Gram-negative bacteria but also exhibited anti-fungi efficacy. GHa4R also showed antibacterial activity against methicillin-resistant S. aureus (MRSA-1-3) clinical isolates, which was even better than vancomycin (Table 2). As shown in Table 2, the MBC of GHa4R against S. aureus was equal to the MIC value, indicating that the derived peptide had stronger bactericidal effect than GHa.

Table 2.

MICs and MBCs of the peptides against the tested strains.

Species	Strains	MIC/MBC (μM)
		GHa	GHa4R	Kan	Van
Gram+	S. aureus	12.5/25	6.2/6.2	6.2	0.05
	S. mutans	25/50	6.2/6.2	6.2	-
	B. subtilis	>100/>100	12.5/25	>100	-
	MRSA	100/>100	6.2/6.2	>100	0.4
	MRSA-1	>100	6.2/12.5	>100	25
	MRSA-2	>100	6.2/12.5	>100	3.1
	MRSA-3	>100	12.5/25	6.2	50
Gram-	E. coli	25/50	50/100	12.5	-
	D31	>100/>100	50/50	25	-
	PAO1	>100/>100	>100/>100	25	-
	PA	>100/>100	12.5/12.5	>100	-
Fungi	C. albicans	50/>100	12.5/25	>100	-

Abbreviations: Kan: Kanamycin; Van: Vancomycin; - means the MIC assay for vancomycin was not conducted.

3.3. Determination of Growth Inhibition Curves and Time-Killing Curves

The effects of different concentrations of GHa4R on the growth of S. aureus were evaluated (Figure 2A). The growth curve of S. aureus contains an adaptation period, logarithmic period, stationary period, and decline period. When the concentration of GHa4R was 6.2 μM (1 × MIC), the growth of S. aureus was completely inhibited. After being treated with 0.8-1.6 μM GHa4R, the growth of S. aureus remained almost unaffected, and the growth curves were the same as in the untreated group. When the bacteria were exposed to 3.2 μM GHa4R, the growth of S. aureus was inhibited initially, and the logarithmic period lagged for about 4 h. Once the bacteria reached the stationary phase, there was no significant difference between the treated and untreated groups. The bactericidal activity of GHa4R against S. aureus showed in a concentration and time dependence model (Figure 2B). GHa4R killed the bacteria completely in 90 min at 2 × MIC (12.5 μM). Meanwhile, the bacteria treated with 1 × MIC GHa4R could not completely be eliminated at 180 min, but the number of bacterial colonies decreased by about four orders of magnitude. The wild peptide GHa did not kill all the bacteria, even at the concentration of 2 × MIC (25 μM).

Figure 2.

The growth curves and time-killing curves of S. aureus treated with or without GHa4R. (A) The growth curves of GHa and GHa4R. (B) The time-killing curves of GHa and GHa4R.

3.4. Investigation of GHa4R Stability

After GHa4R was treated at 25°C and 50°C for 30 min, the MICs were measured (Table S2 ^{(130.9KB, pdf)} ). The MIC of GHa4R against S. aureus after treatment at the tested temperature was consistent with that treated at 37°C, indicating that GHa4R had good thermal stability. After incubating GHa4R with the solution with pH 5 or 7.4, the MIC of GHa4R was the same as that of water (Table S2 ^{(130.9KB, pdf)} ).

The stability of GHa4R against S. aureus in different metal cations is shown in Table S3 ^{(130.9KB, pdf)} . GHa4R showed excellent stability in the presence of monovalent cations (Na⁺, K⁺) and divalent cations (Ca²⁺, Mg²⁺) at physiological concentrations.

The different storage times and temperatures had no effect on the antibacterial activity of GHa4R against S. aureus (Table S3 ^{(130.9KB, pdf)} ). The MIC of GHa4R against S. aureus was unchanged after 30 and 45 days of storage at 25°C, 4°C, and -20°C, indicating that GHa4R had excellent stability.

3.5. Propidium Iodide (PI) Uptake Assay

We detected the membrane permeability of the GHa4R by PI-uptake assay. PI cannot penetrate the intact cell membrane. When the cell membrane is damaged, PI will enter into bacteria and bind to DNA, resulting in an increase in PI fluorescence. As shown in (Figure 3), the intensity of PI fluorescence treated with GHa4R increased in a concentration-dependent manner. Compared to the untreated control, the fluorescence of bacteria treated with 25 μM (4 × MIC) GHa4R increased dramatically and reached the maximum fluorescence unit (RFU) in 10 min. The PI uptake was also observed on the bacteria treated by GHa, but the increase in fluorescence was mild. S. aureus did not proliferate in all groups within 120 min. The results indicated that GHa4R interfered with bacterial membrane integrity to exert its antibacterial activity.

Figure 3.

The membrane permeability and morphology of S. aureus induced by the GHa4R were detected. The real-time growth of the bacteria treated by (A) GHa and (B) GHa4R was monitored. The membrane permeability of (C) GHa and (D) GHa4R was analyzed by using fluorescence spectrometry. The morphology of S. aureus treated in the (E) absence and presence of (F) GHa4R was observed by using SEM. The arrows indicate the changes induced by GHa4R in cell morphology.

3.6. Morphological Observation by Scanning Electron Microscope (SEM)

The morphological changes of bacteria incubated with the peptides were observed by SEM. In the absence of GHa4R, the outer surface of the bacteria was smooth and intact (Figure 3E). After the bacteria were exposed to a 4 × MIC concentration of GHa4R (Figure 3F), the morphology of some bacterial cells was altered. Those cells were ruptured completely, and the cytoplasmic contents were expelled. The other bacterial cells underwent morphological changes, leading to a rough cell surface. Some protuberances were observed on most of the treated bacteria. Combined with the PI assay, these results showed that the membrane integrity of S. aureus was interrupted by GHa4R, resulting in bacterial death.

3.7. Drug Resistance Assay

The measurement of drug resistance induced in vitro lasted for 30 days (Table S4 ^{(130.9KB, pdf)} ). The MIC of GHa4R against S. aureus remained unchanged for 30 days, indicating that it was difficult for S. aureus to develop drug resistance to GHa4R. On the contrary, the MIC of vancomycin against S. aureus increased by one time on 20 days, indicating that S. aureus developed resistance to vancomycin rapidly.

3.8. Effect of the GHa4R on Biofilms

The ability of GHa4R to inhibit biofilm formation was studied, which was represented by the minimum biofilm inhibitory concentration (MBIC) of the peptides [11]. MBIC is defined as the lowest concentration of the peptides required to inhibit biofilm formation. GHa4R could effectively inhibit the biofilm formation of S. aureus in a concentration-dependent manner. The minimum concen-tration required to inhibit 50% biofilm formation (MBIC50) of GHa4R was 6.2 μM, and the inhibition rate was up to 90%, indicating that GHa4R had a remarkable ability to inhibit biofilm formation. The MBIC50 value of GHa4R is equal to the MIC value for planktonic cells. Therefore, this inhibitory effect may be due to the antibacterial effect on these cells.

The activity of GHa4R to eradicate biofilm was determined, represented by the minimum biofilm eradication concentration (MBEC) of the peptides [10, 11]. MBEC is defined as the minimum concen-tration of the peptides required for the eradication of mature biofilm. GHa4R could effectively eradicate the 24 h-biofilm of S. aureus, showing a concentration-dependent manner (Figure 4). The minimum concentration required to eradicate 50% biofilm (MBEC50) of GHa4R was 25 μM, with a destruction rate of 87%, indicating that GHa4R could destroy the 24 h-biofilm of S. aureus at higher concen-trations.

Figure 4.

The effect of GHa4R on biofilms of S. aureus. The antibiofilm activity of (A) GHa and (B) GHa4R during biofilm formation. The biofilm eradication effect of (C) GHa and (D) GHa4R. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. nontreated controls. (E-L) The antibiofilm activity of GHa4R was observed by using a fluorescence microscope. (E–H) GHa4R inhibited the biofilm formation of S. aureus. The bacteria were treated with (E) 0, (F) 1.6, (G) 3.1, and (H) 6.2 μM GHa4R. (I-L) GHa4R eradicated the 24 h-biofilm of S. aureus. The 24 h-biofilms were exposed to (I) 0, (J) 25, (K) 50, and (L) 100 μM GHa4R. The biofilms were stained with SYTO and colored green. The black area means the biofilms were absent or destroyed.

3.9. Fluorescence Microscope Analysis

The efficacy of GHa4R in inhibiting the formation of S. aureus biofilm and eradicating biofilm was observed using a fluorescence microscope. In the untreated group, the biofilms were dense and thick. In the assay of inhibiting biofilm formation (Figures 4E-H), with the increase in GHa4R, the biofilm formation was decreased, and fewer scatted biofilm clusters were observed. When the GHa4R concentration was 6.2 μM (Figure 4H), the biofilm formation was almost completely inhibited.

4. DISCUSSION

According to the bioinformatics statistics on AMPs, we found that the positively charged amino acids, lysine and arginine, occur more frequently in natural AMPs rather than histidine [10]. GHa is composed of 13 amino acid residues containing 2 histidine residues [9]. In order to investigate the effect of different positively charged amino acid residues on the antibacterial activity of AMPs, the histidine in position 4 from N-terminal was substituted by arginine to obtain a derived peptide GHa4R. Although GHa4R exhibited better activity, it was only twice stronger as GHa (12.5 versus 6.2 µM), and the MBC of GHa4R was also 6.2 µM, indicating GHa4R has much stronger bactericidal activity than GHa (MBC of 25 µM). Bacalum et al. reported that the substitution of the N-terminal arginine by histidine of an arginine-rich peptide slightly reduced the antimicrobial activity against S. aureus; replacement of both N- and C-terminal arginine or in position 5 (of 9) reduced more antimicrobial activity [16]. These studies demonstrated that compared to histidine, arginine residue in AMPs significantly contributed to their antimicrobial activity. Generally, AMPs with strong antibacterial activity usually have higher amphiphilicity or hydrophobic distance; their GRAVY is positive, while the Boman index is negative or close to zero [13]. Compared to GHa, the GRAVY value of GHa4R was reduced, and its hydrophobic moment was almost unchanged. Meanwhile, the BI value was increased from -1.49 to -0.7. Many researchers have proposed a variety of antimicrobial action mechanisms of AMPs. However, a large number of studies have shown that most AMPs prefer to target the cell membrane [17, 18]. Positively charged residues contribute to positive charges interacting with negative charges distributed on the envelopes and membranes of bacteria by electrostatic attractions. Arginine replacement provides a positive charge on GHa4R, leading to strong attractions between the peptide and bacterial membranes. GHa4R increased the membrane permeability of bacteria and achieved a bactericidal effect by destroying the bacterial cell membrane. The cation-π interaction plays a very important role in various proteins bound to cation ligands or substrates [19]. In GHa4R, the cation-π interaction between phenylalanine at N-terminal and arginine at position 4 may occur, which might be beneficial to the insertion of GHa4R into the bacterial cell membrane and enhance the antibacterial activity. Bacteria were less prone to developing drug resistance against GHa4R than vancomycin. GHa4R inhibits bacteria growth by disturbing cell membrane integrity, which makes it difficult for pathogenic bacteria to develop drug resistance. As the composition of the cell membrane of pathogenic bacteria is relatively stable, changing the constitution of a cell membrane is a big challenge for bacteria to develop drug resistance [20].

It is reported that more than 70% of lethal bacterial infections are associated with the formation of biofilms [21, 22]. GHa4R showed a significant ability to inhibit biofilm formation and eradicate 24 h-biofilms of S. aureus. At sub-inhibitory concentrations, some conventional antibiotics (including aminoglycosides, fluoroquinolones, and tetracyc-lines) stimulate the formation of bacterial biofilms [23, 24], causing aggravated inflammation and infections in patients. GHa4R can significantly inhibit biofilm formation at subinhibitory concentration, suggesting that GHa4R has the potential to develop into a new anti-bacterial agent.

AMPs have high thermal stability [25-27]. The activity of GHa4R to S. aureus did not change after being preheated at high temperatures, indicating its excellent thermal stability. Similarly, the antibacterial activity of GHa4R had no significant changes under different pH and cations (Na⁺, Ca²⁺, Mg²⁺, K⁺). Good acid and cations resistance indicates that GHa4R adapts to different pH body fluid environments. The MIC of GHa4R did not change at different storage temperatures and storage times, showing good environmental stability and long shelf life.

CONCLUSION

In summary, the temporin-derived peptide GHa4R showed higher antibacterial activity and bactericidal efficacy than GHa. Replacing histidine with arginine increased the positive charge and amphiphilicity of the derived peptide, contributing to stronger and faster bactericidal action. GHa4R inhibited the formation of biofilms and had the ability to eradicate 24 h-biofilms of S. aureus. Moreover, GHa4R was stable under different harsh conditions. It was difficult for S. aureus to develop drug resistance against GHa4R. These characteristics make GHa4R a promising antibacterial agent with the potential for the treatment of S. aureus biofilm infections.

LIST OF ABBREVIATIONS

GHa

Temporin-GHa

GHa4R

Temporin-GHa4R

AMPs

Antimicrobial Peptides

MTT

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Bromide

MRSA

Methicillin-resistant Staphylococcus aureus

MIC

Minimum Inhibitory Concentration

MBC

Minimum Bactericidal Concentration

CFU

Colony-Forming Units

TSB

Tryptic Soy Broth

TSA

Tryptic Soy Agar

SDB

Sabouraud Dextrose Broth

RP-HPLC

Reverse Phase High Performance Liquid Chromatography

µH

Hydrophobic Moment

PI

Isoelectric Point

BI

Boman Index

GRAVY

Grand Average Hydropathy

SEM

Scanning Electron Microscopy

AUTHOR’S CONTRIBUTIONS

M. A., R. G., S. X., and J. W. performed the experiments and analyzed the data. M. A. and J. W. wrote the original manuscript. Y. S., R. W., and W. J. performed data curation. S. W. and Y. Z. supervised the project and reviewed the manuscript. Y. Z. got the funding support.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No animals/humans were used in the studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available within the article.

FUNDING

This work was financially supported by the Education Department of Hainan Province (Number Hnjg2021-26) and the National Natural Science Foundation of China (Number 32060130).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s website along with the published article.