Rui-Qi Wang, Chen-Peng Liu, Hong-Ji Yao, Jing-Yan Xiong, Guo-Yu Li, Yong Zhang?

1. Department of Department of Basic Medicine and Life Sciences, Hainan Medical University, Haikou 571199, China

2. Research Center of Pharmaceutical Engineering Technology, Harbin University of Commerce, Harbin 150076, China

ABSTRACT Objective: To design a bi-functional antimicrobial peptide with bactericidal and endotoxin neutralization activity, and explore its bactericidal properties. Methods: The LBP (86-99)peptides and HLF (1-11) peptides were connected by a GGGS flexible 4-peptide linker to obtain the bi-functional antimicrobial peptide, which was named LLH. The secondary structure characteristics of LLH were analyzed by Emboss software. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of LLH against Escherichia coli ATCC25922 and DH5α were determined by the microtiter broth dilution method. The bactericidal kinetics of LLH was characterized and its effect on endotoxin neutralization was determined. The hemolysis of LLH was evaluated. Results:LLH carried a positive charge of +9, exhibited β-folding and β-corner structure, and had strong hydrophobicity. The MIC of LLH against Escherichia coli ATCC25922 and DH5α was 4μM, and the MBC of LLH against Escherichia coli ATCC25922 and DH5α was 8 and 4μM,respectively. LLH showed rapid bactericidal effects and significantly neutralized the endotoxin released in the sterilization process as well as reducing release of endotoxin. LLH showed no significant hemolysis at concentrations up to 400μg/ mL. Conclusion: LLH produces dual effects of rapid sterilization and endotoxin neutralization, and does not induce significant hemolysis.

Keywords:Hybrid antimicrobial peptides Linker Endotoxin Bactericidal Hemolytic?Corresponding author: ZHANG Yong, Ph.D..E-mail: hy0206162@hainmc.edu.cn.

1. Introduction

With the extensive production of antibiotic resistance, bacterial infections are becoming a greater threat to human health. The development of new antibacterial drugs is urgent to alleviate the problem of antibiotic resistance, and decrease the pressure of clinical treatment, especially for gram-negative bacteria infections[1].In addition, one of the main problem during the treatment of gramnegative bacteria is endotoxins released by bacterial killing in clinic,which may induce the inflammatory cytokines production[2], such as the treatment of sepsis[3]. The design of novel anti-bacterial drugs with both bactericidal and neutralizing endotoxin effect is one of the effective ways to solve the endotoxin released problem during the treatment of gram-negative bacterial infections.

LBP (86-99) (RVQGRWKVRASFFK) is an peptide from 86-99 aa of lipopolysaccharide binding protein (LBP), which can bind to endotoxin in serum and exerts a neutralizing effect on endotoxin,but its antibacterial activity against gram-negative bacteria is low[4]. HLF (1-11) (GRRRRSVQWCA) is a human-derived lactoferrin peptide that is non-toxic, stable and has a broad antibacterial spectrum, but has no neutralizing endotoxin effect [5]. In this study,the hybridization of LBP (86-99) and HLF (1-11) was achieved by GGGS linker, the novel hybrid antibacterial peptides was named LLH, which own higher antibacterial activity and endotoxin neutralization effect. This study provided a new idea and reference for the design and development of novel antibacterial drugs against gram negative bacteria.

2. Materials and methods

2.1 Materials

2.1.1 strains of bacteria

E.coli ATCC25922, E.coli ATCC DH5α were from our laboratory frozen.

2.1.2 Reagents and instruments

Yeast dipping powder, tryptone, agar, NaCl and other reagents were purchased from Beijing Solarbio Technology Co., Ltd;Chromogenic matrix limulus assay kit was purchased from Beijing Solarbio Technology Co., Ltd; The main instruments are a biosafety cabinet(HR900-IIA2), bacteria thermostatic incubator (INNOVA44R),multifunctional microplate reader (SynergyHTX), centrifuge, etc.

2.2 Methods

2.2.1 Design and synthesis of bi-functional bybrid peptides

LBP (86-99)(RVQGRWKVRASFFK) is a polypeptide with the function of neutralizing endotoxin, but its antibacterial activity is low; HLF (1-11) (GRRRRSVQWCA) is a lactoferrin polypeptide from human, which own broad-spectrum antibacterial activity,but does not have the function of neutralizing endotoxin. The novel bybrid peptides LBP and LLH were designed by coupling with GGGS tetrapeptide linker (Table 1).The peptides were synthesized by a solid phase synthesis method with a purity of ＞98% by Biotech Bioengineering (Shanghai) Co. The peptides were analyzed by EMBOSS and ExPASy bioinformatics software for prediction of physicochemical parameters, secondary structure and hydrophobicity.

2.2.2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test

The MIC and MBC of peptides were determined by the microtiter broth dilution method MIC test was as a previous study[6]: the peptides and the positive drug ampicillin (Amp) were twofold serial dilutions with gradient concentration of 256, 128, 64, 32, 16, 8, 4,2, 1, 0.5, 0.25μM with sterile LB liquid medium. The MIC plates were prepared using 50 μL of serial concentration gradient solutions of peptides or Amp, and the sterile LB liquid medium was added as a negative control. E.coli ATCC25922 and E.coli ATCCDH5α tested strains were cultured in LB liquid medium and incubated at 37oC with shaking until OD600nm reach to 0.4. E.coli suspension was prepared at a concentration of 0.5 Mackay's turbidimetric standard,then diluted 1000 times with pre-warmed (37oC) sterile LB liquid medium. And 50μL of bacterial suspension was added to MIC plate pre-prepared (initial bacterial concentration was approximately 1.0×105 CFU/ml) and incubated at 37℃ for 16-18 h. The MIC was defined as the lowest concentration of ones at which there was no visible growth.. Minimum bactericidal concentration (short for MBC, the lowest concentration of each peptide when 99.9 %reduction of the initial inoculum was observed) was determined by plating 10-μl samples from wells with no visible growth onto MHA plates.

2.2.3 Time-kill curve assays

As in 1.2.2, 1.0×105cfu/ml of E.coli ATCC 25922 and ATCC DH5a suspensions were prepared, and 50μl bacterial suspension was added to 96-well plate, and LLH hybrid peptide solution was added at final concentrations of 1×, 2× and 4×MIC, and incubated at 37oC. At the same time, the same volume of LB medium and 2×MIC final concentration of Amp was added as blank control and positive control treatment. Samples were taken at 0, 1, 2, 4, 6 and 12h time points, and coated on LB solid medium plates respectively and incubated at 37oC for 18-24h, single colonies were counted and time-kill curve were done.

2.2.4 Endotoxin neutralization test

(1) Sample preparation: prepartion E.coli ATCC25922 bacterial suspension as in 1.2.2 at a final concentration of 1.0×105 CFU/ml(triangular flask dry baked at 250oC for 2h to remove pyrogens,all other consumables were purchased as pyrogens-free). Add 90μl of bacterial suspension to a 96-well cell culture plate and 10μl of LLH hybrid peptide at a final concentration of 4×MIC, add an equal volume of pyrogen-free saline and 2×MIC Amp as control treatment.The group without inoculation of bacteria and drugs and the addition of an equal volume of pyrogen-free saline was used as a blank control. Six replicates wells were done per treatment group, and incubated at 37oC for 2h.

(2) Bacterial endotoxin assay: three parallel wells were selected for each treatment group and the supernatant of the bacterial fluid was collected for endotoxin level determination. The principle of the assay is as follows: the horseshoe crab reagent is a freeze-dried product of the blood deformed cell lysate of the Oriental horseshoe crab, and the bacterial endotoxin can activate the C factor in the horseshoe crab reagent, which activates the prothrombin to form thrombin, then breaks down the chromogenic substrate to produce yellow p-nitroaniline (pNA), which is coupled with the azo reagent to form pNA azo compound (λ=545nm). The amount of pNA azo produced is positively correlated with the concentration of bacterial endotoxin. The method and procedure are described in the chromogenic matrix horseshoe crab kit (T7571, Solebro).

(3) Bacterial count assay: three parallel replicate wells were selected for each treatment group and used for colony counting to detect the number of viable bacteria in the culture medium and to evaluate the antibacterial activity of LLH.

2.2.5 Hemolytic assay

The cytotoxicity of LLH hybrid peptide was determined by the haemolytic assay of rabbit erythrocytes with the following method.LLH hybrid peptide was dissolved in sterile saline at 230μM (800μg/ml) and twofold serial diluted to concentration of 0.1μg/ml. Then 5ml of blood was collected through a rabbit ear marginal vein,centrifuged at 4oC, 1500rpm for 5min, then removed the serum,and collected the red blood cells. Red blood cells were washed 3 times with sterile saline. Prepared 8% erythrocyte suspension with sterile saline, taken 100μl of erythrocyte suspension and a series of concentrations of LLH hybrid peptide solution into a 96-well plate,then incubated at 37oC for 1h, centrifuged at 1500rpm for 5min,transfered the supernatant into an ELISA enzyme plate and detected the UV absorbance value at 540nm. Sterile saline and 0.1% Triton X-100 were used as 0% and 100% haemolytic control respectively.The formulation for calculating the haemolysis is as follows:

Hemolysis (%)=[(Abs540nmhybrid peptide LLH-Abs540nmsterile saline)/(Abs540nm0.1 % Triton X-100-Abs540nmsterile saline)]×100%

2.2.6 Statistical analysis

Graphpad Prism 5.0 was used for statistical analysis and mapping of relevant data in this study. T-test was used for pairwise comparison between different treatment groups, and two-way ANOVA test was used for comparison between multiple groups. P < 0.05 was considered as significant difference.

3. Results

3.1 Design of peptides

The HLF and LBP were used as parent peptides, and LBPLinker-HLF (LLH) hybird peptide was designed and obtained by GGGS tetrapeptide linker. The molecular weight, charge number,isoelectric point, amphiphilicity and hydrophobicity parameters of the relevant peptides are shown in Table 1, from which it can be seen that, compare to the parent peptide, LLH carries an increased charge number to +9 and an increased amphiphilicity index to 45.33, in addition, its hydrophobicity parameter of GRAVY value was reduced.

3.2 Secondary structural features of LLH hybrid peptide

The secondary structure of LLH was predicted by the GOR method of EMBOSS/garnier software. The results showed that LLH mainly showed β-folded and β-turned structures (Figure 1A).The hydrophobicity of the sequence was analysed by EMBOSS/Pepwindowall and showed that the 10-20 aa peptide segment of LLH showed strong hydrophobicity, and its two ends showed hydrophilic characteristics (Figure 1B).

Figure 1 Secondary structure and hydrophobicity prediction of LLH Note: (A) Secondary structure; (B) Hydrophobicity

Table 1 Sequence and physicochemical parameters of peptides

3.3 Antimicrobial effects of LLH

Two E.coli strains (ATCC25922, ATCCDH5a) were selected to evaluate the antibacterial activity of LBP (86-99) and HFL (1-11),and the results are shown in Table 2. Both LBP (86-99) and HFL(1-11) showed antibacterial activity against-E.coli ATCC25922 with MICs of 16 and 64μM respectively, while only LBP(86-99) showed some antibacterial activity against ATCCDH5a with an MIC of 8μM. The MICs of LLH hybrid peptide against E.coli ATCC25922 and ATCCDH5a strains were all 4μM, with significantly enhanced antimicrobial activity compare to the parent peptide (Table 2). The results of the MBC assay are shown in Table 2. The MBC values of LLH hybrid peptide against E.coli were all 1-2 times higher than the MIC values, indicated that LLH hybrid peptide has a strong bactericidal effect. Compared to the traditional antibiotic of Amp(Table 2), LLH hybrid peptide had better activity than Amp.

Table 2 MIC and MBC of LLH

3.4 Bactericidal kinetic features of LLH

The bactericidal kinetic features of LLH hybrid peptide was investigated by time-kill curve assays in vitro, and E.coli ATCC25922 and E.coli ATCC DH5a were selected as representative strains. The initial inoculum of E.coli ATCC25922 and E.coli ATCC DH5α were 4.54±0.09 log10CFU/ml and 4.91±0.11 log10CFU/ml reapectively.The E.coli ATCC25922 increased to 8.27±0.10 log10CFU/ml and E.coli ATCC DH5α increased to 8.41±0.09 log10CFU/ml after 12h incubation without LLH intervention, respectively (Figure 2). After 1h treatment with 1×MIC, 2×MIC and 4×MIC of LLH, the number of E.coli ATCC25922 decreased by 1.1, 1.5 and 2.2 log10CFU/ml, respectively (Figure 2A), while the number of E.coli ATCCDH5a decreased by 0.6, 1.4 and 1.9 log10CFU/ml,respectively (Figure 2B). Those indicated that more than 90% of E.coli were killed after the 1h treatment. After 12h of treatment,E.coli ATCC25922 were reduced by 3.2, 3.5 and 3.5 log10CFU/ml,respectively (Figure 2A), while E.coli ATCCDH5a were reduced by 3.5, 3.9 and 3.9 log10CFU/ml, respectively (Figure 2B). Compared to the LB medium group without LLH treatment, the LLH group with 4×MIC showed 7.2 log10CFU/ml decrease, and indicated that LLH showed strong bactericidal activity against E.coli and did not rebound after 12h (Figure 2).

Figure 2 time-kill curves of LLH

3.5 Neutralizing endotoxin effect of LLH hybrid peptide

E.coli ATCC25922 was selected as the test organism for further investigations. After 2h treatment with 4×MIC LLH hybrid peptide, the number of E.coli ATCC25922 decreased significantly by 3.5 log10CFU/ml and the number of Amp at 2×MIC decreased by 2.59 log10CFU/ml, suggesting that both 4×MIC LLH hybrid peptide and 2×MIC Amp exhibited bactericidal effect (Figure 3A).Under the same treatment conditions, we measured the endotoxin levels in the supernatant of bacterial culture broth, and the results showed that the endotoxin level was significantly lower in LLH hybrid peptide group at 4×MIC compared to the normal group with ddH2O, while the endotoxin level was significantly higher in the 2×MIC Amp group than the 4×MIC LLH hybrid peptide group (Figure 3B).Endotoxin levels were also significantly higher in the Amp-treated group compared to the normal group (Figure 3B).

Figure 3 Neutralizing endotoxin effect of LLH

3.6 Hemolytic assays of LLH hybrid peptide

We further investigated the haemolysis of LLH hybrid peptide. The results shown that less than 1% hemolytic activity was observed about LLH hybrid peptide at various concentration of 0-115 μM(400μg/ml) (Figure 4), therefore, LLH hybrid peptide exhibited no toxic to mammalian erythrocytes and can be used to sterilize mammals and has potential clinical application value.

Figure 4 haemolytic activity of LLH

4. Discussion

Antibiotic resistance has always been an important challenge for clinical anti-infection treatment, especially for gram-negative infections, which pose a significant threat to human health[7,8]. In order to relieve the pressure of traditional antibiotic resistance, it is urgent to develop new antibacterial drugs with high anti-resistance,high activity and low toxicity. Antimicrobial peptides are considered as potential new antibacterial drugs with unique bactericidal mechanism, high efficiency and rapid bactericidal activity and less susceptible to drug resistance[9]. They have been widely concerned and high expectations.

The treatment of gram-negative infections, especially those associated with sepsis and bacteremia, is not sufficient for clinical purposes[10,11]. The growth of gram-negative bacteria and killing bacteria by antibacterial drugs are often cause the releasing of endotoxins, further induce the production of inflammatory factors,and these inflammatory factors further cause many diseases,such as sepsis, and threat to patients' life [12]. Killing bacteria and neutralizing endotoxin are important challenges for the treatment of gram-negative bacteria. In this study, based on these dual function,the GGGS flexible linker was used to realize the hybird of two peptides with endotoxin neutralization effect and killing bacteria effect respectively, so as to realize the dual function effectively without significant hemolysis.

Small peptide flexible linker is an effective way for the design and development of novel multifunctional peptide or protein drugs [12-14].These linkers, mostly in the form of (GGGGS)n or (G)n, are widely used in the fusion peptides or proteins construction with different functions, such as LHP7[14] and Syn-GNU7 hybrid peptide [12].Considering that the parent peptides are all short peptide, we chose the GGGS tetrapeptide linker to minimise the effect of the flexible hinge region linker on the parent peptide structure and function. Our results showed that the novel LLH peptides exhibited endotoxin neutralization effect and antibacterial activity. Our results also further confirm that a design strategy to achieve a multifunctional peptide or protein by a flexible linker is feasible.

The novel LLH hybird antimicrobial peptide exhibited endotoxin neutralization (LPS binding) effect, and similar to the LBP(86-89)peptide, at the same time, showed significantly stronger anti-E.coli activity (MIC, 4–8 μM) than the parent peptides of LBP and HLF.secondary structure analysis showed that the LLH hybrid peptide own +9 positive charge, and the amphipathicity and hydrophobicity were located between the two parent peptides. Many studies have shown that the positive charge of antimicrobial peptides is closely related to its antibacterial activity, and the more positive charge, the stronger binding effect of antimicrobial peptides on bacterial membranes [15,16]. At the same time, the balance between amphiphilicity and hydrophobicity is also a key factor in the antimicrobial activity of the antimicrobial peptide [15]. Therefore,we speculate that the enhanced antimicrobial activity of LLH hybrid peptide may be related to the increase of its positive charge and the balance of amphiphilicity and hydrophobicity, the exact mechanism of which needs to be further investigated.

Haemolysis is an important parameter in response to drug toxicity to mammalian red blood cells. One of the limiting factors for the development of antimicrobial peptides as clinical antimicrobial agents is cytotoxicity [17]. Although some antimicrobial peptides have broad antimicrobial activity and unique antimicrobial properties, their indiscriminate disruption of cell membranes tends to disrupt erythrocyte membranes and predispose them to haemolysis[18]. Scientists can improve the bactericidal activity of antimicrobial peptides while reducing haemolysis by modifying them, for example, by adding flexible linker [19], side chains [20], net charge[21] and hydrophobicity [22]. Our novel bifunctional hybrid peptide LLH, which neutralises endotoxin and potentiates antimicrobial activity, achieved by flexible linker linkage, did not show significant haemolysis, indicated that it is safe for clinical application.

In conclusion, we design a new LLH hybrid peptides by a flexible GGGS linker connection two parent peptides of LBP (86-99) and HFL (1-11), and got a endotoxin neutralization and bacteria killing dual functions peptides, provided an ideas and reference for the design of novel drugs against gram-negative bacterial infection.Meanwhile, LLH showed significantly endotoxin neutralization effect, strongly and rapidly killing E.coli activity (MIC, 4 -8μM) and no hemolytic activity. These results suggested that LLH is a potential antimicrobial drug for the treatment of E.coli infection.

Author contribution: Zhang Yong was responsible for experimental design, data processing and article writing; Li Guoyu is responsible for manuscript proofreading and experimental guidance; Wang Ruiqi, Liu Chenpeng, Yao Hongji and Xiong jinyan were responsible for experiment implementation and data collection.