Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata

doi:10.21203/rs.2.17979/v2

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Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata

https://doi.org/10.21203/rs.2.17979/v2

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published 01 Aug, 2020

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Background: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages.

Results: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis . The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala 23 -Pro 24 , while the mature AC-1 included 39 amino acid residues. Chemically-synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella , Escherichia coli ( E. c oli ), Klebsiella pneumonia ( K. pneumonia ), and Pseudomonas aeruginosa ( P . aeruginosa ). Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, and MgCl 2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. c oli gradually increased with the increase of AC-1 concentration, and AC-1 could cause significant deformation , severe edema, cytoplasmic lysis, cell membrane damage of E. c oli , and reduce intracellular electron density. Additionally, the fusion protein AC-1 expressed in E. coli was digested by enterokinase protease to obtain the AC-1, which showed similar antimicrobial activity against E. c oli with chemically-synthesized AC-1.

Conclusions: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

Applied & Industrial Microbiology

General Microbiology

Antimicrobial peptide

Armyworm

Antimicrobial activity

Cecropin

The long-term overuse of conventional antibiotics has led to an increase in multidrug-resistant bacteria in animals and humans, including methicillin-resistant Staphylococcus aureus (MRSA), highlighting the need for useful alternatives to antibiotics for controlling and treating the resulting bacterial diseases [1]. Antimicrobial peptides (AMPs) are important components of natural immunity that are widely distributed in insects, mammals, amphibians, fish, plants, and bacteria [2-4]. AMPs exert activities against microorganisms, including bacteria, viruses, parasites, and fungi [5, 6], and have thus received much attention as potential antibiotic substitutes.

Researchers have accordingly discovered many novel AMPs in different species using various techniques, with the aim of identifying useful AMPs to substitute for traditional antibiotics to prevent and control bacterial infections. Insects do not display adaptive immunity but do possess an effective self-defense system similar to mammalian innate immunity [7, 8]. AMPs are an important part of the insect defense system, and can directly kill pathogenic microorganisms or activate other immune pathways to clear pathogens [9]. Over 200 kinds of AMPs have been identified in insects to date, including about 30 kinds of cecropins. In the current study, we investigated a novel insect cecropin in the armyworm, Mythimna separate, named armyworm cecropin 1 (AC-1), predicted and analyzed its physicochemical characteristics and structure. We also evaluated the hemolytic activities, cytotoxicity, and in vitro antimicrobial activities of chemically synthesized AC-1. Additionally, the AC-1 was expressed using E. coli prokaryotic expression system.

Identification of AC-1

Transcriptome sequencing of the armyworm was performed to obtain a gene pool, which was compared with the published AMP sequence. A novel cecropin was identified in the armyworm, and its gene and amino acid sequences are shown in Figure 1. The gene sequence of the novel AMP was confirmed by the PCR amplification and gene sequencing. The open reading frame of the novel AMP was 192 bp in length and was translated into 63 amino acid residues. A conserved cleavage site was predicted in the signal peptide at Ala₂₃-Pro₂₄using the SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/). Multi-sequence alignment indicated that the cleavage site at Ala₂₃-Pro₂₄was conserved among cecropins from many insects (Figure 2). The mature peptide therefore comprised 39 amino acid residues, and was named armyworm cecropin-1 (AC-1). The amino acid sequence of AC-1 was highly similar to cecropins from other insects (Figure2).

Physicochemical characteristics and secondary structure of AC-1

The physicochemical characteristics of AC-1 were predicted and are shown in Table 1. AC-1 included 22 kinds of basic amino acid residues, and its secondary structure predicted using the online PEP-FOLD3 software suggested that AC-1 might adopt two α-helix conformations (Lys₅-Met₁₃, and Ala₂₇-Gly₃₈) (Figure 3). The predicted secondary structure indicated an α-helix content of AC-1 of 58.97%, consistent with the predicted secondary structure of AC-1. In order to further analyze the secondary structure of AC-1, the circular dichroism (CD) spectra of AC-1 in phosphate buffer saline (PBS) was detected and analyzed by CD spectrometer. In the physiological environment (20 mmol/L PBS, pH 7.4 ), AC-1 mainly existed in three structural forms, anti-parallel (44.5%), β-Turn (22.6%), and random coil (32.6%) (Table 2).

Hemolytic and cytotoxic activities of AC-1

The hemolytic activities of AMPs need to be assessed prior to their use in clinical practice. Chemically synthesized AC-1 showed low hemolytic capacity against chicken red blood cells, and the hemolysis rate was only 14.47±1.03% even after treatment with AC-1 at 500 μg/mL for 1 h (Figure 4A). The cytotoxicity of AC-1 was determined on ST cells. The viability of the ST cells was not significantly influenced by AC-1, and the cell survival rate remained >90% even after treatment with at 500 μg/mL AC-1for 1 h (Figure 4B). These results indicated that AC-1 had low hemolytic and cytotoxic activities.

Antimicrobial activity of AC-1

The antimicrobial activities of chemically synthesized AC-1 were evaluated in vitro by detecting the minimum inhibitory concentrations (MICs) using a standard two-fold broth microdilution method. AC-1 exhibited antimicrobial activities against standard strains of Salmonella, Escherichia coli (E. coli), and Klebsiella pneumonia (K. pneumonia) with MICs of <20.00 μg/mL, but did not inhibit Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus) at 250.00 μg/mL (Table 3). AC-1 still showed antimicrobial activities against clinically isolated Salmonella and E. coli with MICs of 31.25 and 25.00 μg/mL, respectively (Table 3).

Thermal- and salt-resistant stabilities of AC-1

We further evaluated the thermal- and salt-resistant stabilities of AC-1 by exposure to different temperatures, and NaCl, KCl, and MgCl₂ concentrations for 1 h. Treatment at temperatures ranging from 4-60°C had no obvious influence on the antimicrobial activities of AC-1 against Salmonella; however, its activity was lower at 80°C and 100°C, compared with 4°C (Figure 5A). The antimicrobial activities of AC-1 against Salmonella decreased with increasing NaCl, KCl, and MgCl₂ concentrations from 0 mM to 250 mM, but the ability of AC-1 to inhibit Salmonella growth was not significantly decreased even at a final concentrations of 250 mM NaCl, 250mM KCl, and 200 mM MgCl₂ for 1 h (Figure 5B-D). These results suggested that AC-1 had good thermal- and salt-resistant stabilities.

Antibacterial effect of AC-1 on E. coli

In order to further study antibacterial activities of AC-1, time killing curve of AC-1 against E. coli was determined by plate count method. The results showed that bactericidal effect of AC-1 on E. coli gradually increased with the increase of AC-1 concentration, and within 60 min, the bactericidal effect gradually increased as the action time increased (Figure 6). TEM observation suggested that compared with the control group (Figure 7A), AC-1 resulted in significant deformation, severe edema, cytoplasmic lysis, cell membrane damage of E. coli, and decreased intracellular electron density (Figure 7B). These results indicated that AC-1 showed effectively antibacterial activity on E. coli.

AC-1 expression in E. coli

The chemical synthesis of peptide was expensive, therefore AC-1 expression in E. coli was studied using recombinant DNA technology. The results showed that the fusion protein AC-1 was expressed mainly in inclusion bodies at 20 or 37 °C. Through an Ni-NTA gravity column, the fusion protein AC-1 was purified using the imidazole eluent (50 mmol/L imidazole). The MIC of the fusion protein AC-1 against E. coli was determined, and the results suggested that the fusion protein AC-1 had no antibacterial activity on E. coli. Therefore, the fusion protein AC-1 was digested by enterokinase protease to get the AC-1. Similarly, the MIC of the AC-1 was also detected as 7.8 µg/mL against E. coli ATCC25922, indicating that AC-1 could be prepared by E. coli prokaryotic expression system.

Several methods can be used to identify novel AMPs. Qi et al. found two novel AMPs in the frog Odorrana livida by PCR amplification, using primer pairs based on the highly conserved sequence of known cathelicidins [10]. Zhou et al. identified five novel AMPs from Hylarana guentheri by isolation and purification [11], and Ma et al. identified 34 AMPs from Rana nigrovittataby peptidomics and genomics [12]. Yang et al. also identified a novel cathelicidin from the Chinese giant salamander Andrias davidianus using transcriptome sequencing and PCR amplification [13]. In the current study, we identified a novel cecropin from the armyworm by the transcriptome sequencing and sequence alignment analysis. Sequencing of the PCR-amplified product confirmed the identity of the novel armyworm cecropin gene. Developments in high-throughput sequencing techniques have led to the discovery of increasing amounts of genetic data among which underlying AMPs are being increasingly identified.

Multi-sequence alignment and biological software analysis showed that the amino acid sequence of AC-1 was highly homologous to cecropins from other insects, and its precursor included the conserved signal peptide Ala-Pro cleavage site upstream of the mature peptide [14, 15]. In addition, AC-1 included the RWK and FKKIE(L)KVG structural domains that are conserved in cecropins from lepidopterous insects. Cecropins usually have a small molecular mass, positive charge, and α-helix conformation [15]. The physicochemical characteristics and predicted secondary structure of AC-1 were consistent with those of cecropins. The theoretical pI value of AC-1 was 10.38, which was >9 suggesting that AC-1 would have a positive charge under physiological conditions [10].

In this study, we evaluated the antimicrobial, hemolytic, and cytotoxic activities, and the thermal- and salt-resistant stabilities of chemically synthesized AC-1. The chemically synthesized AC-1 peptide is currently expensive, and the synthesized peptide may have reduced antimicrobial activities. It is necessary to develop an efficient method for producing clinically applied peptides. A prokaryotic or eukaryotic expression method therefore needs to be developed to produce AC-1 for clinical applications. Wang et al. produced the cecropin pxCECA1 in E. coli [15], while Pei et al. generated the antimicrobial peptide MDAP-2 using an E. coli prokaryotic expression system [16]. Li et al. expressed the peptide CGA-N46 in Bacillus subtilis DB1342 [17]. Luiz et al. produced the abaecin peptide [18] and Li et al. expressed the antimicrobial peptide fowlicidin-2 both in Pichia pastoris [19]. The fusion protein AC-1 was expressed using an E. coli prokaryotic expression system, and the AC-1 obtained by using enterokinase protease digestion exhibited similar antimicrobial activity with the chemically synthesized AC-1.

In conclusion, we identified a novel cecropin AC-1 from armyworms, which showed effective antimicrobial activities, and low hemolytic and cytotoxic activities, as well as good thermal- and salt-resistant stabilities, implying that AC-1 may be clinically useful for preventing and treating bacterial diseases. The AC-1 was prepared by an E. coli prokaryotic expression system and enterokinase protease digestion. However, further studies are needed to determine the antimicrobial mechanisms and in vivo antimicrobial activities of AC-1.

Identification of antimicrobial peptide

Third instar larvae of the armyworm, Mythimna separata were ground in liquid nitrogen and total RNA was extracted for transcriptome sequencing. The known AMP gene sequence was selected to align the transcriptome sequence using MegAlign software. The primers were designed on the basis of the armyworm gene acquired by sequence alignment (sense primer: 5′-TTTGAATTAAGAACAAT-3′; antisense primer: 5′-CTATTTTCCTAAAGCTT-3′). The gene was amplified by PCR using the above primers with a Premix LA Taq (Takara, Dalian, China) according to the manufacturer’s instructions. The PCR conditions were as follows: denaturation at 94 °C for 4 min, 36 cycles of denaturation at 94 °C for 40 s, annealing at 57 °C for 35 s, and elongation at 68 °C for 25 s, and a final elongation at 68 °C for 8 min. The PCR-amplified products were cloned into the pMD18-T vector (Takara, Dalian, China) and positive plasmids were sequenced.

Multi-sequence alignment of cecropins from different insects

The amino acid sequence of the AC-1 precursor was derived from the nucleotide sequence and subjected to multi-sequence alignment with the respective cecropins of different insects from the protein database at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/protein/?term=cecropin) using Vector NTI Advance^® 11.5.3 software.

Physicochemical characteristics and structure prediction of AC-1

The physicochemical characteristics of AC-1 were predicted by the ExPASy Bioinformatics Resource Portal (http://www.expasy.org/tools/) and its secondary structure was predicted using a novel online computational framework PEP-FOLD3.5 (http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) [20]. The secondary structure components of AC-1 were calculated using an online SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html).

In order to further analyze the secondary structure of AC-1, the spectra of AC-1 (0.2 mg/mL) in 20 mmol/L PBS (pH 7.4) was detected by a CD spectrometer (Chirascan, Applied Photophysics Limited, United Kingdom). CD spectra of AC-1 was recorded between 190 and 260 nm of scanning spectrum at 1 nm intervals at room temperature with 0.5 s of response time, 1.0 nm of step size.

Hemolytic and cytotoxic activities of AC-1

AC-1 (purity>98%) was synthesized by Shanghai Gil Biochemical Co., Ltd., China. The synthesized AC-1 was purified by reverse high performance liquid chromatography (Figure S1) and detected by mass spectrum (Figure S2). Its hemolytic activity was tested using blood drawn from chickens and treated with sodium citrate anticoagulant. The treated blood was centrifuged at 3000 × g for 10 min and washed three times in phosphate buffered saline (PBS). The red blood cells were counted and then diluted to 2×10⁷/mL, and 100 μL of chicken red blood cell suspension was mixed with 100 μL of different concentrations of AC-1 (the final concentrations of AC-1: 50, 100, 200, 300, 400, and 500 μg/mL). Triton X-100 solution was selected as a positive control and PBS as a negative control. After incubation for 1 h at 37 °C, the mixture was centrifuged at 3000 × g for 10 min and the absorbance of the supernatants was then detected at 405 nm (OD₄₀₅). The hemolysis ratio was calculated by the formula: hemolysis ratio=(A_405peptide-A_405PBS)/(A_405Triton-A_405PBS)×100%. Each experiment was repeated three times.

The cytotoxicity of AC-1 was evaluated by CCK-8 cell counting kit (Vazyme, Nanjing, China) in swine testis (ST) cells as described previously, with minor modifications [21]. A total of 100 μL of cells (about 5×10⁴/mL) per well was added into 96-well cell-culture plates and then incubated for 24 h at 37 °C. Different concentrations of AC-1 (the final concentrations of AC-1: 100, 200,300, 400, and 500 μg/mL) were added to the cells with further incubation for 12 h at 37 °C, followed by the addition of 10 μL of CCK-8 reagent into each well. The cell-culture plates were incubated for 1 h at 37 °C and the absorbance was determined at 450 nm using an automatic microplate reader. Each experiment was repeated three times.

Antimicrobial assay of AC-1

The antimicrobial activity of AC-1 was analyzed by determining the minimum inhibitory concentration (MIC) against different bacteria, as described previously, with minor modifications [22]. Ampicillin was used as the control. The synthesized AC-1 was dissolved in PBS and added into 96-well microtiter plates at two-fold dilutions. All the bacterial strains were cultured in Luria-Bertani (LB) broth at 37 °C to exponential phase. The bacterialsolutions were diluted to 2×10⁶ colony forming units (CFUs)/mL and then added to 96-well microtiter plates at 50 μL per well, and 100 μL of AC-1/bacteria solution was fully mixed and incubated for 16 h at 37 °C. Resazurin (10 μL 6 mM) was then added to each well and incubated for a further 3 h. The color change was observed in each well. Ampicillin and kanamycin were used as positive controls and PBS and LB broth as negative controls. The MIC was recorded as the concentration of the peptide in the last well that remained blue.

Thermal- and salt-resistant stabilities of AC-1

In order to evaluate the thermal- and salt-resistant stabilities of AC-1,we determined the antimicrobial activities of the treated AC-1 against Salmonella according to the inhibition zone method. For the thermal-resistant stability, the 1mg/mL AC-1 was incubated for 1h at 4, 20, 40, 60, 80, and 100°C, respectively. For the salt-resistant stability, the 1 mg/mL AC-1 was incubated for 1 h at 0, 50, 100, 150, 200, and 250 mM of NaCl, KCl, and MgCl₂, respectively. Salmonella was cultured in LB broth at 37 °C to exponential phase, and diluted to 2×10⁹ CFUs/mL. Then, 100 μL of diluted bacterial solution was fully mixed with 100 mL of sterilized LB solid medium and poured into a sterile culture dish. After cooling, the culture dish was punched using a diameter-same hole puncher. The treated AC-1 solution was added into each well. Ampicillin was used as a positive control and PBS as a negative control. The culture dishes were incubated at 37 °C for 12 h and the diameters of the inhibition zones were measured using vernier calipers. Each experiment was carried out in triplicates.

Time killing curve of AC-1 against E. coli

Time killing curve of AC-1 against E. coli was determined according to the method described previously [23]. E. coli in the logarithmic growth phase was centrifuged to collect the precipitate, diluted with sterile LB liquid medium to 2×10⁷ CFU/mL. Then, 400 μL of bacterial solution and 400 μL of AC-1 solution was thoroughly mixed, and the final concentration of AC-1 was 1 MIC and 4 MIC, respectively. NaCl solution was used as negative control. The mixed solution was set at 37 °C for 0, 10, 20, 30, 40, 50, 60 min, respectively. The mixed solution was centrifuged to collect the bacterial. The bacterial precipitation was washed and suspended by PBS, and then 50 µL of bacterial solution was serially diluted by 10 times. For each dilution, 100 µL of bacterial solution was spread on LB plates to culture for 14 h, and then calculated the number of bacteria. Each experiment was carried out in triplicates.

Transmission electron microscopy

E. coli in the logarithmic growth phase was centrifuged to collect the precipitate. The bacterial precipitation was washed by sterile PBS for three time, and diluted with sterile PBS to 2×10⁷ CFU/mL. Then, 600 µL of bacterial solution and 600 µL of AC-1 solution was thoroughly mixed, and the final concentration of AC-1 was 4 MIC respectively. The mixed was bathed in water at 37 °C for 1 h. E. coli was treated by NaCl solution as negative control. Two groups of E. coli were fixed, dehydrated, and stained by the method described previously [24], and examined under transmission electron microscopy (TEM) (HITACHI, HT7700, Japan).

Expression of AC-1 in E. coli

The recombinant AC-1 gene included the gene of 39 amino acid residues from the mature peptide AC-1 and the enterokinase cleavage site gene at 5′-terminus of AC-1 gene. The recombinant AC-1 gene was synthesized, and cloned into pET-32a(+) by the restriction enzymes Kpn I and Hind III. The recombinant plasmid pET-32a(+)-AC-1 was transformed into E. coli BL21 (DE3) to express the fusion protein AC-1 by isopropyl-β-D-thiogalactoside (IPTG) induction. The fusion protein AC-1 was purified by an Ni-NTA gravity column as described previous [25], and then digested using enterokinase protease. The digested solution was again passed through an Ni-NTA gravity column, and the flowthrough was collected, dialysed, and concentrated to obtain the AC-1 as described previous [25].

Statistical analysis

Data were analyzed using GraphPad Prism 6 software. A value of p < 0.05 was considered significant and p < 0.01 was considered highly significant.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that there are no competing interests.

Funding

This work was supported by the following funding: the National Natural Sciences Foundation of China (31802170), Colleges Key Scientific Research Projects of Henan Province (19B230001).

Authors’ contributions

K.Q.L. and Y.C.Z. designed the study and analyzed the data. L.L.Z. amplified and analyzed the AC gene. M.L.Z. performed the physicochemical characterizations and structure analysis. X.L.L. performed hemolytic and cytotoxic activities experiments. Y.J.T. performed the multi-sequence alignment analysis. Z.Q.S. performed the antimicrobial activity and thermal-resistant and salt-resistant stability experiments. Y.W.S. participated in experiment design. KQL, Y.C.Z., X.P.W. and Y.J.T. wrote and revised the paper. All authors approved the final manuscript.

Acknowledgment

We thank PhD Shuai Wang from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences for help to predict the peptide structure.

Author details

Academician Workstation of Animal Disease Control and Nutrition Immunity in Henan Province, Henan Joint International Research Laboratory of Veterinary Biologics Research and Application, Anyang Institute of Technology, No.73 Huanghe Road, Anyang, Henan 225009, People’s Republic of China.

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Table 1 Physicochemical characterizations of the AC-1

Peptide

Grand average of hydropathicity

Number of

amino acids (n)

Net

charge

Theoretical pI

Molecular weight (Da)

AC-1

- 0.321

10.38

4262.13

Table 2 Percentages of the secondary structural elements of AC-1 in PBS (20 mmol/L, pH 7.4)

	190-260 nm	195-260 nm	200-260 nm	205-260 nm	210-260 nm
Helix	6.9%	6.9%	7.6%	8.6%	10.7%
Anti-parallel	44.5%	40.3%	53.7%	52.1%	44.4%
Parallel	2.7%	3.6%	4.4%	4.8%	5.2%
Beta-Turn	22.6%	23.0%	24.7%	24.5%	22.1%
Random.Coil	32.6%	34.1%	31.6%	29.6%	33.5%
Total Sum	109.3%	107.9%	121.9%	119.6%	115.9%

Table 3 Minimum inhibitory concentration (μg/mL) of the AC-1 and ampicillin against microorganisms

Microorganisms		MIC (µg/mL)
Microorganisms		AC-1 Ampicillin
Gram negative bacteria	Escherichia coli ATCC25922	7.80 (1.83 µmol/L) 0.50
	Escherichia coli clinical strain	25.00(5.87 µmol/L) >250.00
	Salmonella ATCC13076	12.50 (2.93 µmol/L) 1.00
	Salmonella clinical strain	31.25(7.33 µmol/L) >125.00
	Klebsiella pneumonia ATCC27853	15.63 (3.67 µmol/L) -
	Pseudomonasaeruginosa ATCC700603	125.00 (29.33 µmol/L) >62.50
Gram positive bacteria	Bacillus cereus ATCC11778	>250.00 (58.66 µmol/L) -
Gram positive bacteria	Staphylococcus aureus ATCC29213	>250.00 (58.66 µmol/L) 0.50

>, no activity detected at the concentration indicated. -, not assayed. Gram positive bacteria

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Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata

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