- Email: [email protected]
Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli
Protein Expression and Purification 100 (2014) 48–53
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli Germán Alberto Téllez ⇑, Jhon Carlos Castaño-Osorio Group of Molecular Immunology, Faculty of Health Sciences, Quindío University, Cra 15 calle 12 norte, Armenia Quindío, Colombia
a r t i c l e
i n f o
Article history: Received 16 April 2014 and in revised form 6 May 2014 Available online 20 May 2014 Keywords: Antimicrobial peptide Cecropin Insect Drug resistance Recombinant fusion proteins Chromatography
a b s t r a c t Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12 h induction with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 lM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 lM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Antimicrobial peptides or host defense peptides are effector molecules of the innate immune system and are produced in a wide variety of organisms from bacteria, plants, insects and mammals. Their diverse mechanisms of action, including pore formation, carpet-like saturation of the membrane, immunomodulatory effects and LPS neutralization, make them an attractive target for the development of new antimicrobial compounds [1]. The cecropins are a family of cationic host defense peptides that adopt an a-helical structure. Peptides from this family have been found in a wide variety of insects and mammals. They recruit and promote elements of innate immunity [2]. Moreover, the cecropins have a wide spectrum of antimicrobial activity, especially against Gram-negative bacteria, Gram-positive bacteria and protozoa, such as Leishmania and Plasmodium falciparum, as well as activity against yeast such as Candida albicans [3]. The cecropins have been shown to have good in vitro activity against tumor cell lines and low toxicity against normal eukaryotic cells with weak hemolytic activity [4–6]. Lucilin is a 3.8 kDa cecropin antimicrobial peptide found in the maggots of Lucilia sericata as a partial genetic sequence in the mRNA. These maggots are used as debridement therapy for necro⇑ Corresponding author. Address: Cra 15 calle 12 norte, Universidad del Quindío, Facultad de ciencias de la salud, Armenia Quindío, Colombia. Tel.: +56 6 7359392. E-mail address: [email protected] (G.A. Téllez). http://dx.doi.org/10.1016/j.pep.2014.05.004 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
tic and infected wounds and their antimicrobial activity is explained partially by the production of antimicrobial peptides such as Lucifensin in different secretions and tissues of the maggot [7]. The antimicrobial activity and toxicity of the Lucilin peptide is unknown. The Vibrio cholerae MARTX toxin cysteine protease domain (CPD)1 is described as a self-cleavage tag that has been shown to enhance the expression, integrity and solubility of intractable proteins in Escherichia coli [8]. In this study, a recombinant analog of the Lucilin peptide fused to the CPD tag was produced in E. coli, purified by Nickel IMAC (immobilized metal affinity chromatography), and its antimicrobial activity assessed against a clinical isolated multidrug resistance E. coli strain. The toxicity profile of the lucilin-CPDHis8 was also investigated against human erythrocytes and Vero cells. Materials and methods Reagents, strains and plasmids The plasmid pJExpress404 with the GWLK-Lucilin-CPDHis8 synthetic gene was purchase from DNA2.0 (dna2.0, California, USA). E. coli DH10B was used for plasmid storage and amplification, 1 Abbreviations used: CFU, colony forming units; MIC, minimum inhibitory concentration; ESBL, extended spectrum b-lactamase; IMAC, ion metal affinity chromatography; CPD, cysteine protease domain; ELISA, enzyme-linked immune assay; His, histidine; LPS, lipopolysaccharide.
G.A. Téllez, J.C. Castaño-Osorio / Protein Expression and Purification 100 (2014) 48–53
whereas E. coli BL21 cells (GE Healthcare, Pittsburgh, USA) were used as the expression host. E. coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 9027), and the clinical isolates Vibrio alginolyticus, Salmonella typhi and Bacillus cereus were donated by the FIDIC (Fundación Instituto de Inmunología de Colombia, Bogota, Colombia). The clinical isolates E. coli ESLB, E. coli, Enterobacter cloacae and Staphylococcus aureus were donated by the San Juan de Dios Hospital (Armenia Quindío, Colombia). The Vero cells (ATCC CCL81) were conserved by our laboratory. Polypropylene 96-well plates were purchase from Corning (Corning, NewYork, USA). Construction of expression vector The plasmid pjExpress404 with the ampicillin resistance marker and the T5 promoter was used to clone the genetic sequences. The genes were designed and codon optimized for E. coli using the program Genedesigner [9]. The self-catalytic CPD amino acid sequence was taken from residues 3442 to 3650 of the RTX toxin RtxA of the Vibrio cholerae strain. N16961 (GenBank accession No NP_231094.1) and the antimicrobial peptide was design based on the Lucilin antimicrobial GenBank entry (GenBank accession No ADH04298), with the addition of GWLK at the N-terminus to increase the hydrophobic momentum and the net charge of the peptide [10].
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assay as described previously with some modifications [2]. The E. coli DH10B, E. coli (ATCC 35218), P. aeruginosa (ATCC 9027) and the clinical isolates Vibrio alginolyticus, Salmonella tiphy, Bacillus cereus, E. coli (ESBL), Enterobacter cloacae, Pseudomonas aeruginosa and Staphylococcus epidermidis were used as test bacteria. The cleavage protein in solution with the InP6 was tested against E. coli DH10B. The bacteria were grown overnight in Muller Hilton medium (Scharlau 02-136-500, Barcelona, Spain) and adjusted to an Optical Density of 0.4 (at 570 nm), a 1:1000 dilution of the cells was used as the inoculum. Ninety microliters of bacteria and 10 ll of the recombinant protein were mixed to a final protein concentration between 0.2 and 200 lg/ml (0.007–7.1 lM), the solution was incubated at 37 °C for 12 h and then 10 ll of resazurin (Acrosorganics 418900050, Geel, Belgium) (44 lM final concentration) was added, the plate was incubated for additional 2 h and the absorbance values at 570 and 603 nm were measured. A delta value (i.e., A570– A603) was calculated for each well; the average value of the blanks was subtracted from each sample, and the growth percentage was calculated relative to a control. Each concentration was measured in triplicate. The minimum inhibitory concentration was defined as the concentration that inhibited at least 50% of the growth of the bacterial [11]. Cytotoxic activity
Expression and purification The recombinant plasmid pJexpress404-GWLK-Lucilin-CPDHis8 was transformed into E. coli BL21 cells. The recombinant E. coli pJexpress404-GWLK-Lucilin-CPD-His8 cells were grown overnight in 2 ml of Miller’s Luria Bertani (LB) medium (Life Technologies 12795084, New York, USA) with 100 lg/ml ampicillin at 37 °C and 150 rpm. The inoculum was refreshed with 200 ml of Miller’s LB medium containing 100 lg/ml ampicillin, the cells grown until mid-log phase (A570 = 0.4–0.5) and IPTG (Promega V3951, Wisconsin, USA) was added to a final concentration of 0.5 mM. The cells were cultivated for an additional 12 h at 37 °C and 150 rpm, and harvested by centrifugation at 2700g for 15 min at 4 °C. Aliquots were taken at 0, 1, 3, 6, 9, 12 and 24 h post induction with IPTG and the protein production was assessed by an ELISA using a monoclonal anti-histidine antibody (Sigma H1029, Missouri, USA). The cell pellet was re-suspended in binding buffer (0.5 M NaCl, 150 mM imidazole, 20 mM NaH2PO4, pH 8, supplemented with 0.5% Triton-X100) and lysed by sonication using an ultrasonic processor GE 50 with 10 pulses of 30 s on ice and centrifuged at 12,000g for 18 min at 4 °C. The supernatant was collected and loaded onto an equilibrated 5 ml Hitrap Ni-Sepharose column. The column was wash with 10 column volumes of the binding buffer without TritonX100 and the protein eluted with elution buffer (0.5 M NaCl, 0.4 M imidazole, 20 mM NaH2PO4, pH 8). The purified protein was desalted using a 5 ml Hitrap Sephadex G25 column in PBS (2.5 mM NaH2PO4, pH 8) and the protein concentration was measured by UV absorbance at 260 and 280 nm in a Take3 plate EPOCH spectrophotometer (BIOTEK, Vermont, USA).
Vero cells (ATCC:CCL-81) were grown in Dulbecco’s Modified Eagle Medium (Life Technologies 12100-046, New York, USA) supplemented with 10,000 units/ml penicillin and streptomycin, 1 L-glutamine and 2% (v/v) fetal bovine serum (Eurobio, Les Ulis, France). Cells were cultured in polypropylene 96 well microplates (Costar 3879, New York, USA) with 22,500 cells per well. The cells were incubated for 24 h with protein concentrations ranging from 0.08 to 51.49 lM (25–1450 lg/ml) at 37 °C and 5% CO2 humidity. At the end of this incubation period, resazurin was added to each well at a final concentration of 44 lM and the cells incubated for a further 2 h at 37 °C with 5% CO2. The absorbance at 603 and 570 nm were measured. A delta value was calculated (i.e., A570–A603) and the average of the controls were subtracted from each well value. The percentage cell viability was calculated by comparison with the untreated control cells. Human erythrocytes hemolytic activity
The purified protein was self-cleaved by induction of the fusion protein in 100, 500 and 1000 lM of inositol hexakisphosphate (iP6) solution (Sigma–Aldrich 80180, Missouri, USA) for 2 h at 37 °C and 150 rpm.
Four milliliters of human blood was taken in a dry tube with ethylene-diaminetetraacetic acid and centrifuged at 800g for 10 min at room temperature. The erythrocytes were washed three times with a 1 PBS stock solution (130 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM K2HPO4, pH 7.4). An erythrocyte dilution of 1:250 was performed from the erythrocyte stock solution and incubated at 37 °C for 15 min (work solution). In polypropylene 96 well microplates, the peptides were added to final concentrations between 56.8 and 0.106 lM (1600–3 lg/ml) and 100 ll of the erythrocyte work solution was added to these wells. The culture was then incubated for 2 h at 37 °C and centrifuged at 2000g for 15 min. The supernatants were taken and the absorbance measured at 540 nm. The absorbance of the hemoglobin from the erythrocytes incubated with 1% (v/v) Triton-X100 was taken as the control of 100% hemolysis, and the percentage of hemolysis was calculated as (A540–A540 nm blank)⁄100/(control Triton A540–A540 nm blank) [12].
Antimicrobial activity
Molecular modeling of the GWLK-Lucilin-CPD-His8
Antimicrobial activity of the complete fused recombinant protein GWLK-Lucilin-CPD-His8 was tested by the broth microdilution
The 3D molecular model of the GWLK-Lucilin-CPD-His8 was constructed using the program I-Tasser [13].
Self-cleavage of the CPD tag
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Statistical analysis All experiments were perform in triplicates and expressed as the mean value with the standard deviations as error bars. Results Cloning and expression of the recombinant GWLK-Lucilin-CPD-His8 protein The GWLK-Lucilin synthetic gene and the CPD were codon optimized for E. coli expression and the gene cloned into the plasmid pjexpress404, with the antimicrobial peptide N-terminal of the CPD and the poly-histidine tag located at the C-terminus, i.e., GWLK-Lucilin-CPD-His8. The expected molecular weight of the fusion protein is 28159.22 Da and the calculated isoelectric point is 7.05 (Fig. 1). The recombinant protein was expressed in E coli BL21 cells with a peak expression after 12 h induction with 0.5 mM IPTG, as assessed by an anti-histidine tag ELISA. This growth point corresponded with the initial stage of the cells entering into the stationary phase (Fig. 2). Purification of the recombinant protein Approximately 70 mg of the GWLK-Lucilin-CPD-His8 protein was purified from 1 L of cultured cells using IMAC with a Nickel Sepharose column. The wash step of the chromatography process was optimized up to a concentration of 150 mM imidazole, and the GWLK-Lucilin-CPD-His8 protein was eluted with 0.4 M imidazole with few contaminants present, as assessed by tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (tSDS–PAGE). A major protein band with a relative mass of 28 kDa was observed in the tSDS–PAGE analysis corresponding to the target fusion protein. A weaker band of 23 kDa was also observed corresponding to CPD-His8. Inducible cleavage of the GWLK-Lucilin-CPD-His8 The self-cleavage of the protein was induced in solution following the desalting step, the incubation of the GWLK-Lucilin-CPD-
Fig. 2. (A) Production of the recombinant protein GWLK-Lucilin-CPD-His8 in E. coli BL21 evaluated by the anti-histidine tag ELISA and the bacterial growth kinetics (Abs. 570 nm). A growing production of the protein was observed in the ELISA with a behavior following the growth kinetic with a peak expression at 12 h after the induction, the growth kinetics was not affected by the production of the protein.
His8 with 100 lM InP6 for 2 h at 37 °C was enough to see the cleavage reaction monitored by tSDS–PAGE giving bands with molecular masses of 27 and 23 kDa and a double band of 4 kDa, which corresponds to the GWLK-Lucilin-CPD-His8 fusion protein, CPD-His8 and GWLK-Lucilin, respectively, higher concentrations of InP6 produced multiple bands of low molecular weight and did not improved the amount of cleavage protein (Fig. 3).
Antimicrobial activity of the GWLK-Lucilin-CPD-His8 The GWLK-Lucilin-CPD-His8 protein and the cleaved protein GWLK-Lucilin plus CPD-His8 were assessed for antimicrobial activity against E. coli DH10B. The completed fusion protein was active with a MIC of 0.2 lM but the cleavage protein was not active (Fig. 4). The GWLK-Lucilin-CPD-His8 protein showed activity against the multidrug resistance bacteria, namely E. coli ESBL, E. cloacae, E. coli and Salmonella typhy; with MICs from 0.2 to 1.7 lM (Table 1).
A
B
GWLK-Lucilin
His8
CPD
Fig. 1. Design of the GWLK-Lucilin-CPD-His8 protein. (A) Molecular modeling GWLK-Lucilin in black and CPD-His8 in grey. (B) Optimized DNA sequence and codon map.
G.A. Téllez, J.C. Castaño-Osorio / Protein Expression and Purification 100 (2014) 48–53
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Fig. 3. 12% SDS–PAGE analysis of the purification and induced cleavage of the fusion protein GWLK-Lucilin-CPD-His8. M: BenchMark™ pre-stained protein ladder (Life Technologies 10748-010); S: Soluble Lysed bacteria; FT: Flow through; W: Wash; E: Elusion; D: Desalting of the protein; I: Purified protein after cleavage with 100 lM inP6 (I100), 500 lM inP6 (I500) and 1000 lM inP6 (I1000).
ing the average MIC of 0.89 lM and the higher concentration used in the cytotoxic assay.
Discussion
Fig. 4. Antimicrobial activity of GWLK-Lucilin-CPD-His8 protein and the cleaved protein against E. coli DH10B. The cleaved mixture of InP6 100 lM GWLK-Lucilin and CPD show no antimicrobial activity at any tested concentration, while the completed fusion protein GWLK-Lucilin-CPD-His8 show a strong antimicrobial activity with a MIC of 0.2 lM.
Table 1 Minimum inhibitory concentration of the protein GWLK-Lucilin-CPD-His8. Microorganism
E. Coli CI E. Coli ESBL Enterobacter cloacae E Coli ATCC 35218 Salmonella typhy E coli DH10B Bacillus cereus P. aeruginosa ATCC 9027 Vidrio alginolyticus Staphylococcus epidermidis
MICs
lM
lg/ml
1.7 0.8 1.4 0.7 0.6 0.2 NA NA NA NA
50 23.8 38.5 19.3 18.0 6.5 NA NA NA NA
NA: no activity detected.
Hemolytic and cytotoxic activity The purified GWLK-Lucilin-CPD-His8 protein showed no hemolytic activity against human erythrocytes at the evaluated concentrations. The cellular assay showed no toxicity against Vero cells after 24 h incubation with different concentrations of the protein (up to 56.4 lM) thereby giving a therapeutic index >63 when tak-
There is an urgent need for the development of new and active antimicrobial compounds against multidrug resistant microorganisms, this is particularly important for combating Gram-negative bacteria, such as E. coli, because these microorganisms are emergent pathogens of significant threat given the limited therapeutic options currently available in the clinic and the growing incidence of patients affected by these microorganisms [14]. In this work, a recombinant fusion protein was produced with activity against pathogenic clinical isolates with multidrug resistance profiles such as E. coli, E. cloacae and S. typhi, thereby providing insights on the possible use of these molecules for the development of new therapeutic alternatives. The complete recombinant fused protein GWLK-Lucilin-CPDHis8 was active against Gram-negative bacteria but the cleaved protein was not, this activity may be explained by the conformational structure of the protein where the peptide is positioned at the N-terminus of the CPD and this segment is believed to adopt a random coil structure giving the possibility of interaction for peptide with the bacterial membrane (Fig.1). The N-terminal region of the peptide is believed to be the primary region responsible for the membrane interaction because of its hydrophobic character and polarity as seen for other cecropins [15]. The protection of the C-terminus of the peptide from degradation by bacterial proteases likely increases the stability of the peptide when compared with the cleaved product. This finding may aid in the construction of new chimeric recombinant molecules with higher antimicrobial activity and different functional domains at the C-terminus that may provide new functionalities to the antimicrobial peptides. It remains unknown why the cleaved protein was inactive. Nonetheless, it is possible that the stability of the cleaved peptide was compromised, or that the inP6 interacted with the peptide, or the cleaved peptide aggregated, thereby limiting its antimicrobial activity; however, these possibilities require further investigation. E. coli is still the preferred host for recombinant protein expression, because of its high expression level, simplicity and low cost. However, difficulties have been encountered in the expression of antimicrobial proteins because of their cytotoxicity to host bacte-
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Acknowledgments This work was supported by the Grant AMFRI 24-10 of the Red de Universidades del eje cafetero, ALMA MATER and the call 511 for doctorate study in Colombia. Administrative department of science technology and innovation COLCIENCIAS. Appendix A. Amino acids sequences of the peptide and proteins A.1. Lucilin (GenBank: ADH04298) KLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKG A.2. CPD >gi|15641462|ref|NP_231094.1| RTX toxin RtxA [Vibrio cholerae O1 biovar El Tor str. N16961] LADGKILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVA KAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHG RDHSETNNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSL VSDDKQKGFGHQFINAMDANGLRVDVSVRSSELAVDEAGRKHTKDAN GDWÊ LADGKILHNQNVNSW Fig. 5. Hemolytic and cytotoxic activity of the GWLK-Lucilin-CPD-His8 protein. (A) Cytotoxic activity of the GWLK-Lucilin-CPD-His8 on Vero cells after 24 h incubation. Show no decrease in the cell viability greater than 80 % in general with a slight decrease at 1.29 lM. (B) Hemolytic activity towards human erythrocytes. The GWLK-Lucilin-CPD-His8 show no hemolytic activity at any concentration tested.
rial cells, susceptibility to proteolytic degradation and low-production yields. The production of antimicrobial peptides in E. coli using other fusion partners such as thioredoxin or small ubiquitinrelated modifier (SUMO) have been described [16,17]. The CPD domain has been used for the production of cathelicidin-derived antimicrobial peptides with a production yield varying between 5 and 20 mg/L. In this work, we had a yield of 70 mg/L. A reason for this high yield may be because the codon optimization used for the production of the synthetic gene. We were also successfully in producing and purify a soluble active recombinant antimicrobial protein based on the Lucilin peptide fused with the CPD in E. coli BL21. However, the purification via a one-step approach using incolumn cleavage of the protein was not successful; i.e., a chromatographic peak corresponding to the peptide after column cleavage was not observed. This may be, because of the slower elution of the peptide or the formation of protein aggregates, and it would be necessary to further purify the cleaved peptide by HPLC, as described previously by Wright [10]. The GWLK-Lucilin-CPD-His8 protein was confirmed to have no hemolytic activity against human erythrocytes and no toxicity towards Vero cells (Fig. 5). This protein may be a potentially safe substitute for traditional antibiotics against human pathogenic bacteria. However, the safety of GWLK-Lucilin-CPD-His8 for potential use in therapeutics will be explored further. Conclusions In the current study, we have successfully produced and purify a recombinant protein GWLK-Lucilin-CPD-His8, which showed strong antimicrobial activities against drug resistance Gram-negative bacteria. This suggests that GWLK-Lucilin-CPD-His8 can be a useful candidate for therapeutic design and usage. This project has laid the foundation for further studies examining the biological functions of a chimeric recombinant Lucilin and to explore ways to design genetically engineered antimicrobial drugs.
A.3. GWKL-LUCILIN-CPD-His8 MGWLKKLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKGLADG KILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANL AGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSET NNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQ KGFMGWLKKLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKGLADG KILHNQNVNSWGPITVTPT
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Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli Germán Alberto Téllez ⇑, Jhon Carlos Castaño-Osorio Group of Molecular Immunology, Faculty of Health Sciences, Quindío University, Cra 15 calle 12 norte, Armenia Quindío, Colombia
a r t i c l e
i n f o
Article history: Received 16 April 2014 and in revised form 6 May 2014 Available online 20 May 2014 Keywords: Antimicrobial peptide Cecropin Insect Drug resistance Recombinant fusion proteins Chromatography
a b s t r a c t Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12 h induction with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 lM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 lM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Antimicrobial peptides or host defense peptides are effector molecules of the innate immune system and are produced in a wide variety of organisms from bacteria, plants, insects and mammals. Their diverse mechanisms of action, including pore formation, carpet-like saturation of the membrane, immunomodulatory effects and LPS neutralization, make them an attractive target for the development of new antimicrobial compounds [1]. The cecropins are a family of cationic host defense peptides that adopt an a-helical structure. Peptides from this family have been found in a wide variety of insects and mammals. They recruit and promote elements of innate immunity [2]. Moreover, the cecropins have a wide spectrum of antimicrobial activity, especially against Gram-negative bacteria, Gram-positive bacteria and protozoa, such as Leishmania and Plasmodium falciparum, as well as activity against yeast such as Candida albicans [3]. The cecropins have been shown to have good in vitro activity against tumor cell lines and low toxicity against normal eukaryotic cells with weak hemolytic activity [4–6]. Lucilin is a 3.8 kDa cecropin antimicrobial peptide found in the maggots of Lucilia sericata as a partial genetic sequence in the mRNA. These maggots are used as debridement therapy for necro⇑ Corresponding author. Address: Cra 15 calle 12 norte, Universidad del Quindío, Facultad de ciencias de la salud, Armenia Quindío, Colombia. Tel.: +56 6 7359392. E-mail address: [email protected] (G.A. Téllez). http://dx.doi.org/10.1016/j.pep.2014.05.004 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
tic and infected wounds and their antimicrobial activity is explained partially by the production of antimicrobial peptides such as Lucifensin in different secretions and tissues of the maggot [7]. The antimicrobial activity and toxicity of the Lucilin peptide is unknown. The Vibrio cholerae MARTX toxin cysteine protease domain (CPD)1 is described as a self-cleavage tag that has been shown to enhance the expression, integrity and solubility of intractable proteins in Escherichia coli [8]. In this study, a recombinant analog of the Lucilin peptide fused to the CPD tag was produced in E. coli, purified by Nickel IMAC (immobilized metal affinity chromatography), and its antimicrobial activity assessed against a clinical isolated multidrug resistance E. coli strain. The toxicity profile of the lucilin-CPDHis8 was also investigated against human erythrocytes and Vero cells. Materials and methods Reagents, strains and plasmids The plasmid pJExpress404 with the GWLK-Lucilin-CPDHis8 synthetic gene was purchase from DNA2.0 (dna2.0, California, USA). E. coli DH10B was used for plasmid storage and amplification, 1 Abbreviations used: CFU, colony forming units; MIC, minimum inhibitory concentration; ESBL, extended spectrum b-lactamase; IMAC, ion metal affinity chromatography; CPD, cysteine protease domain; ELISA, enzyme-linked immune assay; His, histidine; LPS, lipopolysaccharide.
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whereas E. coli BL21 cells (GE Healthcare, Pittsburgh, USA) were used as the expression host. E. coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 9027), and the clinical isolates Vibrio alginolyticus, Salmonella typhi and Bacillus cereus were donated by the FIDIC (Fundación Instituto de Inmunología de Colombia, Bogota, Colombia). The clinical isolates E. coli ESLB, E. coli, Enterobacter cloacae and Staphylococcus aureus were donated by the San Juan de Dios Hospital (Armenia Quindío, Colombia). The Vero cells (ATCC CCL81) were conserved by our laboratory. Polypropylene 96-well plates were purchase from Corning (Corning, NewYork, USA). Construction of expression vector The plasmid pjExpress404 with the ampicillin resistance marker and the T5 promoter was used to clone the genetic sequences. The genes were designed and codon optimized for E. coli using the program Genedesigner [9]. The self-catalytic CPD amino acid sequence was taken from residues 3442 to 3650 of the RTX toxin RtxA of the Vibrio cholerae strain. N16961 (GenBank accession No NP_231094.1) and the antimicrobial peptide was design based on the Lucilin antimicrobial GenBank entry (GenBank accession No ADH04298), with the addition of GWLK at the N-terminus to increase the hydrophobic momentum and the net charge of the peptide [10].
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assay as described previously with some modifications [2]. The E. coli DH10B, E. coli (ATCC 35218), P. aeruginosa (ATCC 9027) and the clinical isolates Vibrio alginolyticus, Salmonella tiphy, Bacillus cereus, E. coli (ESBL), Enterobacter cloacae, Pseudomonas aeruginosa and Staphylococcus epidermidis were used as test bacteria. The cleavage protein in solution with the InP6 was tested against E. coli DH10B. The bacteria were grown overnight in Muller Hilton medium (Scharlau 02-136-500, Barcelona, Spain) and adjusted to an Optical Density of 0.4 (at 570 nm), a 1:1000 dilution of the cells was used as the inoculum. Ninety microliters of bacteria and 10 ll of the recombinant protein were mixed to a final protein concentration between 0.2 and 200 lg/ml (0.007–7.1 lM), the solution was incubated at 37 °C for 12 h and then 10 ll of resazurin (Acrosorganics 418900050, Geel, Belgium) (44 lM final concentration) was added, the plate was incubated for additional 2 h and the absorbance values at 570 and 603 nm were measured. A delta value (i.e., A570– A603) was calculated for each well; the average value of the blanks was subtracted from each sample, and the growth percentage was calculated relative to a control. Each concentration was measured in triplicate. The minimum inhibitory concentration was defined as the concentration that inhibited at least 50% of the growth of the bacterial [11]. Cytotoxic activity
Expression and purification The recombinant plasmid pJexpress404-GWLK-Lucilin-CPDHis8 was transformed into E. coli BL21 cells. The recombinant E. coli pJexpress404-GWLK-Lucilin-CPD-His8 cells were grown overnight in 2 ml of Miller’s Luria Bertani (LB) medium (Life Technologies 12795084, New York, USA) with 100 lg/ml ampicillin at 37 °C and 150 rpm. The inoculum was refreshed with 200 ml of Miller’s LB medium containing 100 lg/ml ampicillin, the cells grown until mid-log phase (A570 = 0.4–0.5) and IPTG (Promega V3951, Wisconsin, USA) was added to a final concentration of 0.5 mM. The cells were cultivated for an additional 12 h at 37 °C and 150 rpm, and harvested by centrifugation at 2700g for 15 min at 4 °C. Aliquots were taken at 0, 1, 3, 6, 9, 12 and 24 h post induction with IPTG and the protein production was assessed by an ELISA using a monoclonal anti-histidine antibody (Sigma H1029, Missouri, USA). The cell pellet was re-suspended in binding buffer (0.5 M NaCl, 150 mM imidazole, 20 mM NaH2PO4, pH 8, supplemented with 0.5% Triton-X100) and lysed by sonication using an ultrasonic processor GE 50 with 10 pulses of 30 s on ice and centrifuged at 12,000g for 18 min at 4 °C. The supernatant was collected and loaded onto an equilibrated 5 ml Hitrap Ni-Sepharose column. The column was wash with 10 column volumes of the binding buffer without TritonX100 and the protein eluted with elution buffer (0.5 M NaCl, 0.4 M imidazole, 20 mM NaH2PO4, pH 8). The purified protein was desalted using a 5 ml Hitrap Sephadex G25 column in PBS (2.5 mM NaH2PO4, pH 8) and the protein concentration was measured by UV absorbance at 260 and 280 nm in a Take3 plate EPOCH spectrophotometer (BIOTEK, Vermont, USA).
Vero cells (ATCC:CCL-81) were grown in Dulbecco’s Modified Eagle Medium (Life Technologies 12100-046, New York, USA) supplemented with 10,000 units/ml penicillin and streptomycin, 1 L-glutamine and 2% (v/v) fetal bovine serum (Eurobio, Les Ulis, France). Cells were cultured in polypropylene 96 well microplates (Costar 3879, New York, USA) with 22,500 cells per well. The cells were incubated for 24 h with protein concentrations ranging from 0.08 to 51.49 lM (25–1450 lg/ml) at 37 °C and 5% CO2 humidity. At the end of this incubation period, resazurin was added to each well at a final concentration of 44 lM and the cells incubated for a further 2 h at 37 °C with 5% CO2. The absorbance at 603 and 570 nm were measured. A delta value was calculated (i.e., A570–A603) and the average of the controls were subtracted from each well value. The percentage cell viability was calculated by comparison with the untreated control cells. Human erythrocytes hemolytic activity
The purified protein was self-cleaved by induction of the fusion protein in 100, 500 and 1000 lM of inositol hexakisphosphate (iP6) solution (Sigma–Aldrich 80180, Missouri, USA) for 2 h at 37 °C and 150 rpm.
Four milliliters of human blood was taken in a dry tube with ethylene-diaminetetraacetic acid and centrifuged at 800g for 10 min at room temperature. The erythrocytes were washed three times with a 1 PBS stock solution (130 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM K2HPO4, pH 7.4). An erythrocyte dilution of 1:250 was performed from the erythrocyte stock solution and incubated at 37 °C for 15 min (work solution). In polypropylene 96 well microplates, the peptides were added to final concentrations between 56.8 and 0.106 lM (1600–3 lg/ml) and 100 ll of the erythrocyte work solution was added to these wells. The culture was then incubated for 2 h at 37 °C and centrifuged at 2000g for 15 min. The supernatants were taken and the absorbance measured at 540 nm. The absorbance of the hemoglobin from the erythrocytes incubated with 1% (v/v) Triton-X100 was taken as the control of 100% hemolysis, and the percentage of hemolysis was calculated as (A540–A540 nm blank)⁄100/(control Triton A540–A540 nm blank) [12].
Antimicrobial activity
Molecular modeling of the GWLK-Lucilin-CPD-His8
Antimicrobial activity of the complete fused recombinant protein GWLK-Lucilin-CPD-His8 was tested by the broth microdilution
The 3D molecular model of the GWLK-Lucilin-CPD-His8 was constructed using the program I-Tasser [13].
Self-cleavage of the CPD tag
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Statistical analysis All experiments were perform in triplicates and expressed as the mean value with the standard deviations as error bars. Results Cloning and expression of the recombinant GWLK-Lucilin-CPD-His8 protein The GWLK-Lucilin synthetic gene and the CPD were codon optimized for E. coli expression and the gene cloned into the plasmid pjexpress404, with the antimicrobial peptide N-terminal of the CPD and the poly-histidine tag located at the C-terminus, i.e., GWLK-Lucilin-CPD-His8. The expected molecular weight of the fusion protein is 28159.22 Da and the calculated isoelectric point is 7.05 (Fig. 1). The recombinant protein was expressed in E coli BL21 cells with a peak expression after 12 h induction with 0.5 mM IPTG, as assessed by an anti-histidine tag ELISA. This growth point corresponded with the initial stage of the cells entering into the stationary phase (Fig. 2). Purification of the recombinant protein Approximately 70 mg of the GWLK-Lucilin-CPD-His8 protein was purified from 1 L of cultured cells using IMAC with a Nickel Sepharose column. The wash step of the chromatography process was optimized up to a concentration of 150 mM imidazole, and the GWLK-Lucilin-CPD-His8 protein was eluted with 0.4 M imidazole with few contaminants present, as assessed by tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (tSDS–PAGE). A major protein band with a relative mass of 28 kDa was observed in the tSDS–PAGE analysis corresponding to the target fusion protein. A weaker band of 23 kDa was also observed corresponding to CPD-His8. Inducible cleavage of the GWLK-Lucilin-CPD-His8 The self-cleavage of the protein was induced in solution following the desalting step, the incubation of the GWLK-Lucilin-CPD-
Fig. 2. (A) Production of the recombinant protein GWLK-Lucilin-CPD-His8 in E. coli BL21 evaluated by the anti-histidine tag ELISA and the bacterial growth kinetics (Abs. 570 nm). A growing production of the protein was observed in the ELISA with a behavior following the growth kinetic with a peak expression at 12 h after the induction, the growth kinetics was not affected by the production of the protein.
His8 with 100 lM InP6 for 2 h at 37 °C was enough to see the cleavage reaction monitored by tSDS–PAGE giving bands with molecular masses of 27 and 23 kDa and a double band of 4 kDa, which corresponds to the GWLK-Lucilin-CPD-His8 fusion protein, CPD-His8 and GWLK-Lucilin, respectively, higher concentrations of InP6 produced multiple bands of low molecular weight and did not improved the amount of cleavage protein (Fig. 3).
Antimicrobial activity of the GWLK-Lucilin-CPD-His8 The GWLK-Lucilin-CPD-His8 protein and the cleaved protein GWLK-Lucilin plus CPD-His8 were assessed for antimicrobial activity against E. coli DH10B. The completed fusion protein was active with a MIC of 0.2 lM but the cleavage protein was not active (Fig. 4). The GWLK-Lucilin-CPD-His8 protein showed activity against the multidrug resistance bacteria, namely E. coli ESBL, E. cloacae, E. coli and Salmonella typhy; with MICs from 0.2 to 1.7 lM (Table 1).
A
B
GWLK-Lucilin
His8
CPD
Fig. 1. Design of the GWLK-Lucilin-CPD-His8 protein. (A) Molecular modeling GWLK-Lucilin in black and CPD-His8 in grey. (B) Optimized DNA sequence and codon map.
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Fig. 3. 12% SDS–PAGE analysis of the purification and induced cleavage of the fusion protein GWLK-Lucilin-CPD-His8. M: BenchMark™ pre-stained protein ladder (Life Technologies 10748-010); S: Soluble Lysed bacteria; FT: Flow through; W: Wash; E: Elusion; D: Desalting of the protein; I: Purified protein after cleavage with 100 lM inP6 (I100), 500 lM inP6 (I500) and 1000 lM inP6 (I1000).
ing the average MIC of 0.89 lM and the higher concentration used in the cytotoxic assay.
Discussion
Fig. 4. Antimicrobial activity of GWLK-Lucilin-CPD-His8 protein and the cleaved protein against E. coli DH10B. The cleaved mixture of InP6 100 lM GWLK-Lucilin and CPD show no antimicrobial activity at any tested concentration, while the completed fusion protein GWLK-Lucilin-CPD-His8 show a strong antimicrobial activity with a MIC of 0.2 lM.
Table 1 Minimum inhibitory concentration of the protein GWLK-Lucilin-CPD-His8. Microorganism
E. Coli CI E. Coli ESBL Enterobacter cloacae E Coli ATCC 35218 Salmonella typhy E coli DH10B Bacillus cereus P. aeruginosa ATCC 9027 Vidrio alginolyticus Staphylococcus epidermidis
MICs
lM
lg/ml
1.7 0.8 1.4 0.7 0.6 0.2 NA NA NA NA
50 23.8 38.5 19.3 18.0 6.5 NA NA NA NA
NA: no activity detected.
Hemolytic and cytotoxic activity The purified GWLK-Lucilin-CPD-His8 protein showed no hemolytic activity against human erythrocytes at the evaluated concentrations. The cellular assay showed no toxicity against Vero cells after 24 h incubation with different concentrations of the protein (up to 56.4 lM) thereby giving a therapeutic index >63 when tak-
There is an urgent need for the development of new and active antimicrobial compounds against multidrug resistant microorganisms, this is particularly important for combating Gram-negative bacteria, such as E. coli, because these microorganisms are emergent pathogens of significant threat given the limited therapeutic options currently available in the clinic and the growing incidence of patients affected by these microorganisms [14]. In this work, a recombinant fusion protein was produced with activity against pathogenic clinical isolates with multidrug resistance profiles such as E. coli, E. cloacae and S. typhi, thereby providing insights on the possible use of these molecules for the development of new therapeutic alternatives. The complete recombinant fused protein GWLK-Lucilin-CPDHis8 was active against Gram-negative bacteria but the cleaved protein was not, this activity may be explained by the conformational structure of the protein where the peptide is positioned at the N-terminus of the CPD and this segment is believed to adopt a random coil structure giving the possibility of interaction for peptide with the bacterial membrane (Fig.1). The N-terminal region of the peptide is believed to be the primary region responsible for the membrane interaction because of its hydrophobic character and polarity as seen for other cecropins [15]. The protection of the C-terminus of the peptide from degradation by bacterial proteases likely increases the stability of the peptide when compared with the cleaved product. This finding may aid in the construction of new chimeric recombinant molecules with higher antimicrobial activity and different functional domains at the C-terminus that may provide new functionalities to the antimicrobial peptides. It remains unknown why the cleaved protein was inactive. Nonetheless, it is possible that the stability of the cleaved peptide was compromised, or that the inP6 interacted with the peptide, or the cleaved peptide aggregated, thereby limiting its antimicrobial activity; however, these possibilities require further investigation. E. coli is still the preferred host for recombinant protein expression, because of its high expression level, simplicity and low cost. However, difficulties have been encountered in the expression of antimicrobial proteins because of their cytotoxicity to host bacte-
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Acknowledgments This work was supported by the Grant AMFRI 24-10 of the Red de Universidades del eje cafetero, ALMA MATER and the call 511 for doctorate study in Colombia. Administrative department of science technology and innovation COLCIENCIAS. Appendix A. Amino acids sequences of the peptide and proteins A.1. Lucilin (GenBank: ADH04298) KLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKG A.2. CPD >gi|15641462|ref|NP_231094.1| RTX toxin RtxA [Vibrio cholerae O1 biovar El Tor str. N16961] LADGKILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVA KAAANLAGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHG RDHSETNNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSL VSDDKQKGFGHQFINAMDANGLRVDVSVRSSELAVDEAGRKHTKDAN GDWÊ LADGKILHNQNVNSW Fig. 5. Hemolytic and cytotoxic activity of the GWLK-Lucilin-CPD-His8 protein. (A) Cytotoxic activity of the GWLK-Lucilin-CPD-His8 on Vero cells after 24 h incubation. Show no decrease in the cell viability greater than 80 % in general with a slight decrease at 1.29 lM. (B) Hemolytic activity towards human erythrocytes. The GWLK-Lucilin-CPD-His8 show no hemolytic activity at any concentration tested.
rial cells, susceptibility to proteolytic degradation and low-production yields. The production of antimicrobial peptides in E. coli using other fusion partners such as thioredoxin or small ubiquitinrelated modifier (SUMO) have been described [16,17]. The CPD domain has been used for the production of cathelicidin-derived antimicrobial peptides with a production yield varying between 5 and 20 mg/L. In this work, we had a yield of 70 mg/L. A reason for this high yield may be because the codon optimization used for the production of the synthetic gene. We were also successfully in producing and purify a soluble active recombinant antimicrobial protein based on the Lucilin peptide fused with the CPD in E. coli BL21. However, the purification via a one-step approach using incolumn cleavage of the protein was not successful; i.e., a chromatographic peak corresponding to the peptide after column cleavage was not observed. This may be, because of the slower elution of the peptide or the formation of protein aggregates, and it would be necessary to further purify the cleaved peptide by HPLC, as described previously by Wright [10]. The GWLK-Lucilin-CPD-His8 protein was confirmed to have no hemolytic activity against human erythrocytes and no toxicity towards Vero cells (Fig. 5). This protein may be a potentially safe substitute for traditional antibiotics against human pathogenic bacteria. However, the safety of GWLK-Lucilin-CPD-His8 for potential use in therapeutics will be explored further. Conclusions In the current study, we have successfully produced and purify a recombinant protein GWLK-Lucilin-CPD-His8, which showed strong antimicrobial activities against drug resistance Gram-negative bacteria. This suggests that GWLK-Lucilin-CPD-His8 can be a useful candidate for therapeutic design and usage. This project has laid the foundation for further studies examining the biological functions of a chimeric recombinant Lucilin and to explore ways to design genetically engineered antimicrobial drugs.
A.3. GWKL-LUCILIN-CPD-His8 MGWLKKLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKGLADG KILHNQNVNSWGPITVTPTTDGGETRFDGQIIVQMENDPVVAKAAANL AGKHAESSVVVQLDSDGNYRVVYGDPSKLDGKLRWQLVGHGRDHSET NNTRLSGYSADELAVKLAKFQQSFNQAENINNKPDHISIVGCSLVSDDKQ KGFMGWLKKLGKKIERVGQHTRDATIQTIGVAQQAVNVAATLKGLADG KILHNQNVNSWGPITVTPT
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