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A Bacteriocin with Broad Antimicrobial Spectrum Abbas Tanhaeian1, Mohammad Sadegh Damavandi2 , Davood Mansury3* and Kiarash Ghaznini4*

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A Bacteriocin with Broad Antimicrobial Spectrum Abbas Tanhaeian1, Mohammad Sadegh Damavandi2 , Davood Mansury3* and Kiarash Ghaznini4* Tanhaeian et al. AMB Expr (2019) 9:6 https://doi.org/10.1186/s13568-018-0729-6 ORIGINAL ARTICLE Open Access Expression in eukaryotic cells and purifcation of synthetic gene encoding enterocin P: a bacteriocin with broad antimicrobial spectrum Abbas Tanhaeian1, Mohammad Sadegh Damavandi2 , Davood Mansury3* and Kiarash Ghaznini4* Abstract Due to the emergence of multidrug-resistant bacteria, treatment options for infectious diseases are decreasing. Bac- teriocins are small antimicrobial peptides produced by numerous bacteria that ofer alternative therapeutic strategies to combat multidrug-resistant bacterial infections. We evaluated the cloning, functional expression, and antimicrobial activities of enterocin P (EntP), a class II bacteriocin member, in Chinese hamster ovary (CHO) cells. A synthetic gene matching CHO cell codon usage was designed from the known mature amino acid sequence of EntP and cloned into the protein expression vector pcDNA™3.1( ). CHO cells were transformed with the recombinant plasmid and cultured, and the recombinant protein was purifed+ by afnity chromatography. Antimicrobial activities of the recom- binant EntP were evaluated on Gram-positive, Gram-negative, and multidrug-resistant pathogens. Recombinant EntP inhibited growth of a variety of bacteria, including pathogenic species known to cause nosocomial infections, often with multidrug-resistant strains. In addition, recombinant EntP demonstrated broad antimicrobial activities in both high salt medium and human plasma and was stable at high temperatures. The broad antimicrobial activity and stability of EntP make it an attractive therapeutic candidate, particularly for treatment of multidrug-resistant bacterial infections. Keywords: Bacteriocin, Antimicrobial peptides, CHO cell, Expression, Enterocin P Introduction lack of new antimicrobial agents will inevitably result in Multidrug-resistant pathogenic bacteria are a major increased resistance to existing antibiotics (White et al. threat to global health and are rising to dangerously high 2011). Te use of broad-spectrum antibiotics can damage levels worldwide (Jasovsky et al. 2016). Te World Health the human microbiota, which is critical in host health. Organization 2017 fact sheet warns that new antimicro- Signifcant evidence demonstrates that administration of bial resistance mechanisms are emerging and spreading broad-spectrum antibiotics may lead to impaired micro- globally, threatening our ability to treat common infec- biota and compositional and functional disturbances. Te tious diseases. Several pathogenic bacteria have devel- potential link between the use of broad-spectrum antibi- oped resistance to multiple antibiotics. In addition, a otics and increasing incidence of atopic and autoimmune diseases is a specifc reason for concern (Krishnamoorthy et al. 2018; Willing et al. 2011). Consequently, new anti- *Correspondence: [email protected]; mansuryd@varastegan. microbials are needed. An option that can no longer be ac.ir; [email protected] ignored is a subgroup of antimicrobial peptides (AMPs) 3 Department of Medical Laboratory Sciences, Varastegan Institute for Medical Sciences, Mashhad, Iran known as bacteriocins. 4 Antimicrobial Resistance Research Center, Mashhad University Bacteriocins are a heterogeneous group of ribosomally- of Medical Sciences, Mashhad, Iran synthesized AMPs or complex proteins secreted by Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Tanhaeian et al. AMB Expr (2019) 9:6 Page 2 of 9 bacteria that exhibit antimicrobial activity against similar searches using the NCBI protein BLAST (http://www. or closely-related species through numerous mechanisms ncbi.nlm.nih.gov/BLAST /) were performed to identify (Micenkova et al. 2014; Shabir et al. 2018). Bacteriocins the template AMPs. To analyze nucleotide sequences, produced by Gram-positive bacteria are typically clas- determine the correct nucleotide sequence from protein, sifed into three classes: Te Class I bacteriocins, also and design restriction enzyme digestion sites for clon- known as lantibiotics, include lanthionine, methyllan- ing, CLC Genomics Workbench Version 3.6.5 software thionine, dehydroalanine, and 2-aminoisobutyric acid; (Redwood City, CA, USA) was used. Ten the I-TASSER the Class II bacteriocins are small non-lanthionine- simulations (Roy et al. 2010; Yang et al. 2015), were con- containing proteins and are divided into fve subclasses ducted to generate structure predictions for EntP, where (IIa/b/c/d/e); and the Class III bacteriocins are large homologous templates were excluded from the thread- heat-labile proteins, often with enzymatic activity (Cotter ing template library. I-TASSER simulations predicted the et al. 2013). Bacteriocins have net positive charges, which B-factor profle (BFP), the 3D model, and the estimated makes them useful for various applications. Bacteriocins global accuracy for EntP. have been shown to be active against human and animal bacterial pathogens, either of the same species (narrow Strains and vectors spectrum), or other species (broad spectrum). Bacteri- Escherichia (E.) coli DH5α cells were grown in LB broth ocins are highly selective, are efective against multidrug- (Sigma Chemical Co., St. Louis, MO, USA) at 37 °C with ™ resistant pathogens, and are non-toxic, which makes shaking. CHO cells, pcDNA 3.1(+) vector, BamHI and them important lead compounds for drug development HindIII restriction enzymes, and kits for plasmid extrac- (Bowdish et al. 2005; Mathur et al. 2018). tion were purchased from Termo Fisher Scientifc Bacteriocins produced by Enterococcus spp. are termed (USA). enterocins. Most enterocins are class II type. Tey are generally stable over a wide range of temperatures, pHs, Construction of the expression vector and transformation and following lyophilization. Tese compounds have To increase recombinant EntP expression, a His tag selective antimicrobial activity, and use this feature to was generated by GenScript’s OptimumGene software attack Clostridium (C.) perfringens, Staphylococcus (S.) (Genscript®, USA) and then chemically synthesized by aureus, and especially species of the food-borne patho- Generay Biotech (Shanghai, China), according to the genic bacteria including Clostridium (C.) botulinum and codon usage of CHO cells. Te optimized gene con- Listeria (L.) monocytogenes (Franz et al. 2007; Hu et al. struct encoding a 44-mer EntP peptide plus a 6-His tail 2010). Enterocins are usually cationic amphiphilic mol- was ligated into the multiple cloning site of the plasmid ecules that kill bacterial cells by inserting and accumulat- pcDNA3.1(+) with BamHI and HindIII restriction sites ing in the membrane, causing increased permeability and at the 5′ and 3′ ends, respectively (GenScript, USA). loss of barrier functions (Montalban-Lopez et al. 2012). Ligations were performed with T4 DNA ligase (Roche Short, non-toxic, and highly efective AMPs are needed Molecular Biochemicals, Mannheim, Germany). After to overcome antibiotic resistance issues. Here we inves- verifcation of the correct nucleotide sequence, E. coli tigated the expression, purifcation, and antibacterial DH5α cells were transformed with the construct using efects enterocin P (EntP), a class II cateriocin and used CaCl2 and grown on LB agar contain-ing 100 μg/ml of Chinese hamster ovary (CHO) cells as a production ampicillin. Colony-PCR was performed to identify colo- platform. CHO cells are widely used for recombinant nies containing the recombinant vec-tor. Te recombi- biopharmaceutical protein production because they are nant vector, named pcDNA3.1(+)-EntP, was extracted efective protein producers, are suitable for large-scale using a plasmid extraction kit (Termo Fisher Scientifc, production, and can be adapted to grow in serum-free USA) and used to transform CHO cells. and chemically-defned culture media. Also, this mam- malian expression system is tolerant to pressure, temper- Cell culture and transfection ature, pH, and oxygen level changes during manipulation CHO cells were maintained in Dulbecco’s Modifed (Kim et al. 2012; Lai et al. 2013). Eagle’s Medium (DMEM) supplemented with 8–12 mM l-glutamine with 100 μg/ml penicillin/streptomycin, and Materials and methods 10% heat-inactivated fetal bovine serum (FBS) (Invitro- In‑silico analysis tools gen, USA), at 37 °C with 5% CO2 and a relative humidity Te frst step for in silico analysis was to search in the of 95%. Te culture media was renewed every third day Antimicrobial Peptide Database (APD) (http://aps.unmc. and cultures were passaged every 4–5 days. Viable cells edu/AP/main.php) for sequences with high similarities to were counted using 0.5% trypan blue (Sigma Aldrich, 5 the target bacteriocin. EntPBLAST sequence homology USA). Approximately 2.5 × 10 CHO cells were seeded Tanhaeian et al. AMB Expr (2019) 9:6 Page 3 of 9 into 35 mm wells and cultured in DMEM until 60–80% overnight at 4 °C. To identify the recombinant EntP pep- confuent before transfection. CHO cells were transiently tide the membranes were incubated for 60 min at room transfected with 10 μg of pcDNA3.1(+)-EntP in
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