Glycin-rich antimicrobial peptide YD1 from B. amyloliquefaciens, induced morphological alteration in and showed affinity for plasmid DNA of E. coli

Natural products are extensively used in our society. More than 60% of approved drugs and drug candidates either are natural goods and/or derived from them. About 100,000 secondary metabolites (organic compounds, not directly involved in the growth, development, or reproduction of an organism) with molecular weight less than 2500 Da have been characterized; half from microbes and the other half from plants (Strobel and Daisy 2003). AMPs and “bacteriocins” have gained attention for their potential application in controlling pathogenic bacteria and infectious diseases. AMPs are ubiquitously produced by a wide range of microorganisms. Bacillus, a genus of significant interest to human health, produces a varied array of AMPs with several different basic chemical structures. Several peptides with biological activities have been identified from Bacillus and are under active investigation for their antimicrobial effects (Dischinger et al. 2009; Teixeira et al. 2009; Wu et al. 2005). Bacteriocin or bacteriocin-like substances have also been described for some other important pathogens such as Listeria monocytogenes and Streptococcus pyogenes (Cherif et al. 2001).

The history of use of fermented vegetables as a source of beneficial bacteria is extensive. For over 2000 years, Koreans have consumed salted and fermented vegetables such as kimchi. The primary ingredients of kimchi are Baechu cabbage and radish; however, other vegetables such as green onion, leek, and cucumber are also used for preparing various types of kimchi. Kimchi contains high levels of vitamins, minerals, dietary fibers, and other functional ingredients. Many previous studies have reported that kimchi has anticancer, antimicrobial, antioxidant, antiatherosclerotic, antidiabetic, and antiobesity effects among others (Islam and Choi 2009; Kim et al. 2007, 2011).

The rapid development and spread of bacterial resistance and the emergence of multi-drug resistant pathogens have created a need for the discovery of new classes of antimicrobial agents against novel microbial targets while evading existing resistance mechanisms. Elucidation of the mechanism(s) of action of antimicrobial drugs helps to characterize the interaction of the pathogen with both the chemical and the host, design improved antimicrobials, determine effective combinations of drugs, and understand the development of microbial resistance. The exploration of action mechanisms of developmental compounds, not originated from target-based discovery, should be undertaken early in their development stage to facilitate the modification of the drug scaffold for improved selectivity of action and pharmaceutical profile. The production of AMPs from bacteria isolated from fermented foods and their mechanisms of action have not been extensively studied.

In the present work, we designed a two-step study. Firstly, we isolated the strain from kimchi, identified, characterized, and purified the AMP, and further characterized the AMP designated as YD1. Secondly, upon confirmation of the novelty of purified YD1, we investigated its antimicrobial mechanism.

Screening and characterization of novel AMPs are attractive owing to their potential applications for therapeutic usage and in the food industry. In the present study, we identified, purified, and characterized an AMP from strains isolated from Korean fermented kimchi. Furthermore, we conducted a preliminary investigation of the antimicrobial mechanism of the purified AMP. Till date, a few AMPs producing strains have been isolated from kimchi (Mah et al. 2001; Teixeira et al. 2009; Wu et al. 2005). In our present study, we have reported the isolation of AMP producing bacterial strain, CBSYD1, from kimchi and studied the influence of several growth media and culture conditions on the production of AMP, and determined the optimized media (1% peptone, 2% maltose, and 0.01% CaCl₂) that promotes greater production of YD1 over the commercially available media such as MRS and MH (Fig. 2). Growth media temperature and nutrients played a major role in the production of bacteriocin (Todorov et al. 2006). Bacteriocin activity was significantly higher at 37 °C, consistent with the results from Lisboa et al. on bacteriocin, produced by B. amyloliquefaciens, (Lisboa et al. 2010) and bacteriocin was very much stable at pH 4-9 and up to 80 °C, consistent with results by Todorov reports (Todorov et al. 2006). The protease enzymes failed to alter the antimicrobial activity of the YD1, which was not unusual (Korenblum et al. 2005). The homogeneity of YD1 was obtained by a two-step purification procedure, which resulted in 40-fold purification with 12% activity recovery. The purified YD1 has lower molecular weight compared to a bacteriocin-like substance (BLS) and some other Bacillus AMPs (Teixeira et al. 2009). YD1 was shown to be effective against both gram-positive and gram-negative bacteria as presented in Table 1. YD1 displayed a better antagonistic effect than reference antibiotics (bacitracin and vancomycin) against MDR pathogens such as MRSA and VRE. Antimicrobial effects of YD1 were prominent in comparison to other reports of AMPs against MDR, and non-MDR pathogens of Bacillus have been published (Dischinger et al. 2009; Sandiford and Upton 2012; Zheng and Slavik 1999).

The amino acid residues of YD1 were Ala-Pro-Lys-Gly-Val-Gln-Gly-Pro-Asn-Gly. APD search revealed that the closest (40%) similarity of YD1 was with an AMP Leucrocin I (AP02344) isolated from white blood cell extracts of crocodile, Crocodylus siamensis (Pata et al. 2011). Furthermore, sequence alignment suggested other AMPs (Table 4) showed ≤40% similarity. Sequences comparison with other AMP sequences revealed that YD1 possesses unique characteristics. Most of the AMPs are minuscule and strongly cationic (Yeung et al. 2011). Characteristics of the YD1 protein suggests that it is an AMP include: (1) small size (0.924 kDa) with 10 amino acids; (2) cationic character (net charge +1); and (3) theoretical pI of 8.80 (calculated according to amino acids sequences by ProtParam; http://web.expasy.org/protparam). Owing to its broad spectrum of antimicrobial activity and unique mechanism of action. Initial results from our study showed synergism between YD1 and erythromycin, a protein synthesis translocation inhibitor, indicating a relationship between their mechanisms of action (Odds 2003) and the property of YD1 to interact with intracellular macromolecules rather than the cell wall machinery. Using E. coli (10⁶ cfu/mL bacterial suspension) as a model organism, antimicrobial mechanism of action study was performed. TEM images showed morphological alteration in E. coli when treated with 5x MIC (40 µg/mL) of YD1 (Fig. 5a); while >88% of spheroplasts remained intact (Fig. 5b), 80% of E. coli cells died at the same YD1 concentration (Fig. 5c). This antibacterial action might result from effects on cellular metabolism. Kang et al. (2015) and Li et al. (2014) also reported the similar observation of morphological alteration of E. coli cells when treated with berberin and α-terpineol respectively (Kang et al. 2015; Li et al. 2014). In Fig. 5a, we demonstrated that YD1 possesses cell-penetrating and translocation ability, likely responsible for its antimicrobial activity without damaging bacterial cell wall or causing without cytotoxicity in RAW 264.7 macrophage cells (Fig. 5d). A recent review by Katrin and Neundorf (2011) enlisted the commonly used cell-penetrating peptides (CPP) and AMPs (Splith and Neundorf 2011). Furthermore, the positively charged residues in the α-helical sequence, and Arg-rich peptide, associate with the lipid phosphate groups to neutralize the Arg residue and allow the peptide translocation across the membrane (Amand et al. 2008; Boman 1995; Su et al. 2009). Recently, a report by Xie et al. (2011) suggested that the position of Pro residue in buforin II was more important than the overall α-helical content for the translocation (Xie et al. 2011). Consistent with these previous reports, YD1 AMP, a positively charged peptide, contains 3 glycines ([G^4,7,10]) and 2 proline ([P^2,8]) residues out of 10 amino acids. The antimicrobial potency of YD1 might not be determined solely by the coil-shaped secondary structure, but other factors such as the type of positive charge or the location of Pro might also be vital. In 2012, Jang and his co-workers (Jang et al. 2012) reported two cell-penetrating motifs Q-F-P–V-G and Q-W–P-V-G for peptides Buforin IIb and Buf IIIa, respectively. Analogously, Q-G-P-N-G is the likely expected cell-penetrating motif for YD1. Moreover, I-TASSER prediction analysis has suggested the presence of ligand binding sites in YD1 sequence for a nucleic acid (NUC) (Fig. 6b) which was consistent with the DNA binding affinity result shown in Fig. 5c.

In spite of the several proposed molecular processes regulated by AMPs, it is still unclear which, if any, of the hypothesized mechanisms, is responsible for their biological activity (Pálffy et al. 2009). Based on our findings, the antimicrobial mechanism of YD1 may be driven by cell-penetration and translocation inside the cell via a voltage-gated ion channel possibly (currently in process) and followed by interaction with bacterial plasmid DNA ultimately leading to bacterial cell death (Fig. 6c). After elucidation of complete structural information of the YD1 peptide (currently in progress), we will study the in-depth antimicrobial mechanism of action and synthesize AMP analogs.

In summary, the results presented here demonstrate the activity of the novel AMP YD1 purified from Bacillus amyliquefaciens subsp. plantarum FZB42 isolated from Korean fermented kimchi. A broad-spectrum glycine-rich, low-molecular-weight AMP YD1 exhibits a unique antimicrobial mechanism of action characterized by its affinity for bacterial DNA, without damaging bacterial cell wall. Potent antimicrobial effects of YD1, in particular against resistant pathogens such as MRSA, VRE, and also E. coli, were observed. Our results collectively suggest that YD1 serve as a promising candidate for developing therapeutic agents for bacterial infections and endotoxin shock.