AAC Accepts, published online ahead of print on 24 January 2011 Antimicrob. Agents Chemother. doi:10.1128/AAC.01097-10 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK Soo Youn Jun,1 Gi Mo Jung,1 Jee-Soo Son,2 Seong Jun Yoon,1 Yun-Jaie Choi,2 and Sang Hyeon Kang1* D o w n 5 lo a d e d iNtRON Biotechnology, Inc., Room 703, JungAng Induspia V, 138-6, Sangdaewon-dong, f r o m Jungwon-gu, Seongnam-si, Gyeonggi-do 462-120, Korea,1 and Laboratory of Animal h t t p : / Cell Biotechnology, Department of Agricultural Biotechnology, Seoul /a a c . a National University, Building 200-#4204, Sillim-9 dong, Seoul 151-742, Korea2 s m . o r 10 g / o n * Corresponding author. Mailing address: iNtRON Biotechnology, Inc., Room 703, M a r c h JungAng Induspia V, 138-6, Sangdaewon-dong, Jungwon-gu, Seongnam-si, Gyeonggi- 3 0 , 2 do 462-120, Korea. Phone: 82-31-739-5352; Fax: 82-31-739-5353. E-mail: 0 1 9 b [email protected] y g u e 15 st 1 In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately two-fold lower minimum inhibitory concentration against several Staphylococcus aureus strains and higher bacterial cell wall hydrolyzing activity D o w n 5 compared to LysK. The amino acid residue change contributing the most to this lo a d e enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th d f r o m residue. h t t p : / /a a c . a Keywords Phage endolysin, LysK, SAL-1, Minimum inhibitory concentration, s m . o r 10 Staphylococcus aureus g / o n M a r c h 3 0 , 2 0 1 9 b y g u e 15 st 2 Staphylococcus aureus is a highly virulent human pathogen, and S. aureus infections are a significant cause of morbidity and mortality, particularly in settings such as hospitals, nursing homes and infirmaries (24). In addition to the more severe consequences of contact with S. aureus, this pathogen is also responsible for many cases D o w n 5 of food poisoning (14). Many recent isolates of S. aureus show innate resistance to lo a d e d currently available antibiotics (18), leading to challenges in managing S. aureus f r o m infections. h t t p : / Since the discovery of bacteriophages, their destructive effect on their host /a a c . a organisms has been exploited as a way of treating infectious bacteria (10). In addition, s m . o r 10 phage endolysins, also termed lysins, have been proposed as potent antibacterial agents g / o n (15, 17). Phage endolysins derived from bacteriophages are bacteriophage-encoded M a r c h peptidoglycan hydrolases that have evolved to rapidly break down the bacterial cell wall, 3 0 , 2 thereby allowing the release of phage progeny (30). Many phage endolysins have shown 0 1 9 b promise in preclinical trials involving animal models of human diseases (3, 5, 7, 16, 20, y g u e 15 27), and they thus constitute a promising route for the discovery and development of st novel antibacterial therapeutic agents. The phage endolysin LysK from staphylococcal phage K (22) is a valuable endolysin due to its broad-spectrum activity against the staphylococcal genus (23). A 3 large number of articles have been published on this phage endolysin (2, 6, 11) due to its potency and promise as an antibacterial agent. The present study was prompted by the serendipitous observation that phage endolysin SAL-1, derived from the bacteriophage SAP-1 recently isolated in our D o w n 5 laboratory, had a significantly lower minimum inhibitory concentration (MIC) than lo a d e d phage endolysin LysK in spite of the high level of amino acid sequence similarity f r o m between the two proteins. Based on sequencing studies, we determined that phage h t t p : / endolysin SAL-1 only differs from LysK at three residues: isoleucine instead of valine /a a c . a at the 26th residue, glutamine instead of glutamic acid at the 114th residue and histidine s m . o 10 instead of glutamine at the 486th residue. rg / o n This article describes a comparative study of the newly discovered phage M a r c h endolysin SAL-1 and the well-studied phage endolysin LysK. 3 0 , 2 An S. aureus bacteriophage was isolated from an environmental sample by a 0 1 9 b conventional method (29) and designated bacteriophage SAP-1. Bacteriophage SAP-1 y g u e 15 was deposited in the Korean Collection for Type Culture (KCTC), the Biological st Resource Center (BRC), and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) (Accession No.: KCTC 11153BP). The gene encoding the endolysin of bacteriophage SAP-1 was identified by comparison with reported 4 sequences and subcloned into the pBAD-TOPO vector (Invitrogen, Carlsbad, CA, USA) by conventional PCR cloning methods. E. coli TOP10 (Invitrogen) cells were transformed with the resulting plasmid (named pBAD::SAL-1). The C-terminal His- tagged phage endolysin SAL-1 was produced by culturing the resulting transformed D o w n 5 cells, and it was purified according to our previously reported method (29) with one lo a d e d modification: cation-exchange chromatography was replaced with conventional nickel f r o m affinity chromatography. h t t p : / To compare SAL-1 with LysK and to determine the amino acid residue /a a c . a change(s) that contributed the most to the difference in antibacterial activity, s m . o r 10 recombinant phage endolysin LysK and several derivatives of phage endolysin SAL-1 g / o n were prepared as C-terminal His-tagged proteins. The expression plasmids for LysK M a r c h and SAL-1 derivatives were constructed by conventional site-directed mutagenesis with 3 0 , 2 the SAL-1 expression plasmid pBAD::SAL-1. LysK and SAL-1 derivatives were 0 1 9 b produced and purified by the same method used in the production and purification of y g u e 15 recombinant SAL-1. All of the prepared phage endolysins and derivatives are st summarized in Table 1. All assays were performed immediately after determining the concentration and integrity of the phage endolysins as well as the quality of the phage endolysin 5 preparation by conventional SDS-PAGE and zymogram assays (Fig. 1). The protein concentration was measured using a SMARTTM BCA assay kit (iNtRON Biotechnology, Gyeonggi, Korea), and the final protein concentration-adjusted solutions were analyzed by 12% SDS-PAGE. D o w n 5 The antibacterial activity of phage endolysins SAL-1 and LysK toward various lo a d e d bacterial strains, including ten S. aureus strains (SA1, SA2, SA3, CCARM3095, f r o m CCARM3547, CCARM0027, CCARM3090, CCARM3271, ATCC31886 and h t t p : / ATCC35556) and nine non-S. aureus strains (one S. saprophyticus strain (SS1), one S. /a a c . a epidermidis strain (SE1), one Enterococcus faecalis strain (EFA1), one Enterococcus s m . o r 10 faecium strain (EFA2), two Salmonella spp. strains (Salmonella gallinarum SG120 and g / o n Salmonella enteritidis SE51), one Streptococcus mutans strain (CCARM0079), one M a r c h Streptococcus mitis strain (CCARM0143) and one E. coli strain (E1)), was assessed 3 0 , 2 using a conventional clearing-formation assay (29). The strains SA1, SA2, SA3, SS1, 0 1 9 b SE1, EFA1, EFA2, SG120, SE51 and E1 were isolated from environmental samples, y g u e 15 and the other strains were obtained from either the American Type Culture Collection st (ATCC, Rockville, MD, USA) or from the Culture Collection of Antimicrobial Resistant Microbes (CCARM, Korea). The MICs of all of the prepared phage endolysins for the ten S. aureus strains were determined as previously described (1) 6 with some modifications. More detailed dilution steps were utilized for determining more precise MIC values within the predetermined MIC range for each case. In spite of their high amino acid sequence similarity, we found significant differences between the MICs of SAL-1 and LysK toward the ten S. aureus strains D o w n 5 (Table 2). SAL-1 had MICs that were approximately two-fold lower than those of LysK, lo a d e d although their antibacterial ranges were the same among the tested strains (data not f r o m shown); each could lyse all ten S. aureus strains and two staphylococcal strains (S. h t t p : / saprophyticus and S. epidermidis strain), but none could lyse any of the tested strains /a a c . a outside of the Staphylococcus genus. s m . o r 10 Using various derivatives prepared by systematic site-directed mutagenesis, g / o n the amino acid residue change contributing the most to the difference in MICs was M a r c h determined (Table 2). The results indicated that the change from glutamic acid to 3 0 , 2 glutamine at the 114th residue was the primary amino acid residue change contributing 0 1 9 b to the difference between SAL-1 and LysK. The enzymatic activity assay measured the y g u e 15 rapid drop in optical absorbance of a suspension of S. aureus when exposed to phage st endolysin, as previously described (13). Five S. aureus strains (SA1, CCARM0027, CCARM3271, ATCC31886 and ATCC35556) were used. First, 5 µg of purified SAL-1, one of several derivatives of SAL-1 or LysK, was added to each 1-ml S. aureus 7 suspension in phosphate-buffered saline (adjusted to an optical density of 1.0 at 650 nm (OD )). Then, the OD was read at 30-s intervals using a Cary100 spectrophotometer 650 650 (Varian, Palo Alto, CA, USA) for 30 min. The time to reach half of the starting absorbance (TOD , equivalent to one-half log drop in the initial number of viable D 50 o w n 5 bacteria) of the bacterial suspension was measured for each strain. The experiment was lo a d e d repeated three times, and the average is reported. The bacterial cell wall hydrolyzing f r o m activity of SAL-1 was found to be approximately two-fold higher against all S. aureus h t t p : / strains tested than that of LysK (Table 3), indicating that the significant reduction in /a a c . a MIC is mainly attributable to enhanced enzymatic activity. The results presented in s m . o 10 Table 3 indicate that the change from glutamic acid to glutamine at the 114th residue was rg / o n the major amino acid residue change contributing to the enhanced enzymatic activity of M a r c h SAL-1. 3 0 , 2 S. aureus is highly resistant to many commonly used antibiotics, and there has 0 1 9 b been an explosion in the prevalence of MRSA (methicillin-resistant S. aureus) in y g u e 15 hospitals to the point that it is now endemic (12). Therefore, the discovery and/or st development of new antibacterial agents that are effective at treating MRSA infections should be quite beneficial. Due to their specific antibacterial activity, the endolysins of bacteriophages that target pathogens of interest have been proposed as potent 8 antibacterial agents that offer several advantages over conventional antibiotics (17). The novel phage endolysin SAL-1 was found to have a lytic activity spectrum similar to that of LysK against all S. aureus strains tested in this study, including both clinically isolated MRSA strains and environmentally isolated S. aureus strains. D o w n 5 However, SAL-1 had higher enzymatic activity in hydrolyzing the cell wall than LysK, lo a d e d and SAL-1 had reduced MICs for the tested S. aureus strains. In addition, our results f r o m indicate that the change from glutamic acid to glutamine at the 114th residue is the major h t t p : / amino acid residue change contributing to this enhanced enzymatic activity. /a a c . a The phage endolysins SAL-1 and LysK have two catalytic domains, a CHAP s m . o r 10 domain (cysteine, histidine-dependent amidohydrolases/peptidase) and a central g / o n amidase-2 domain (N-acetylmuramoyl-L-alanine amidase), as well as a C-terminal M a r c h SH3b cell-binding domain (4, 19, 21, 23, 26). The 114th residue that was identified as 3 0 , 2 the major contributor to the lower MICs is located in the CHAP domain. Many 0 1 9 b published reports have indicated that a single residue change in a functional domain can y g u e 15 drastically modify biological activity (8, 9, 25, 28). Therefore, the changes in the st enzymatic activity that we observed in this study are well supported by the current literature and general scientific knowledge. Intensive structural analysis will be required to gain further insight into the 9 chemical structure and charge requirements for endolysin’s biological activity and to fully understand the enhanced activity provided by the single residue difference (i.e., Derivative 5 in Table 2) relative to LysK. However, it is reasonable to conclude based on our findings that the newly discovered phage endolysin SAL-1 is a more powerful D o w n 5 endolysin than the currently used and highly potent antibacterial LysK (2). lo a d e d f r o m Acknowledgments This work was supported by grants from the Technology h t t p : / Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, /a a c . a Forestry and Fisheries, Republic of Korea and from the Korean Health Technology s m . o r 10 R&D Project (A101858), Ministry for Health, Welfare & Family Affairs, Republic of g / o n Korea. M a r c h 3 0 , 2 0 1 9 b y g u e 15 st 10