Characteristics and Lytic Activity of Phage-Derived Peptidoglycan Hydrolase, LysSAP8, as a Potent Alternative Biocontrol Agent for Staphylococcus aureus원문보기
Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine....
Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, and Zn2+) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.
Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, and Zn2+) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
Based on the putative endolysin gene of SAP8, the primers SAP8_F #5 (5’-GGAATTCCATATGTTAAT GACAAAA-AATCAA-3’) and SAP8_R #4 (5’-CCGCTCGAGAAT CGTGCTAAA-3’) were designed.
In the experiment conducted to assess the effects of different concentrations of NaCl on the endolysin, lytic activity of LysSAP8 varied significantly with change in NaCl concentrations (Fig. 6C). In the absence of NaCl, the efficacy of LysSAP8 for lysis was approximately 50% of that in buffer supplemented with 500 mM NaCl, which was considered the optimum NaCl concentration.
The predicted ORFs were annotated using Blastp based on the e-value < 1E-05, and classified into six groups based on their functions: packaging, structure, host lysis, DNA manipulation, DNA regulation, and additional function. The G+C content was predicted using the DNA/RNA GC content calculator from ENDMEMO (http://www.endme mo.com/bio/gc.php), and the tRNAscan-SE 1.21 (The Lowe Lab, University of California Santa Cruz, USA) was used to search for the tRNA [27, 28].
The predicted ORFs were annotated using Blastp based on the e-value < 1E-05, and classified into six groups based on their functions: packaging, structure, host lysis, DNA manipulation, DNA regulation, and additional function.
To test the susceptibility of LysSAP8 at various factors, the endolysin was inoculated into the cell preparation (final conc. 1 μM), which was then incubated for 30 min with the following conditions: temperature (4°C, 15°C, 25°C, 30°C, 37°C, and 43°C), pH (50 mM citrate buffer pH 3–5, 50 mM imidazole-HCl buffer pH 6, and 50 mM tris-HCl buffer pH 7-9), NaCl (0, 500, 750, and 1,000 mM), and divalent metal ions (1 mM: Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, and Zn2+, and EDTA for control).
데이터처리
Statistically significant differences were evaluated using an unpaired one-tailed t-test for three independent experiments. pvalues less than 0.
이론/모형
The phage lysate was purified using a modified polyethylene glycol precipitation method [26]. Purified phage solution was deposited onto formvar-carbon 200 mesh TH copper grids (Ted Pella, Inc.
aureus, were aligned using ClustalW [30]. These endolysin alignments were constructed into a phylogenetic tree by the neighbor-joining method with 1000 bootstrap replicates using MEGA 7 (MEGA) [31]. The NCBI database was accessed to identify all endolysin sequences.
성능/효과
aureus isolates including MRSA, 73 strains showed susceptibilities to LysSAP8. Additionally, lysis activity of LysSAP8 against most of the other staphylococcal species (S. epidermidis, S. hominis, S. sciuri, S. warneri, S. xylosus) was also observed except for S. condiment PS10-3, S. warneri CCBK901, S. epidermidis KCCM 35494 and 40416. However, LysSAP8 killed neither the other gram-positive (Bacillus, Enterococccus, Lactobacillus, Listeria, Streptococcus) nor gramnegative (Escherichia, Enterobacter, Klebsiella, Salmonella, Vibrio) strains.
aureus was isolated from feces. The morphological analysis of SAP8 by transmission electron microscopy revealed that SAP8 has a long non-contractile tail approximately 300 nm in length, exhibiting typical characteristics representative of the Siphoviridae family with the adopted elongated head (Fig. 1).
Thus, it was determined that LysSAP8 was composed of two EADs and one CBD. These results showed that LysSAP8, as a LysK-like lysin, belongs to the type IV category, based on the grouping of staphylococcal endolysins [37]. Moreover, similar to LysK, LysSAP8 was determined to be approximately 53 kDa in molecular weight and composed of three domains [21, 22].
Interestingly, phage SAP8 could not infect any of the tested MRSA which were susceptible to LysSAP8. These results suggested that antibiotic resistant-bacteria might be susceptible to endolysin LysSAP8, even if they were insensitive to phage SAP8 which was the source of LysSAP8.
참고문헌 (42)
Knox J, Uhlemann AC, Lowy FD. 2015. Staphylococcus aureus infections: transmission within households and the community. Trends Microbiol. 23: 437-444.
Choi SW, Lee JC, Kim J, Kim JE, Baek MJ, Park SY, et al. 2019. Prevalence and risk factors for positive nasal methicillin-resistant Staphylococcus aureus carriage among orthopedic patients in Korea. J. Clin. Med. 8(5): pii: E631.
Hennekinne J-A. 2018. Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, pp. 129-146. In Fetsch A (ed.), Staphylococcus aureus, 1st Ed. Academic Press, Cambridge.
Hyeon JY. 2013. A foodborne outbreak of Staphylococcus aureus associated with fried chicken in Republic of Korea. J. Microbiol. Biotechnol. 23: 85-87.
Papadopoulos P, Papadopoulos T, Angelidis AS, Boukouvala E, Zdragas A, Papa A, et al. 2018. Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus (MRSA) along the production chain of dairy products in north-western Greece. Food Microbiol. 69: 43-50.
Lin DM, Koskella B, Lin HC. 2017. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J. Gastrointest Pharmacol. Ther. 8: 162-173.
Jamal M, Bukhari S, Andleeb S, Ali M, Raza S, Nawaz MA, et al. 2019. Bacteriophages: an overview of the control strategies against multiple bacterial infections in different fields. J. Basic Microbiol. 59: 123-133.
Wittebole X, De Roock S, Opal SM. 2014. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5: 226-235.
Chang Y, Yoon H, Kang DH, Chang PS, Ryu S. 2017. Endolysin LysSA97 is synergistic with carvacrol in controlling Staphylococcus aureus in foods. Int. J. Food Microbiol. 244: 19-26.
Jasim HN, Hafidh RR, Abdulamir AS. 2018. Formation of therapeutic phage cocktail and endolysin to highly multidrug resistant Acinetobacter baumannii: in vitro and in vivo study. Iran J. Basic Med. Sci. 21: 1100-1108.
Zhang L, Li D, Li X, Hu L, Cheng M, Xia F, et al. 2016. LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation. Sci. Rep. 6: 29344.
Abaev I, Foster-Frey J, Korobova O, Shishkova N, Kiseleva N, Kopylov P, et al. 2013. Staphylococcal phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lyticdomain secondary translational start site. Appl. Microbiol. Biotechnol. 97: 3449-3456.
Gu J, Xu W, Lei L, Huang J, Feng X, Sun C, et al. 2011. LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection. J. Clin. Microbiol. 49: 111-117.
Haddad Kashani H, Schmelcher M, Sabzalipoor H, Seyed Hosseini E, Moniri R. 2018. Recombinant endolysins as potential therapeutics against antibiotic-resistant: current status of research and novel delivery strategies. Clin. Microbiol. Rev. 31: e00071-00017.
Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, van Raaij MJ. 2014. Crystal structure of the lytic CHAP(K) domain of the endolysin LysK from Staphylococcus aureus bacteriophage K. Virol J. 11: 133-133.
Melo LDR, Brandao A, Akturk E, Santos SB, Azeredo J. 2018. Characterization of a new Staphylococcus aureus kayvirus harboring a lysin active against biofilms. Viruses 10(4). pii: E182.
Kim NH, Park WB, Cho JE, Choi YJ, Choi SJ, Jun SY, et al. 2018. Effects of phage endolysin SAL200 combined with antibiotics on Staphylococcus aureus infection. Antimicrob. Agents Chemother. 62. pii: e00731-18.
Lu L, Cai L, Jiao N, Zhang R. 2017. Isolation and characterization of the first phage infecting ecologically important marine bacteria Erythrobacter. Virol J. 14(1): 104.
Khan Shawan MM, Hasan MA, Hossain MM, Hasan MM, Parvin A, Akter S, et al. 2016. Genomics dataset on unclassified published organism (patent US 7547531). Data Brief. 9: 602-605.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and clustal X version 2.0. Bioinformatics 23: 2947-2948.
Kumar S, Nei M, Dudley J, Tamura K. 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform. 9: 299-306.
Cabrita LD, Bottomley SP. 2004. Protein expression and refolding - a practical guide to getting the most out of inclusion bodies, pp. 31-50. In El-Gewely MR (ed.), Biotechnology Annual Review Volume 10., 10th Ed. Elsevier, Amsterdam.
Schmelcher M, Shen Y, Nelson DC, Eugster MR, Eichenseher F, Hanke DC, et al. 2015. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection. J. Antimicrob. Chemother. 70: 1453-1465.
Won G, Hajam IA, Lee JH. 2017. Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by coexpression of lambda phage holin-endolysin and X174 gene E. Sci. Rep. 7: 45139.
Larpin Y, Oechslin F, Moreillon P, Resch G, Entenza JM, Mancini S. 2018. In vitro characterization of PlyE146, a novel phage lysin that targets Gram-negative bacteria. PLoS One 13: e0192507.
Dong H, Zhu C, Chen J, Ye X, Huang YP. 2015. Antibacterial activity of Stenotrophomonas maltophilia endolysin P28 against both gram-positive and gram-negative bacteria. Front Microbiol. 6: 1299.
Chang Y, Kim M, Ryu S. 2017. Characterization of a novel endolysin LysSA11 and its utility as a potent biocontrol agent against Staphylococcus aureus on food and utensils. Food Microbiol. 68: 112-120.
Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM. 2009. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol. Lett. 294: 52-60.
Heselpoth RD, Yin Y, Moult J, Nelson DC. 2015. Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling. Protein Eng. Des. Sel. 28: 85-92.
Gupta R, Prasad Y. 2011. P-27/HP endolysin as antibacterial agent for antibiotic resistant Staphylococcus aureus of human infections. Curr. Microbiol. 63: 39-45.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.