Cloning, Recombinant Production and Functional Analysis of Staphylococcal Phage Endolysins

Yıl 2023, Cilt: 2 Sayı: 2, 14 - 22, 30.06.2023

Serap Pektaş , Osman Birol Özgümüş , İnci Durukan , Ümit Uzun , Ersin Karataş , Ali Osman Kılıç

https://doi.org/10.59518/farabimedj.1255123

Öz

Staphylococci are pathogens that cause serious infections in humans and animals. Nosocomial infections caused by staphylococci, particularly methicillin-resistant Staphylococcus aureus (MRSA) strains, are mostly transmitted through healthcare workers, patients, or contaminated materials and food. In recent years, studies have been carried out to develop alternative antimicrobial strategies due to the inadequacy of existing antibiotics in the prevention of systemic, skin and implant-related biofilm infections caused by these multi-antibiotic resistant strains. One of these new approaches is the development of products containing the bacteriophage endolysin, which is particularly effective against multi-antibiotic-resistant bacteria. In this study, endolysin genes of bacteriophages (prophages) integrated into the chromosomes of Staphylococcus strains were amplified by polymerase chain reaction (PCR) and cloned into pET SUMO and pET-30b(+) vectors and produced recombinantly in E. coli. Anti-staphylococcal and antibiofilm activity of recombinant endolysins against S. aureus, S. epidermidis, and S. haemolyticus strains isolated from clinical specimens, were demonstrated using turbidity reduction, biofilm removal in microwell plates by crystal violet method, and capacity of endolysins to kill biofilm-forming bacteria by confocal microscopy imaging by live-dead staining. The combination of endolysin was shown to reduce bacterial culture turbidity by at least 50% at 60 minutes and biofilms by approximately 70% at 12 hours. These results show that endolysins have the potential to be used in the prevention of staphylococcal infections.

Anahtar Kelimeler

Bacteriophage, Anti-biofilm, Endolysin, Confocal Microscopy, Staphylococcus

Destekleyen Kurum

Karadeniz Teknik Üniversitesi

Proje Numarası

TSA-2019-8344

Teşekkür

This work was supported by Office of Scientific Research Projects of Karadeniz Technical University.

Stafilokkal Faj Endolizinlerinin Klonlanması, Rekombinant Olarak Üretilmesi ve Fonksiyonel Analizleri

Öz

Stafilokoklar insanlarda ve hayvanlarda ciddi enfeksiyonlara neden olan patojenlerdir. Stafilokokların, özellikle metisiline dirençli Staphylococcus aureus (MRSA) suşlarının neden olduğu nozokomiyal enfeksiyonlar çoğunlukla sağlık çalışanları, hastalar veya kontamine olmuş maddeler ve yiyecekler yoluyla bulaşır. Son yıllarda bu çoklu antibiyotik dirençli suşların neden olduğu sistemik, deri ve implant ilişkili biyofilm enfeksiyonlarının önlenmesinde mevcut antibiyotiklerin yetersizliği nedeniyle alternatif antimikrobiyal stratejiler geliştirmeye yönelik çalışmalar yapılmaktadır. Bu yeni yaklaşımlardan biri, çoklu antibiyotiğe dirençli bakterilere karşı özellikle etkili olan bakteriyofaj endolizin enzimini içeren ürünlerin geliştirilmesidir. Bu çalışmada, Staphylococcus suşlarının kromozomlarına entegre olmuş, bakteriyofajların (profaj) endolizin genleri polimeraz zincir reaksiyonu (PCR) ile çoğalaltılarak pET SUMO ve pET-30b(+) vektörlerine klonlandı ve E. coli’de rekombinant olarak üretildi. Rekombinant endolizinlerin, klinik örneklerden izole edilen S. aureus, S. epidermidis ve S. haemolyticus suşlarına karşı anti-stafilokokal ve antibiyofilm etkileri bulanıklılık azaltma, mikrokuyucuklu plaklarda kristal viyole yöntemi ile biyofilm uzaklaştırma ve konfokal mikroskopi yöntemi ile endolizinlerin biyofilmi oluşturan bakterileri öldürme kapasiteleri canlı ve ölü bakteri görüntüleme yöntemi ile saptandı. Endolizin kombinasyonunun bakteri kültür bulanıklılığını 60 dakikada en az %50 ve biyofilmleri 12 saatte yaklaşık %70 oranında azalttığı gösterildi. Bu sonuçlar endolizin enzimlerinin stafilokokal enfeksiyonların önlenmesinde kullanılma potansiyeline sahip olduğunu göstermektedir.

Anahtar Kelimeler

Konfokal Mikroskopi, Staphylococcus, Bakteriyofaj, Biyofilm, Endolizin, Bacteriophage, Anti-biofilm, Endolysin, Confocal Microscopy

Proje Numarası

TSA-2019-8344

Kaynakça

• Sakr A, Brégeon F, Mège JL, Rolain JM, Blin O. Staphylococcus aureus nasal colonization: An update on mechanisms, epidemiology, risk factors, and subsequent ınfections. Front Microbiol. 2018; 9: 2419. DOI: 10.3389/fmicb.2018.02419.
• Otto M. Staphylococcus epidermidis--the 'accidental' pathogen. Nat Rev Microbiol. 2009; 7(8): 555-67. DOI: 10.1038/nrmicro2182.
• Adesanya O, Oduselu T, Akin-Ajani O, Adewumi OM, Ademowo OG. An exegesis of bacteriophage therapy: An emerging player in the fight against anti-microbial resistance. AIMS Microbiol. 2020; 6(3): 204-230. DOI: 10.3934/microbiol.2020014.
• Loeffler JM, Nelson D, Fischetti VA. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science. 2001; 294(5549): 2170-2. DOI: 10.1126/science.1066869.
• Pastagia M, Euler C, Chahales P, Fuentes-Duculan J, Krueger JG, Fischetti VA. A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and -sensitive Staphylococcus aureus strains. Antimicrob Agents Chemother. 2011; 55(2): 738-44. DOI: 10.1128/AAC.00890-10. Linden SB, Zhang H, Heselpoth RD, et al. Biochemical and biophysical characterization of PlyGRCS, a bacteriophage endolysin active against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol. 2015; 99(2): 741-52. DOI: 10.1007/s00253-014-5930-1.
• Proença D, Fernandes S, Leandro C, et al. Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains. Microb Drug Resist. 2012; 18(3): 322-32. DOI: 10.1089/mdr.2012.0024.
• Schuch R, Nelson D, Fischetti VA. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature. 2002; 418(6900):884-9. DOI: 10.1038/nature01026.
• Briers Y, Volckaert G, Cornelissen A, et al. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL. Mol Microbiol. 2007; 65(5): 1334-44. DOI: 10.1111/j.1365-2958.2007.05870.x.
• Lu R, Liu B, Wu L, Bao Het al. A broad-spectrum phage endolysin (LysCP28) able to remove biofilms and ınactivate Clostridium perfringens strains. Foods. 2023; 12(2): 411. DOI: 10.3390/foods12020411.
• Sass P, Bierbaum G. Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus. Appl Environ Microbiol. 2007; 73(1): 347-52. DOI: 10.1128/AEM.01616-06.
• Gutiérrez D, Ruas-Madiedo P, Martínez B, Rodríguez A, García P. Effective removal of staphylococcal biofilms by the endolysin LysH5. PLoS One. 2014; 9(9): e107307. DOI: 10.1371/journal.pone.0107307.
• Meng X, Shi Y, Ji W, et al. Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis. Appl Environ Microbiol. 2011; 77(23): 8272-9. DOI: 10.1128/AEM.05151-11.
• Gilmer DB, Schmitz JE, Euler CW, Fischetti VA. Novel bacteriophage lysin with broad lytic activity protects against mixed infection by Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2013; 57(6): 2743-50. DOI: 10.1128/AAC.02526-12.
• Arroyo-Moreno S, Cummings M, Corcoran DB, Coffey A, McCarthy RR. Identification and characterization of novel endolysins targeting Gardnerella vaginalis biofilms to treat bacterial vaginosis. NPJ Biofilms Microbiomes. 2022; 8(1): 29. DOI: 10.1038/s41522-022-00285-0.
• Abdelrahman F, Easwaran M, Daramola OI, et al. Phage-Encoded Endolysins. Antibiotics (Basel). 2021; 10(2): 124. DOI: 10.3390/antibiotics10020124.
• Zhang Y, Cheng M, Zhang H, et al. Antibacterial effects of phage lysin LysGH15 on Planktonic cells and biofilms of diverse Staphylococci. Appl Environ Microbiol. 2018; 84(15): e00886-18. DOI: 10.1128/AEM.00886-18.
• Lu Y, Wang Y, Wang J, et al. Phage endolysin LysP108 showed promising antibacterial potential against methicillin-resistant Staphylococcus aureus. Front Cell Infect Microbiol. 2021; 11:668430. DOI: 10.3389/fcimb.2021.668430.
• Gutiérrez D, Fernández L, Rodríguez A, García P. Are phage lytic proteins the secret weapon to kill Staphylococcus aureus? mBio. 2018; 9(1): e01923-17. DOI: 10.1128/mBio.01923-17.
• Lee C, Kim H, Ryu S. Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends. Crit Rev Food Sci Nutr. 2022; 9: 1-20. DOI: 10.1080/10408398.2022.2059442.
• Hoopes JT, Stark CJ, Kim HA, Sussman DJ, Donovan DM, Nelson DC. Use of a bacteriophage lysin, PlyC, as an enzyme disinfectant against Streptococcus equi. Appl Environ Microbiol. 2009; 75(5): 1388-94. DOI: 10.1128/AEM.02195-08.
• Balaban CL, Suárez CA, Boncompain CA, Peressutti-Bacci N, Ceccarelli EA, Morbidoni HR. Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli. Microb Cell Fact. 2022; 21(1): 40. DOI: 10.1186/s12934-022-01766-9.
• Olsen NMC, Thiran E, Hasler T, et al. Synergistic removal of static and dynamic Staphylococcus aureus biofilms by combined treatment with a bacteriophage endolysin and a polysaccharide depolymerase. Viruses. 2018; 110(8): 438. DOI: 10.3390/v10080438.
• Álvarez A, Fernández L, Gutiérrez D, Iglesias B, Rodríguez A, García P. Methicillin-resistant Staphylococcus aureus in hospitals: Latest trends and treatments based on bacteriophages. J Clin Microbiol. 2019; 57(12): e01006-19. DOI: 10.1128/JCM.01006-19.
• Ghose C, Euler CW. Gram-negative bacterial lysins. Antibiotics (Basel). 2020; 9(2): 74. DOI: 10.3390/antibiotics9020074.
• Murray E, Draper LA, Ross RP, Hill C. The advantages and challenges of using endolysins in a clinical setting. viruses. 2021;13(4):680. DOI: 10.3390/v13040680.
• Rahman MU, Wang W, Sun Q, et al. Endolysin, a promising solution against antimicrobial resistance. Antibiotics (Basel). 2021; 10(11): 1277. DOI: 10.3390/antibiotics10111277.
• Danis-Wlodarczyk KM, Wozniak DJ, Abedon ST. Treating bacterial infections with bacteriophage-based enzybiotics: In vitro, in vivo and clinical application. Antibiotics (Basel). 2021; 10(12): 1497. DOI: 10.3390/antibiotics10121497.
• Keary R, McAuliffe O, Ross RP, Hill C, O'Mahony J, Coffey A. Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase. Bacteriophage. 2014; 4: e28451. DOI: 10.4161/bact.28451.