Pseudomonas aeruginosa PAO1’de QS ilişkili genlerin ekspresyon seviyeleri üzerine Lactobacillus sp. metabolitlerinin anti-quorum sensing etkilerinin belirlenmesi

Yıl 2020, Cilt: 77 Sayı: 3, 311 - 318, 01.09.2020

Didem Kart Suna Sibel Gürpınar Müjde Eryılmaz

Öz

Amaç: Pseudomonas aeruginosa hastane enfeksiyonları ile ilişkili önemli bir patojen olup patojenitesi çoğunlukla quorum sensing QS sistemi ile ilişkilidir. Bu çalışmanın amacı, vajinal Lactobacillus izolatlarının metabolitlerinin anti-QS aktivitelerinin değerlendirilmesi ve metabolitlerin P. aeruginosa PAO1’in QS ilişkili genlerinin transkripsiyonel regülasyonu üzerindeki etkilerinin araştırılmasıdır.Yöntem: Çalışmamızda daha önce 16S rRNA gen dizi analizi ile tanımlanmış olan 13 adet Lactobacillus izolatı kullanılmıştır. Bu izolatların metabolitlerinin, Chromobacterium violaceum CV12472 suşu kullanılarak anti-QS aktiviteleri değerlendirilmiştir. Metabolitlerin QS ile ilişkili genlerin ekspresyonları üzerindeki etkisi kantitatif revers transkriptaz polimeraz zincir reaksiyonu RT-qPZR ile araştırılmıştır. Bulgular: Test edilen tüm metabolitler C. violaceum’un şeffaf zon bölgesi görünümü ile karakterize edilen anti-QS aktivite göstermişlerdir. Metabolitlerle temas sonrasında P. aeruginosa PAO1’de, test edilen tüm quorum sensing ilişkili genler lasI, lasR, rhlR ve mvfR anlamlı bir down-regülasyon göstermişlerdir. Sonuç: Antibakteriyel direncin artması nedeniyle farklı etki mekanizmasına sahip doğal kaynaklardan köken alan, enfeksiyonlara karşı yeni ajanlar gerekmektedir. Çalışmamız, in vitro Pseudomonas biyofilmlerinin kontrolünde Lactobacillus metabolitlerinin olası anti-QS ajanı olarak kullanılabileceklerini vurgulamaktadır

Anahtar Kelimeler

anti-quorum sensing aktivitesi, Lactobacillus sp., metabolitler, RT-qPCR, Pseudomonas aeruginosa

Assessment of the anti-quorum sensing effect of Lactobacillus sp. metabolites on expression levels of QS-related genes in Pseudomonas aeruginosa PAO1

Öz

Objective: Pseudomonas aeruginosa is an important pathogen associated with nosocomial infections and its pathogenicity is mostly linked with the quorum sensing QS system. The aim of this study was to evaluate the anti-QS activity of the metabolites of vaginal Lactobacillus isolates and to investigate the effect of these metabolites on transcriptional regulation of QS related genes in P. aeruginosa PAO1.Methods: In this study, 13 Lactobacillus isolates that were previously identified by 16S rRNA gene sequence analysis were used. Metabolites of these isolates were assessed for the anti-QS activity by using Chromobacterium violaceum CV12472. The influence of metabolites on the expression of QS related genes was also examined by reverse transcription-quantitative polymerase chain reaction RT-qPCR .Results: All tested metabolites exhibited anti-QS activity with the appearance of a non-pigmented zone of C. violaceum. All tested quorum sensing-related genes lasI, lasR, rhlR and mvfR in P. aeruginosa PAO1 showed significant down-regulation after treating with the metabolites Conclusion: New anti-infection agents from natural resources with a different mode of action are necessary due to the increasing occurrence of antibacterial resistance. Our study highlights the possible usage of Lactobacillus metabolites as an anti-QS agent against P. aeruginosa biofilm cells in vitro.

Anahtar Kelimeler

anti-quorum sensing activity, Lactobacillus sp., metabolites, RT-qPCR. Pseudomonas aeruginosa

Kaynakça

• 1. Gomez MI, Prince A. Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis. Curr Opin Pharmacol, 2007; 7(3): 244-51. doi:10.1016/j.coph.2006.12.005. 2007.
• 2. Fuqua WC, Winans SC, Greenberg EP. Quorum Sensing in Bacteria - the Luxr-Luxi Family of Cell Density-Responsive Transcriptional Regulators. J Bacteriol, 1994; 176(2): 269-75.
• 3. Rajkumari J, Borkotoky S, Reddy D, Mohanty SK, Kumavath R, Murali A, et al. Anti-quorum sensing and anti-biofilm activity of 5-hydroxymethylfurfural against Pseudomonas aeruginosa PAO1: Insights from in vitro, in vivo and in silico studies. Microbiol Res, 2019; 226: 19-26. doi:10.1016/j. micres.2019.05.001.
• 4. Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, et al. Structure of the Autoinducer Required for Expression of Pseudomonas-Aeruginosa Virulence Genes. P Natl Acad Sci USA, 1994; 91(1): 197-201. doi: 10.1073/ pnas.91.1.197.
• 5. Sankar Ganesh P, Ravishankar Rai V. Attenuation of quorum-sensing-dependent virulence factors and biofilm formation by medicinal plants against antibiotic resistant Pseudomonas aeruginosa. J Tradit Complement Med, 2018; 8(1): 170-7. doi:10.1016/j.jtcme.2017.05.008.
• 6. Pearson JP, Passador L, Iglewski BH, Greenberg EP. A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Natl Acad Sci USA, 1995; 92(5): 1490-4. doi:10.1073/pnas.92.5.1490.
• 7. Pearson JP, Pesci EC, Iglewski BH. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol, 1997; 179(18): 5756-67. doi:10.1128/jb.179.18.5756-5767.1997.
• 8. Sawa T, Ohara M, Kurahashi K, Twining SS, Frank DW, Doroques DB, et al. In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections. Infect Immun, 1998; 66(7): 3242-9.
• 9. Shukla V, Bhathena Z. Broad Spectrum Anti-Quorum Sensing Activity of Tannin-Rich Crude Extracts of Indian Medicinal Plants. Scientifica, 2016. doi:582 301310.1155/2016/5823013.
• 10. Davoodabadi A, Dallal MMS, Lashani E, Ebrahimi MT. Antimicrobial Activity of Lactobacillus spp. Isolated From Fecal Flora of Healthy Breast-Fed Infants Against Diarrheagenic Escherichia coli. Jundishapur J Microb, 2015; 8(12). doi: e2785210.5812/ jjm.27852.
• 11. Eryilmaz M, Gurpinar SS, Palabiyik IM, Guriz H, Gerceker D. Molecular Identification and Antimicrobial Activity of Vaginal Lactobacillus sp. Curr Pharm Biotechno, 2018; 19(15): 1241-7. doi:10 .2174/1389201020666190110164123.
• 12. Eryılmaz M, Kart D, Gürpınar SS. Investigation of Antibiofilm Activities of Lactobacillus sp. Metabolites Isolated from Vaginal Flora. Journal of Turkish Society of Microbiology, 2019; 49(3): 169- 74.
• 13. Melo TA, dos Santos TF, de Almeida ME, Fontes LAG, Andrade EF, Rezende RP, et al. Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa. BMC Microbiol, 2016. doi:25010.1186/s12866-016-0871-8.
• 14. Shokri D, Khorasgani MR, Mohkam M, Fatemi SM, Ghasemi Y, Taheri-Kafrani A. The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of Pseudomonas aeruginosa. Probiotics Antimicro, 2018; 10(1): 34-42. doi:10.1007/s12602-017-9267- 9.
• 15. Stoyancheva G, Marzotto M, Dellaglio F, Torriani S. Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains. Arch Microbiol, 2014; 196: 645-53.
• 16. Kart D, Tavernier S, Van Acker H, Nelis HJ, Coenye T. Activity of disinfectants against multispecies biofilms formed by Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa. Biofouling, 2014; 30(3): 377-83. doi:10.1080/08927014.2013.8 78333.
• 17. Qu L, She PF, Wang YX, Liu FX, Zhang D, Chen LH, et al. Effects of norspermidine on Pseudomonas aeruginosa biofilm formation and eradication. Microbiologyopen, 2016; 5(3): 402-12. doi:10.1002/ mbo3.338.
• 18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods.,2001; 25(4): 402-8. doi:10.1006/meth.2001.1262.
• 19. Sharma G, Rao S, Bansal A, Dang S, Gupta S, Gabrani R. Pseudomonas aeruginosa biofilm: Potential therapeutic targets. Biologicals, 2014; 42(1): 1-7. doi:10.1016/j.biologicals.2013.11.001.
• 20. Brackman G, Hillaert U, Van Calenbergh S, Nelis HJ, Coenye T. Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia. Res Microbiol, 2009; 160(2): 144-51. doi:10.1016/j.resmic.2008.12.003.
• 21. Musk DJ, Hergenrother PJ. Chemical countermeasures for the control of bacterial biofilms: Effective compounds and promising targets. Curr Med Chem, 2006; 13(18): 2163-77. doi:10.2174/092986706777935212.
• 22. Abraham SVPI, Palani A, Ramaswamy BR, Shunmugiah KP, Arumugam VR. Antiquorum Sensing and Antibiofilm Potential of Capparis spinosa. Arch Med Res, 2011; 42(8): 658-8. doi:10.1016/j. arcmed.2011.12.002.
• 23. Khiralla GM, Mohamed EAH, Farag AG, Elhariry H. Antibiofilm effect of Lactobacillus pentosus and Lactobacillus plantarum cell-free supernatants against some bacterial pathogens. J Biotech Res, 2015; 6: 86-95.
• 24. Ganchev I. Antibiofilm activity of Lactobacillus strains. Sci J Chem, 2018; 6(5):77-82. doi. org:10.11648/j.sjc.20180605.11.
• 25. Hentzer M, Riedel K, Rasmussen TB, Heydorn A, Andersen JB, Parsek MR et al. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. MicrobiolSgm, 2002; 148: 87-102. doi:10.1099/00221287- 148-1-87.
• 26. Soheili V, Tajani AS, Ghodsi R, Bazzaz BSF. AntiPqsR compounds as next-generation antibacterial agents against Pseudomonas aeruginosa: A review. Eur J Med Chem, 2019; 172: 26-35. doi:10.1016/j. ejmech.2019.03.049.
• 27. Li JP, Yang XY, Shi GC, Chang J, Liu ZY, Zeng MY. Cooperation of lactic acid bacteria regulated by the AI-2/LuxS system involve in the biopreservation of refrigerated shrimp. Food Res Int, 2019; 120:679- 87. doi:10.1016/j.foodres.2018.11.025.