Bir antimikrobiyal ajan olarak paraoksonaz1 (laktonaz) enziminin pseudomonas aerugınosa quorum sensıng ilişkili davranışlarına etkisinin incelenmesi
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Date
2014
Authors
Aybey, Aynur
Journal Title
Journal ISSN
Volume Title
Publisher
Uludağ Üniversitesi
Abstract
Pseudomonas aeruginosa'da birçok virulans faktörü üretiminin düzenlenmesinde Quorum sensing (QS)'in rolü gösterilmiştir. P. aeruginosa'da yüksek antibiyotik direnci yeni tedavi seçenekleri arayışına yol açmış ve QS sisteminin inhibisyonu mercek altına alınmıştır. Bakterinin virulansında azalmaya yol açacak QS inhibitörü ajanların bulunması ile P. aeruginosa infeksiyonları tedavisinde yeni yaklaşımlar geliştirilebilir. Bu amaçla çalışmada, P. aeruginosa'da QS sinyal moleküllerini laktonaz aktivitesi ile hidroliz eden insan serum paraoksonaz 1 (hPON1) enzimi kullanılmıştır. hPON1 enzimi, amonyum sülfat çöktürmesi ve hidrofobik etkileşim kromatografisi (Sepharose 4B-L-tirozin-1-Naftilamin) yöntemleri kullanılarak saflaştırılmıştır. Saf enzim, SDS-poliakrilamid jel elektroforezi ile 43 kDa olan tek bir bant olarak saptanmıştır. 3 adet P. aeruginosa suşları büyüme eğrisi için değerlendirilmiş ve durağan faz süreleri tespit edilmiştir. Durağan fazdaki bakteri örnekleri hPON1 enziminin artan konsantrasyonu katılarak (0,1-10 mg/ml) üretilmiştir. Bu suşlar arasında, P. aeruginosa ATCC35032 üremesinin 2,5 mg/ml konsantrasyondan itibaren azaldığı bulunmuştur ve çalışmalara bu suş ile devam edilmiştir. Çalışmada, her bir davranış için farklı konsantrasyonlarda kullanılan hPON1 enziminin bakterinin virulans faktörlerini, hareketliliğini ve biyofilm oluşumunu azalttığı bulunmuştur. Ancak, hPON1 enzimi (0,1-10 mg/ml) virulans faktörlerinden elastaz ve LasA proteaz üzerine azaltıcı yönde bir etki göstermemiş, alkali proteaza karşı ise 0,1 mg/ml gibi düşük konsantrasyonda azaltıcı etkisi saptanmıştır. 0,3-5 mg/ml konsantrasyonları arasında piyosiyanin ve ramnolipid üretimlerinin 1,25 mg/ml'de önemli ölçüde azalmıştır. hPON1 enzimi (0,1-10 mg/ml), biyofilm oluşumu ve olgun biyofilmleri de 1 mg/ml'de %50 oranında azaltmıştır. Olgun biyofilmlerin EPS bileşenini yıkıma uğratmıştır. Enzimin 0,003-30 mg/ml konsantrasyonları arasında kayma, yüzme ve titreme hareketlerine azaltıcı etkisi düşük konsantrasyon olan 0,3 mg/ml'de oldukça yüksek olmuştur.
The role of Quorum sensing (QS) on the regulation of many virulence factor production in Pseudomonas aeruginosa has been shown. High antibiotic resistance in P. aeruginosa has led to the search for new treatment options and inhibition of QS system is scrutinized. By the presence of QS inhibitory agents of lead to a reduction in bacterial virulence, new approaches in the treatment of P. aeruginosa infections can be improved. For this purpose, human serum paraoksonaz1 (hPON1) which is hydrolized signal molecules of P. aeruginosa by using lactonase activity was used. hPON1 was purified by using ammonium sulfate precipitation and hydrophobic interaction chromatography (Sepharose 4B-L-tyrosine-1-Naphthylamine). Purified enzyme was determined as a single band with 43 kDa by SDS-polyacyrilamide gel electrophoresis. 3 P. aeruginosa strains were evaluated for growth curve and stationary phase time was determined. Samples of bacteria in stationary phase by addition of increasing concentrations of hPON1 (0,1-10 mg/ml) were produced. Among strains, growth reduction of P. aeruginosa ATCC35032 in 2,5 mg/ml concentration was found, and studies were continued with these strain. In this study, hPON1 enzyme using different concentrations for each behavior have been found to reduce the virulence factors, motility and biofilm formation. However, hPON1 enzyme (0,1-10 mg/ml) on elastase and LasA protease did not show an effect on the decrease, decreasing effect to against alkaline protease in the lowest concentration as 0,1 mg/ml was determined. It was significantly decreased production of pyocyanin and rhamnolipid in 1,25 mg/ml of hPON1 between 0,3-5 mg/ml concentrations. Biofilm formation and mature biofilm were decreased % 50 by 1mg/ml hPON1 concentration (0,1-10 mg/ml). EPS component on mature biofilm was degraded. Decreasing effect of hPON1 between 0,003-30 mg/ml on swarming, swimming and twitching motility was quite high even in 0,3 mg/ml concentration.
The role of Quorum sensing (QS) on the regulation of many virulence factor production in Pseudomonas aeruginosa has been shown. High antibiotic resistance in P. aeruginosa has led to the search for new treatment options and inhibition of QS system is scrutinized. By the presence of QS inhibitory agents of lead to a reduction in bacterial virulence, new approaches in the treatment of P. aeruginosa infections can be improved. For this purpose, human serum paraoksonaz1 (hPON1) which is hydrolized signal molecules of P. aeruginosa by using lactonase activity was used. hPON1 was purified by using ammonium sulfate precipitation and hydrophobic interaction chromatography (Sepharose 4B-L-tyrosine-1-Naphthylamine). Purified enzyme was determined as a single band with 43 kDa by SDS-polyacyrilamide gel electrophoresis. 3 P. aeruginosa strains were evaluated for growth curve and stationary phase time was determined. Samples of bacteria in stationary phase by addition of increasing concentrations of hPON1 (0,1-10 mg/ml) were produced. Among strains, growth reduction of P. aeruginosa ATCC35032 in 2,5 mg/ml concentration was found, and studies were continued with these strain. In this study, hPON1 enzyme using different concentrations for each behavior have been found to reduce the virulence factors, motility and biofilm formation. However, hPON1 enzyme (0,1-10 mg/ml) on elastase and LasA protease did not show an effect on the decrease, decreasing effect to against alkaline protease in the lowest concentration as 0,1 mg/ml was determined. It was significantly decreased production of pyocyanin and rhamnolipid in 1,25 mg/ml of hPON1 between 0,3-5 mg/ml concentrations. Biofilm formation and mature biofilm were decreased % 50 by 1mg/ml hPON1 concentration (0,1-10 mg/ml). EPS component on mature biofilm was degraded. Decreasing effect of hPON1 between 0,003-30 mg/ml on swarming, swimming and twitching motility was quite high even in 0,3 mg/ml concentration.
Description
Keywords
Biyofilm, Paraoksonaz1, Quorum sensing, Virulans faktörleri, Biofilm, Paraoxonase1, Pseudomonas aeruginosa, Virulence factors
Citation
Aybey, A. (2014). Bir antimikrobiyal ajan olarak paraoksonaz1 (laktonaz) enziminin pseudomonas aerugınosa quorum sensıng ilişkili davranışlarına etkisinin incelenmesi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.