PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection

Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection

Deciphering Physiological Functions of AHL Quorum Quenching Acylases

N-Acylhomoserine lactone (AHL)-acylase (also known as amidase or amidohydrolase) is a class of enzyme that belongs to the Ntn-hydrolase superfamily. As the name implies, AHL-acylases are capable of hydrolysing AHLs, the most studied signaling molecules for quorum sensing in Gram-negative bacteria. Enzymatic degradation of AHLs can be beneficial in attenuating bacterial virulence, which can be exploited as a novel approach to fight infection of human pathogens, phytopathogens or aquaculture-related contaminations. Numerous acylases from both prokaryotic and eukaryotic sources have been characterized and tested for the interference of quorum sensing-regulated functions. The existence of AHL-acylases in a multitude of organisms from various ecological niches, raises the question of what the physiological roles of AHL-acylases actually are. In this review, we attempt to bring together recent studies to extend our understanding of the biological functions of these enzymes in nature

Image_3_PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection.PDF

<p>Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection.</p

Aktivitas Kuersetin sebagai Antihipertensi secara In Silico

Hipertensi merupakan salah satu penyakit tidak menular yang memiliki prevalensi tinggi. Hipertensi disebabkan oleh terbentuknya Angiotensin II dari proses konversi dekapeptida inaktif Angiotensin I oleh angiotensin converting enzyme (ACE) yang akan menyebabkan penyempitan pembuluh darah. Selain itu, hipertensi juga dikaitkan dengan meningkatnya pembentukan reactive oxygen species (ROS). Pengembangan senyawa obat sebagai antihipertensi diperlukan untuk meningkatkan keberhasilan terapi yang belum adekuat. Sebagai salah satu senyawa bahan alam, kuersetin dipilih karena memiliki aktivitas sebagai ACE inhibitor yang diketahui dengan melakukan uji in silico terhadap induksi ACE. Analisis data dilakukan dengan melihat energi ikatan yang dihasilkan dan ikatan yang terbentuk antara senyawa dengan residu asam amino pada protein. Hasil penelitian ini menunjukkan bahwa senyawa kuersetin berpotensi mengatasi hipertensi. Validasi metode menunjukkan konformasi 7 memiliki nilai RMSD yaitu 2,86 Å dan energi ikatan antara protein target (ACE) dengan native ligan-nya yaitu -4.66 kkal/mol. Sedangkan nilai energi ikatan yang diperoleh antara senyawa uji kuersetin dan protein target yaitu -6,32 kkal/mol dimana ikatan hidrogen yang terbentuk menghasilkan residu asam amino ALA356, HIS383, ALA356, TYR523, dan GLU411 berturut-turut melalui gugus O-H, HE2-O, HN-O, HH-O, dan OE1-H. Nilai energi ikatan yang didapat menunjukkan bahwa senyawa uji kuersetin memiliki aktivitas farmakologi sebagai sebagai ACE inhibitor karena energi ikatannya lebih negatif dibandingkan dengan native ligand protein target yaitu lisinopril. Hal ini menunjukkan bahwa kuersetin dapat membentuk ikatan yang lebih stabil dibandingkan dengan lisinopril. Sehingga kuersetin berpotensi dalam pengembangan terapi hipertensi yang berperan sebagai ACE inhibitor

Image_1_PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection.PDF

<p>Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection.</p

Image_5_PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection.pdf

<p>Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection.</p