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Title:	Screening and characterization of anti-infective agents against Staphylococcus aureus using a Caenorhabditis elegans infection model
Authors:	Kong Cin (P54533)
Supervisor:	Sheila Nathan, Prof. Dr.
Keywords:	Staphylococcus aureus Anti-infective Caenorhabditis elegans Dissertations, Academic -- Malaysia
Issue Date:	16-Jul-2014
Description:	With growing concerns over the spread of antibiotic-resistant Staphylococcus aureus strains, the identification of alternative therapeutic molecules has become paramount. One approach to achieve the identification of novel therapeutic molecules is through the discovery of anti-infective agents from natural and synthetic products. In this study, a Caenorhabditis elegans – S. aureus screening platform was developed and used to perform in vivo screens of a collection of natural extracts and synthetic compounds for potential anti-infective properties. Initially, the standard agar-based screening assay was modified into a liquid-based assay to ensure uniform exposure of the infection model to the selected extracts and compounds. A total of 37 extracts and 29 compounds were screened through the optimized screen of which 14 extracts and 14 compounds promoted the survival of S. aureus-infected nematodes by at least 2.8-fold relative to the untreated control. To delineate anti-bacterials from anti-infectives,antimicrobial tests were performed on the 14 extracts and 14 compounds. Seven extracts and one compound did not confer anti-bacterial effects on S. aureus proposing that the S. aureus-infected nematodes were rescued through a means that did not involve death of the pathogen. Of these eight candidates, Orthosiphon stamineus extract (UE-12) significantly reduced intestinal bacterial loads over the course of infection. Furthermore, as noted through observations of a fluorescent transgenic reporter strain infected by S. aureus and treated with UE-12, the repressed expression of the lys-7 defense gene in infected nematodes was restored in the presence of UE-12. In additon, when UE-12 was added to loss-of-function C. elegans mutants, the anti-infective effect was completely impaired in the p38 mitogenactivated protein kinase (MAPK) pathway mutants sek-1 and pmk-1 whilst partially attenuated in the insulin-like signaling pathway daf-16 mutant. This suggests the protective role of UE-12 is mediated via the p38 MAPK and insulin-like signaling pathways. Transcriptome analysis of a selected panel of PMK-1 and DAF-16-regulated genes by quantitative real-time polymerase chain reaction confirmed a significant modulation of these genes in S. aureus-infected nematodes exposed to UE-12. Further analysis on a panel of known bioactive compounds of UE-12 revealed that eupatorin (C18H16O7) is the active molecule contributing to the anti-infective property of UE-12. Taken together, these findings strongly suggest that UE-12 is a promising anti-infective agent that confers a survival advantage against S. aureus infection via modulation of the infected host immune response,Memandangkan kebimbangan masalah penularan strain Staphylococcus aureus yang rintang terhadap antibiotik semakin ketara, pengenalpastian agen terapeutik alternatif telah menjadi amat penting. Salah satu pendekatan untuk mengenalpasti molekul terapeutik baru adalah melalui penerokaan agen anti-infektif daripada sebatian semula jadi dan sintetik. Dalam kajian ini, pletform penyaringan Caenorhabditis elegans-S. Aureus telah dibangunkan dan digunakan dalam penyaringan in vivo koleksi ekstrak semula jadi dan sebatian sintetik untuk mengenalpasti molekul dengan ciri antiinfektif yang berpotensi. Pada mulanya, asai penyaringan piawai bersandar-agar telah diubahsuai kepada asai dalam bentuk cecair untuk memastikan pendedahan sekata ekstrak dan sebatian terpilih kepada model infeksi. Sebanyak 37 ekstrak dan 29 sebatian telah disaring menggunakan asai penyaringan yang telah dioptimumkan dan 14 ekstrak serta 14 sebatian telah meningkatkan kemandirian nematod terinfeksi S.aureus dengan sekurang-kurangnya 2.8 kali ganda berbanding dengan kumpulan kawalan yang tidak dirawat. Dalam usaha mengasingkan agen anti-bakteria daripada agen anti-infektif, ujian anti-mikrob telah dijalankan ke atas 14 ekstrak dan 14 sebatian tersebut. Didapati tujuh ekstrak dan satu sebatian tidak mempamerkan kesan anti-bakteria terhadap S. aureus dan ini mencadangkan nematod terinfeksi S. aureus diselamatkan melalui kaedah yang tidak melibatkan kematian patogen. Antara lapan calon ini, ekstrak Orthosiphon stamineus (UE-12) berjaya mengurangkan beban bakteria dalam usus C. elegans secara signifikan sepanjang proses infeksi. Tambahan pula, melalui pemerhatian ke atas strain pelapor transgen berpendafluor yang diinfeksi S. aureus dan dirawat dengan UE-12, tahap pengekspresan gen pertahanan lys-7 yang ditindas dalam nematod terinfeksi didapati dikembalikan semula dalam kehadiran UE- 12. Apabila UE-12 didedahkan kepada mutan hilang fungsi C. elegans, didapati kesan anti-infektif ditindas sepenuhnya dalam mutan tapak jalan protein kinase p38 bersandarkan mitogen (MAPK) sek-1 dan pmk-1, manakala dalam mutan tapak jalan pengisyaratan mirip insulin daf-16, kesan UE-12 juga telah dilemahkan. Ini mencadangkan peranan perlindungan UE-12 adalah melalui tapak jalan p38 MAPK dan pengisyaratan mirip insulin. Analisis transkriptom secara tindak balas berantai polimerase kuantitatif masa nyata ke atas panel gen terpilih yang dikawal oleh PMK-1 dan DAF-16 juga mengesahkan modulasi gen yang ketara dalam nematod terinfeksi S. aureus yang dirawat UE-12. Analisis lanjutan ke atas panel sebatian bioaktif yang diketahui hadir dalam UE-12 telah mendedahkan bahawa eupatorin (C18H16O7) merupakan molekul aktif yang menyumbang kepada aktiviti anti-infektif UE-12. Secara keseluruhan, hasil kajian ini mencadangkan bahawa UE-12 merupakan agen anti-infektif yang amat berpotensi yang dapat memberikan manfaat kemandirian terhadap infeksi S. aureus melalui modulasi gerak balas imun perumah yang diinfeksi.,Bachelor
Pages:	160
Call Number:	QR201.S68K644 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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