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Title:	Protein hipotetikal BPSL1375 Burkholderia pseudomallei adalah protein yang mempamerkan hemolisis
Authors:	Laziana Ahmad (P55950)
Supervisor:	M. Firdaus Raih, Dr.
Keywords:	Burkholderia pseudomallei Virulence (Microbiology)
Issue Date:	18-Jun-2013
Description:	Burkholderia pseudomallei adalah bakteria patogen intrasel yang menyebabkan penyakit maut, melioidosis. Beberapa faktor virulen yang menyumbang kepada patogenesis penyakit ini telah dicadangkan termasuklah protease, hemolisin dan toksin. Kajian ini dijalankan untuk mengenal pasti residu yang memainkan peranan penting dalam aktiviti protein BPSL1375 dan mengkaji potensi aktiviti BPSL1375 yang lain dengan mendalam. Analisis jujukan nukleotida bpsl1375 menunjukkan perkongsian domain yang dimiliki oleh protein hemolisin putatif. Manakala pencarian homologi mempamerkan padanan dengan ahli superfamili N-asiltransferase, protein AHL synthase. Pencirian awal ke atas BPSL1375 menunjukkan bahawa BPSL1375 berupaya melakukan aktiviti hemolisis. Tujuh residu (Arg54, Phe58, Asp75, Asp78, Arg99, Glu132 dan Arg135) telah terpelihara di dalam analisis penjajaran jujukan berganda antara BPSL1375 dan jujukan AHL synthase. Kesemua residu kecuali Phe58 telah berjaya dimutasi menerusi mutagenesis terarah tapak. Protein rekombinan jenis liar dan protein rekombinan mutan telah diekspres di dalam Escherichia coli BL21 (DE3) dan ditulen melalui kromatografi afiniti. Protein rekombinan mutan R135K dan R99K menunjukkan kehilangan aktiviti selepas satu jam pengeraman pada suhu 37°C mencadangkan bahawa kedua residu ini mempunyai peranan penting dalam aktiviti hemolisis BPSL1375. Protein rekombinan mutan yang lain (R54K, D75E dan E132D) mengalami pengurangan aktiviti hemolisis kepada 14%, 18% dan 22% berbanding 27% yang telah direkod oleh protein rekombinan jenis liar BPSL1375. Protein rekombinan mutan D78E menunjukkan peningkatan aktiviti hemolisis kepada 61% hemolisis, peningkatan sebanyak 2.3 kali berbanding jenis liar BPSL1375. Walaupun mempunyai peratusan hemolisis yang tinggi, kajian kemandirian mencit BALB/c menunjukkan bahawa protein rekombinan jenis liar BPSL1375 dan protein rekombinan mutan D78E tidak menyebabkan kematian kepada mencit. Selain itu, walaupun BPSL1375 mempunyai persamaan dengan AHL synthase, analisis spektrometri jisim tidak mengesan molekul AHL dan seterusnya memberi petunjuk bahawa BPSL1375 tidak terlibat dalam sintesis AHL. Analisis transkripsi berbalik transkrip bps1375 di dalam B. pseudomallei yang dikultur secara in vitro pada persekitaran tertekan menunjukkan bahawa mRNA bpsl1375 hanya ditranskripsi dalam medium NaCl 500 mM dan media M9. Ini menunjukkan bahawa produk gen ini mungkin berperanan dalam adaptasi kepada persekitaran kekurangan nutrien.,Burkholderia pseudomallei is an intracellular bacterial pathogen that causes the fatal disease, melioidosis. A number of virulence factors have been proposed including proteases, hemolysin and toxin. This study was carried out to identify important residues in BPSL1375's activity and to further investigate other potential activity. Sequence analysis of bpsl1375 showed a shared domain with putative hemolysins. Meanwhile, homology search demonstrated hits with N-acyltransferase superfamily members, the AHL synthase. Preliminary characterization of BPSL1375 revealed the protein's ability to cause hemolysis. Seven residues (R54, F58, D75, D78, R99, E132 and R135) were conserved in a diverse set of AHL synthase sequences to BPSL1375. All residues, except for F58, were successfully mutated using site-directed mutagenesis. The recombinant wild type protein and recombinant mutant proteins were expressed in Escherichia coli BL21 (DE3) and purified via affinity chromatography. Recombinant mutant R135K and R99K showed a complete loss of activity after 1 hour incubation at 37°C suggesting that these two residues are important for BPSL1375 hemolysis activity. Other recombinant mutant proteins (R54K, D75E and E132D) reduced hemolytic activity to 14%, 18% and 22% respectively compared to 27% recorded by the recombinant wild type protein BPSL1375. The recombinant mutant D78E resulted in increased hemolytic activity of up to 61% hemolysis, a 2.3-fold increase compared to the recombinant wild type protein BPSL1375. Despite the high percentage of hemolysis, the recombinant wild type BPSL1375 and recombinant mutant D78E were unable to cause mortality to BALB/c mice. The protein also did involved in the synthesis of AHL molecules despite the similarity to AHL synthase as no AHL molecule was detectable by mass spectrometry. Reverse transcription analysis of the bpsl1375 transcript in B. pseudomallei grown in vitro under stressed conditions revealed that the mRNA was only transcribed when grown in NaCl 500 mM and M9 medium, revealing that this protein may play a role in adapting to an environment lacking in rich nutrient.,Master/Sarjana
Pages:	116
Call Number:	QR175.L349 2013 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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