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Title:	Caenorhabditis elegans ugt-29: GFP as a biosensor to identify putative toxic molecules of Burkholderia pseudomallei.
Authors:	Wong Rui Rui (P58604)
Supervisor:	Sheila Nathan, Prof. Dr.
Keywords:	Caenorhabditis elegans Burkholderia pseudomallei Biosensor Virulence factor Dissertations, Academic -- Malaysia
Issue Date:	5-Jul-2016
Description:	The interaction between pathogenic microbes and their hosts forms the underlying basis of host-microbe interaction studies to identify bacterial virulence mechanisms and host defense responses. In this study, the nematode Caenorhabditis elegans was employed as the model host to identify potential virulence factors involved in the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis. A previous study had demonstrated that expression of the C. elegans phase II detoxification enzyme UGT-29 (UDP-glucuronosyltransferase family member) gene was significantly induced in B. pseudomallei-infected worms. This robust induction of ugt-29 reinforced the possibility of toxin-mediated killing of worms by B. pseudomallei. To facilitate the search for this toxin, a ugt-29::GFP transcriptional nematode biosensor was developed as a tool to visualize the ugt-29 gene response. The induction of ugt-29 was found to be pathogen-specific and the magnitude of gene induction positively correlated with the degree of pathogen virulence. RNAi-based gene inactivation that resulted in compromised host protein translation also triggered the expression of ugt-29 in the absence of infection. Thus, induction of ugt-29 expression is most likely a response to the toxin-mediated damage of host cells. The UGT-29 protein is involved downstream of the ZIP-2 immune pathway which is also induced in response to B. pseudomallei infection. These findings support the use of the ugt-29::GFP construct as a biosensor to facilitate the search for vital virulence attributes in B. pseudomallei. The nematode biosensor was used to determine the identity of the bacterial factors responsible for inducing ugt-29 in worms. Exposing the worms to B. pseudomallei diffusible products alone could elicit ugt-29 induction, implying that the factors of interest are secreted extracellularly. By means of membrane ultrafiltration and proteinase K digestion, the factors were determined to be non-proteinaceous compounds of less than 3kDa, further proposing that the factors are most likely metabolites. A liquid chromatography-mass spectrometry (LC-MS) based metabolite identification performed on the B. pseudomallei secreted products identified bactobolin as a potential toxin. This was validated by the observation that ugt-29 was robustly induced in the presence of synthetic bactobolin and that C. elegans exposed to bactobolin had a shortened lifespan compared to untreated worms. In summary, the ugt-29::GFP reporter is a reliable biosensor to screen for virulence determinants of B. pseudomallei. Application of this tool has led to the identification of bactobolin as a potential B. pseudomallei virulence factor. As ugt-29 is expressed in response to toxin-mediated host damage, it is anticipated that more novel toxic bacterial molecules can be identified to aid in understanding the pathogenesis of B. pseudomallei.,Certification of Master's/Doctoral Thesis" is not available
Pages:	140
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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