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Title:	Characterisation of potential caenorhabditis elegans antimicrobial peptides towards Burkholderia pseudomallei
Authors:	Lim Mei Perng (P54534)
Supervisor:	Sheila Nathan, Prof. Dr.
Keywords:	Burkholderia pseudomallei Caenorhabditis elegans Antimicrobial peptides
Issue Date:	2-Feb-2016
Description:	The acquisiton of antibiotic resistance by Burkholderia pseudomallei is now a global problem. An alternative to conventional antibiotics is antimicrobial peptides which present a lower likelihood that a pathogen will develop resistance. In this study, a subset of candidate Caenorhabditis elegans genes that fulfill the following criteria: (i) up-regulated during an infection by B. pseudomallei, (ii) predicted to encode a peptide of less than 150 amino acids, and (iii) predicted to contain a signal peptide, were identified from a previously obtained expression profile of B. pseudomallei-infected nematodes. To confirm if these potential antimicrobial peptides confer protection against B. pseudomallei infection, 34 genes that fulfill the above criteria were individually abrogated in worms by RNAi feeding. Knock-down of 12/34 genes resulted in an enhanced susceptibility to pathogen (Esp) phenotype of C. elegans relative to the control. Lifespan analysis further confirmed that the Esp phenotype is mediated by infection rather than a consequence of loss of worm fitness. The selected peptides were chemically synthesized and assayed for potential antimicrobial properties using the microdilution test. Of these 12 peptides, NLP-31 and Y43C5A.3 were found to exhibit anti-B. pseudomallei activity in a dose-dependent manner with MIC90 values of 256 μM and 512 μM, respectively. Furthermore, both peptides proved to act as broad spectrum peptides as they exerted antibacterial properties on a range of bacterial pathogens. A time-kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC. The SYTOX green assay was performed to determine if the peptides disrupt the bacterial membrane. A lack of fluorescence within peptide-treated cells implied that NLP-31 and Y43C5A.3 do not act by disrupting the B. pseudomallei membrane but rather by a means of intracellular killing. Assessment of DNA-binding ability of these peptides by gel retardation assays revealed that NLP-31 and Y43C5A.3 bind to DNA with varying degrees of affinity and interfere with bacterial viability. The effect of these peptides on bacterial DNA synthesis was examined by microscopy for morphological changes. In contrast to untreated cells, induction of cellular filamentation, a hallmark of inhibition of DNA synthesis, was observed in NLP-31 and Y43C5A.3 treated cells. In addition to displaying antimicrobial activity, the peptides also regulated the expression of inflammatory cytokines in response to B. pseudomallei infection of macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as antimicrobial peptides towards B. pseudomallei based on their dual functions of antimicrobial and immunomodulator.,Ph.D.
Pages:	151
Call Number:	QP552.P4L537 2015 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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