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Title: Potential anti-microbial targets in Acanthamoeba
Author: Sundararaj, Bharath Kanakapura
ISNI:       0000 0004 2743 2516
Awarding Body: University of the West of Scotland
Current Institution: University of the West of Scotland
Date of Award: 2013
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Acanthamoeba castellanii, a causative agent of Acanthamoebic Keratitis (AK) is usually associated with contact lens wear. The present drug regimen induces encystment. Hence there is a need for identification of effective drug targets. This thesis demonstrates the potential of the cellulose biosynthesis pathway, shikimate pathway and methionine biosynthesis pathway as effective drug targets. These biochemical pathways are absent in human hosts. A partially coding gene of cellulose synthase, the ultimate enzyme in the cellulose biosynthesis pathway was amplified from A. castellanii Neff strain. Gene knock down studies showed that the cellulose synthase gene is necessary for A. castellanii (Neff strain) encystation. 2, 6-Dichlorobenzonitrile, which was shown previously to inhibit encystation failed to inhibit in our laboratory conditions. The cellulose biosynthetic pathway could be a potential adjuvant to the present drug regimen. The role of the shikimate pathway, seven enzyme mediated pathway was characterised during encystation and excystation using a known inhibitor, glyphosate [N-(phosphonomethyl) glycine]. Glyphosate inhibited encystation of A. castellanii Neff strain. Glyphosate also inhibited the growth of a recently isolated T4 clinical isolate of A. castellanii. Glyphosate was not effective in inhibiting excystation. The complete coding sequence of seven enzymes of the shikimate pathway- 3- deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, chorismate synthase (CS), 3-dehydroquinate synthase (DHQS), 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase, 3-dehydroquinate dehydratase (DHQD), shikimate 5- dehydrogenase (SDH) and shikimate kinase (SK) have been amplified from A. castellanii Neff and T4 clinical isolate eDNA. The molecular arrangement of the seven enzymes in A. castellanii is novel and not reported in any other organism. A. castellanii Neff strain and the T4 clinical strain possess a four functional AROM like structure encoding DHQS, EPSP synthase, SK and DHQD. CS and DAHP are transcribed by mono functional genes. A. castellanii Neff and the T4 clinical isolate possess a bi-functional novel gene arrangement of SDH and phosphoribo anthranilate synthase (PRAI). PRAI is part of the tryptophan biosynthesis pathway. The shikimate pathway provides a potentially new therapeutic anti-Acanthamoebic target. Herein, is also demonstrate the potential of the methionine biosynthesis pathway as a new drug target. A de novo methionine biosynthesis pathway is present in A. castellanii. Although methionine synthesis is present in human hosts, A. castellanii uses different enzymes to synthesise methionine when compared to human hosts. A complete coding gene of methionine synthase - the ultimate enzyme involved in methionine biosynthesis has been amplified from A. castellanii Neff and T4 clinical isolate cDNA. A. castellanii Neff and T4 clinical isolate growth assays in the absence of methionine and cysteine showed the presence of de novo synthesis of methionine present in A. castellanii. The methionine biosynthetic pathway could be a potential drug target.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available