Bioinformatic characterisation of genes associated with coenzyme A biosynthesis in mycoplasmas and expression and isolation of dephospho-coenzyme A kinase from Mycoplasma sp. Ms02

Ras, Tertius Alwyn (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: The South African ostrich industry is internationally the leading provider of ostrich products. The increasing popularity of ostrich products has resulted in the adjustment of production strategies, which includes intensifying rearing conditions by using feedlot systems. However, this intensive rearing strategy creates an ideal environment for pathogens, such as mycoplasmas, to spread. There are three Mycoplasma species that infect ostriches, which are associated with respiratory diseases. These mycoplasma infections can result in production losses, which not only have an economic impact on the ostrich industry but also significant socio-economic implications. Hence, there is a need for specific and cost-effective treatment against these ostrich-infecting mycoplasmas. The enzymes involved in the biosynthesis pathway of coenzyme A have long been regarded as potential targets for drug development. These enzymes could, therefore, offer a solution to the control of mycoplasma infections in ostriches. Since the coenzyme A biosynthetic pathway is relatively unexplored in mycoplasmas, the first aim of this study was to determine the presence or absence of enzyme-encoding genes involved in this pathway in Mycoplasma species. This was done using a bioinformatics approach. Of the 62 Mycoplasma species investigated, there were eight species (13%) found to have none of the enzyme-encoding genes, while the remaining species had at least one. Additionally, twelve enzyme-encoding gene homologues were identified and their predicted identities confirmed by evaluating the conserved and functional motifs and domains. The enzyme-encoding gene found to be most common amongst the investigated species was that of dephosphocoenzyme A kinase (DPCK), the final enzyme in the biosynthesis pathway. Furthermore, there was no correlation between the number of identified coenzyme A biosynthetic pathway enzyme-encoding genes in a species and the phylogeny of the respective proteins. There was also no correlation with the 16S rRNA phylogenetic groupings. Given the common presence of the DPCK-encoding gene, the second aim of this study was to recombinantly express the DPCK of the ostrich-infecting Mycoplasma sp. Ms02 (Ms02) and isolate the protein using a His-tag. The Ms02 DPCK-encoding gene was successfully amplified, cloned and mutated by site-directed mutagenesis to allow for expression in a nonmycoplasma host. However, the soluble expression and isolation of the Ms02 DPCK protein proved to be challenging. Using a variation of methods, the protein was eventually solubilised using a sarkosyl treatment method. A pure isolate of the Ms02 DPCK protein could, however, not be attained when using immobilised metal affinity chromatography (IMAC) purification. Subsequent activity testing of the isolated DPCK enzyme, using an HPLC-based method, also showed no activity.

AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse volstruisbedryf is internasionaal die grootste verskaffer van volstruisprodukte. Die toenemende gewildheid van volstruisprodukte het gevolglik veroorsaak dat produksiestrategieë aangepas moet word. Een van hierdie strategieë sluit in die intensifisering van boerderypraktyke deur gebruik te maak van voerkraalstelsels. Hierdie intensiewe boerderystrategieë skep egter 'n ideale omgewing vir patogene, soos mikoplasmas, om in te versprei. Daar is drie Mycoplasma spesies wat volstruise infekteer, wat geassosieer word met respiratoriese siektes. Hierdie mikoplasma-infeksies kan verliese in produksie veroorsaak, wat nie net 'n ekonomiese impak op die volstruisbedryf het nie, maar ook beduidende sosio-ekonomiese implikasies het. Daar is dus 'n behoefte aan spesifieke en koste-effektiewe behandeling teen hierdie volstruis-infekterende mikoplasmas. Die ensieme wat by die biosintetiese padweg van koënsiem A betrokke is, is lank reeds beskou as potensiële teikens vir geneesmiddelontwikkeling. Hierdie ensieme kan dus 'n oplossing bied vir die beheer van mikoplasma-infeksies in volstruise. Aangesien die koënsiem A biosintetiese padweg relatief onverken is in mikoplasmas, was die eerste doelwit van hierdie studie om die teenwoordigheid of afwesigheid van ensiem-enkoderende gene betrokke by hierdie padweg in Mycoplasma spesies te bepaal. Dit was gedoen deur gebruik te maak van 'n bioinformatika benadering. Van die 62 Mycoplasma spesies wat ondersoek was, was daar agt spesies (13%) wat nie een van die ensiem-enkoderende gene besit het nie, terwyl die oorblywende spesies minstens een gehad het. Daarbenewens is twaalf ensiem-enkoderende geenhomoloë geïdentifiseer en hul voorspelde identiteite bevestig deur die gekonserveerde en funksionele motiewe en domeine te evalueer. Die mees algemene ensiem-enkoderende geen wat gevind was onder die ondersoekde spesies was dié van defosfo-koënsiem A kinase (DPCK), die finale ensiem in die biosintese padweg. Verder was daar geen verband tussen die aantal geïdentifiseerde koënsiem A biosintetiese padweg ensiem-enkoderende gene in 'n spesie en die filogenie van die betrokke proteïene nie. Daar was ook geen korrelasie met die 16S rRNA filogenetiese groeperings nie. Gegewe die algemene teenwoordigheid van die DPCK geen, was die tweede doelwit van hierdie studie om die DPCK proteïen van die volstruis-infekterende Mycoplasma sp. Ms02 (Ms02) uit te druk en die proteïen te isoleer met behulp van ‗n His-merker. Die Ms02 DPCKenkoderende geen is suksesvol geamplifiseer, gekloon en gemuteer deur posisie-gerigte mutagenese om sodoende ekspressie in ‗n nie-mikoplasma gasheer toe te laat. Die oplosbare uitdrukking en isolasie van die Ms02 DPCK proteïen was egter uitdagend. ‗n Variasie van metodes was gebruik, maar die proteïen was eventueel oplosbaar gemaak met behulp van 'n sarkosiel behandelingsmetode. 'n Suiwer isolaat van die Ms02 DPCK proteïen kon egter nie verkry word tydens ge-imobiliseerde metaal affiniteits chromatografie (IMAC) suiwering nie. Daaropvolgende aktiwiteitsbepalings van die geïsoleerde DPCK ensiem, met behulp van 'n HPLC-gebaseerde metode, het ook geen aktiwiteit getoon nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103878
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