The development of a DNA vaccine against Mycoplasma nasistruthionis sp. nov. for use in ostriches

Wium, Martha (2015-12)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches.

AFRIKAANSE OPSOMMING: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is een van drie mikoplasma spesies wat volstruise infekteer. Mikoplasma-infeksies in volstruise veroorsaak kuiken vrektes, vertraagde groei en afgradering van volstruis karkasse. Daar is tans geen geregistreerde mikoplasma entstof beskikbaar vir gebruik in volstruise nie. Hierdie studie het die ontwikkeling van DNS-entstowwe teen Ms03-infeksies in volstruise ondersoek. Vir hierdie doel was die Ms03-genoomvolgorde bepaal en geannoteer. Die entstofkandidaat-geen, oppA, was geïdentifiseer in die genoomvolgorde en gekarakteriseer voordat DNS-entstowwe (wat die oppA-geen bevat) ontwikkel en getoets is. Die Ms03-genoomvolgorde was bepaal en die gegenereerde 172 aaneenlopende volgordes was geannoteer. Hierdie proefskrif bied die eerste voorlopige volgorde en annotering van die Ms03-genoom wat bygedra het tot die kennis van Ms03 as 'n miniatuur geneties onafhanklike organisme. Genoom-replikasie, seldeling, RNS-transkripsie, proteïen-translasie en glikolise in Ms03 stem ooreen met dié prosesse in die naverwante Mycoplasma synoviae 53. Die purien en pirimidien metabolisme was onvolledig en de novo sintese daarvan was nie moontlik in Ms03 nie. Aminosuursintese in Ms03 was meestal afwesig en net die gene wat aspartaat omskep na asparagien en glisien na serien was gevind in die annoteerde genoom. Meer invoerders as uitvoerders was geannoteer, wat dui op die gebrek aan sintesepadweë in Ms03. Dit is tipies van mikoplasmas wat ‘n parasitiese lewensstyle het. Twee oligopeptied-permeases (opp) operons was gevind in die Ms03-genoom. Die potensiaal van die oppA-geen as 'n entstofkandidaat-geen was geëvalueer deur die behoefte van 'n substraatbindingsdomein (OppA) as deel van die OppBCDF-vervoerder binne mikoplasma spesies te ondersoek. 'n Homoloog van die oppA-geen kon geïdentifiseer word vir elke oppBCDF-operon in al die spesies en behoort daarom 'n noodsaaklike rol te speel in die vervoer van oligopeptiede. Alle mikoplasmas (behalwe vir hemoplasmas) het een, twee of drie opp-operons, wat verdeel kan word in drie tipes (Tipe A, B en C). Elke tipe het unieke InterPro en MEME domeine en motiewe wat saam met die filogenetiese ontleding daarop dui dat hulle unieke rolle in oorlewing onder verskillende omstandighede speel. Ms03 het 'n Tipe A en Tipe B opp-operon, die Tipe A oppA is gebruik as entstofkandidaat-geen. Die Tipe A oppA-geen was gekloneer en teikengerigte-mutagenese was gebruik vir kodonregstellings voordat die gemuteerde geen in drie DNS-entstof vektore gesubkloneer was. Die drie DNS-entstowwe (pCI-neo_oppA, VR1012_oppA en VR1020_oppA) was gebruik om volstruise in te ent en die OppA-teenliggaamsreaksie was geanaliseer deur ELISA. Inenting met die VR1020_oppA en pCI-neo_oppA entstowwe het tot 'n primêre immuniteitsreaksie in volstruise gelei. Dit dui daarop dat die OppA proteïen in vivo uitgedruk en immunogenies was. Dit kan beskou word as die eerste stap in die ontwikkeling van 'n DNS-entstof vir die beheer van mikoplasma-infeksies in volstruise.

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