Characterization of two membrane transporter genes in the genome of Plasmodium yoelii

Gomboc, Pika (author), Keše, Darja (mentor), Franke-Fayard, Blandine (co-mentor)

Malaria, an infectious disease caused by parasites from the Plasmodium genus, is one of the leading causes of death and morbidity worldwide and puts almost half of the world population at risk. Due to the emergence and rise of resistance to the insecticides and antimalarial drugs, there is a great need for a vaccine. So far whole sporozoite based immunization has been shown to be the most effective in experimental settings. Genetically attenuated parasites (GAP) have demonstrated significant protection in rodent models of malaria. The ideal GAP would arrest late in the liver stage of the malaria life cycle and provide sterile immunity in the host. However, the route and method of sporozoite administration have not been optimized yet. Intravenous injection (IV) is currently the only effective route of administration, that has been able to induce strong protective immune response. The preferred route is intradermal injection (ID), but several studies have shown that it induces lower immunity compared to IV injection. In order to improve the ID route of administration and determine the suitability of chosen genes as vaccine candidate genes, we aimed to generate mutant parasite lines of two promising membrane transporter genes, mfs6 and ctr2 of P. yoelii. We generated fluorescently tagged and deletion mutants of candidate genes with different biomolecular methods for functional analysis. We also characterized the expression and localization of fluorescently tagged proteins in the blood stage. These lines can be further used to determine expression and localization of tagged genes in the liver stage by infecting hepatocytes in vitro. Deletion mutant parasite lines need to be fully confirmed or further genetically characterized before use for route of administration experiments. Generated transgenic lines can then be used for studying route of administration and generation of GAP vaccine candidates in murine models of malaria.

• Language: English
• Keywords: vaccine, vaccine development, infectious diseases, malaria, Plasmodium, genetically attenuated parasites, membrane transport, mfs6, ctr2, biomolecular methods, fluorescent tagging
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [P. Gomboc]
• Year: 2017
• PID: 20.500.12556/RUL-91091
• UDC: 616-022.1:616.936:543.9
• COBISS.SI-ID: 4765304
• Publication date in RUL: 19.03.2017

Secondary language

• Language: Slovenian
• Title: Opredelitev dveh genov za membranski transport v genomu parazita Plasmodium yoelii

Abstract

Malarija, nalezljiva bolezen, ki jo povzročajo paraziti rodu Plasmodium, je ena od glavnih vzrokov smrti in obolevnosti po svetu. Ogroža skoraj polovico svetovnega prebivalstva. Zaradi pojava odpornosti proti insekticidom in antimalarijskim zdravilom nujno potrebujemo cepivo. Do sedaj se je za najbolj učinkovito izkazalo poskusno cepljenje s celimi sporozoiti. V mišjih modelih malarije so najboljšo stopnjo zaščite dosegli z genetsko oslabljenimi sporozoiti. Razvoj popolnega genetsko oslabljenega sporozoita bi se ustavil v pozni jetrni stopnji življenjskega kroga parazita in tako sprožil imunski odziv, ki bi zagotovil popolno in trajno zaščito pred okužbo s parazitom malarije. Vseeno pa je za uspešnost samega cepiva potrebno razviti še optimalni način dajanja cepiva. Trenutno je edini preizkušen učinkoviti način vnosa cepiva, ki sproži močan imunski odziv, z intravenozno injekcijo. Vnos z intradermalno injekcijo je bolj zaželjen, vendar pa so testi pokazali, da sproži šibkejši imunski odziv v primerjavi z intravenoznim vnosom. Mutantske parazitske linije mfs6 in ctr2 P. yoelii smo ustvarili zato, da bi izboljšali intradermalni vnos cepiva ter hkrati določili ustreznost izbranih genov kot kandidatnih genov za cepivo proti malariji. S pomočjo različnih biomolekularih metod smo ustvarili parazite s fluorescentno označenima izbranima genoma ter parazite z izbitima genoma. Opredelili smo izražanje in lokalizacijo izbranih genov v krvni stopnji življenjskega kroga parazita. Pridobljene parazitske linije se lahko uporabijo za opredelitev in lokalizacijo označenih genov v jetrni stopnji tako, da se z njimi okuži jetrne celice in vitro. Mutantske linije z izbitimi geni je potrebno najprej potrditi in genetsko opredeliti, preden se jih lahko uporabi v poskusih optimizacije vnosa cepiva. Te transgene parazitske linije se lahko nato uporabi za nadaljnje raziskovanje načina vnosa cepiva, kot tudi za razvoj genetsko oslabljenih parazitov oziroma kandidatnega cepiva v mišjih modelih malarije.

• Keywords: cepiva, izdelava cepiv, infekcijske bolezni, malarija, Plasmodium, genetsko oslabljeni paraziti, membranski transport, mfs6, ctr2, biomolekularne metode, fluorescentno označevanje