Leishmania spp. undergo substantial adaptations from being promastigotes, found in sandflies, to being amastigotes, residing in parasitophorous vacuoles within mammalian macrophages. In the past, microarray studies have sought to elucidate these adaptations using axenic amastigote systems or amastigotes purified from host-cells, raising the question whether the observed transcriptomic signatures were a true reflection of intracellular amastigotes. Moreover, with ever-improving genome annotations being available, it is clear that these studies failed to address the transcriptomic behaviour of a considerable number of transcripts. In the work presented herein, I employed RNA-sequencing to obtain transcriptomic profiles of Leishmania mexicana axenic promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in murine bone-marrow derived macrophages. The intracellular amastigotes were not purified from host cells, but instead sequencing reads assigned to a hybrid L. mexicana - Mus musculus genome and the transcriptomes separated in silico. We were able to map pre-mRNA processing sites, thereby defining transcript boundaries, proposing 184 truncations and 1253 extensions of existing gene models as well as discovering 936 novel genes. Mass-spectrometric evidence was obtained for both proposed extended and novel proteins. Using this improved genome annotation, we generated gene expression profiles for AMA, AXA and PRO, identifying 3832 differentially expressed transcripts between PRO and AMA as well as 2176 between PRO and AXA and 1234 between AXA and AMA. Transcripts differentially expressed between AMA and PRO correlated well with previous reports, were enriched for novel transcripts identified in this study and contained an unprecedented wealth of yet uncharacterised transcripts. Guided by these data, I performed a GFP-tagging screen identifying two proteins which may play an important role in L. mexicana biology, LmxM.16.0500, a member of a small, divergent, amastin-derived gene family, which appears to be released from the cell body of PRO, and LmxM.09.1330 a specific marker of the amastigote flagellar pocket.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658431 |
| Date | January 2014 |
| Creators | Fiebig, Michael |
| Contributors | Gluenz, Eva M.; Gull, Keith |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:ff1a5033-e1a4-40cc-8204-7de904ba7aa2 |
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