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Intracellular trafficking of Leishmania major peptidases

Tonn, Daniela (2010) Intracellular trafficking of Leishmania major peptidases. PhD thesis, University of Glasgow.

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Abstract

Leishmania resides inside mammalian macrophages, from where it is thought to manipulate the host immune system by releasing virulence factors. The cysteine peptidase CPB has been shown to be secreted by the parasite and act as such a virulence factor. CPB is released through the flagellar pocket while being trafficked to the lysosome. Thus, in this project, the intracellular localisations of eight other L. major peptidases were analysed by fluorescence microscopy, after tagging the enzymes with green fluorescent protein (GFP). The candidate peptidases were chosen by bioinformatics analyses and predictions of N-terminal secretory signal peptides and potential transmembrane domains. The aim was to find a peptidase accumulating in the flagellar pocket of the cell, from where it could be secreted. Five candidate peptidases (a ubiquitin hydrolase, a CaaX prenyl protease, a zinc carboxypeptidase and two rhomboid peptidases) localised to the mitochondrion, which was unexpected. Another, a calpain-like peptidase, localised to the flagellum but not to the flagellar pocket. A serine carboxypeptidase was found very close to the flagellar pocket, possibly in small vesicles budding off or fusing with the pocket membrane, but did not co-localise with a flagellar pocket marker. The bioinformatics predictions differed from the experimental results here and, additionally, using different algorithms to predict protein properties resulted in contradictory predictions in several cases. This suggests that generic protein prediction programmes for mammalian or higher eukaryotic proteins can be unreliable and of limited usefulness for Leishmania proteins. This corroborates the notion that Leishmania may use novel, non-classical secretory pathways rather than or in addition to those characterised for higher eukaryotes. The L. major Bem46-like serine peptidase (LmjF35.4020) of the Clan SC (Family S9) was the only candidate peptidase that localised to the flagellar pocket when labelled with GFP. This was an indication that this enzyme may be released from the cell and could act as a virulence factor. Alternatively, it may be a resident protein of the flagellar pocket. Deleting the Bem46 gene in L. major did not have a measurable effect on promastigote growth or on footpad lesion development in mice inoculated with Bem46-deficient cells, so it does not appear to play a role as a major virulence factor. Apart from the secretion of virulence factors, rapid protein turnover, e.g. in the lysosome, is important for the infectivity of Leishmania. To investigate lysosome structure and function in L. major, a potential LMP (lysosomal membrane protein) was identified by bioinformatics. Thus far, no resident membrane proteins of the Leishmania lysosome are known and identifying such a protein would provide a useful marker for the closer investigation of this important organelle. In this project, the location and role of the LMP protein LmjF30.2670 was investigated using GFP-tagging and fluorescence microscopy. The experiments showed that LMP is not lysosomal in L. major, rather, it could be observed localising to a distinct, elongated and sometimes doughnut-shaped structure in close proximity to the kinetoplast. This structure was not directly associated with the flagellar pocket or the cell membrane, its position in the cell was variable within a certain area alongside the kinetoplast, it appeared to duplicate during cell division and it did not co-localise with the endocytic / lysosomal marker FM4-64. Deletion of the LMP gene did not have any effect on promastigote growth in cell culture and only a small and transient slowing effect on the development of mouse footpad lesions after inoculation with LMP-deficient L. major. Lysosomal membrane proteins can be targeted to the lysosome by the protein carrier complex AP3, which binds to tyrosine or dileucine motifs in cargo proteins. LMP contains two such tyrosine motifs at its C-terminus, but disruption of these by site-directed mutagenesis did not affect LMP localisation, suggesting that its trafficking is AP3-independent, which is in accordance with the non-lysosomal localisation of LMP. Finally, the lysosome-like acidocalcisome organelles have previously been shown to rely on the protein carrier complex AP3 for normal structure and function. In AP3-deficient Leishmania, the acidocalcisomes are defective and, at the same time, parasite virulence is markedly reduced (Besteiro et al., 2008o). To analyse how AP3 is important for acidocalcisome morphology and function, a proton pump of the acidocalcisomal membrane, the V-H+-PPase, was investigated by GFP-labelling and fluorescence microscopy. In wild type L. major the V-H+-PPase could be shown to localise to the acidocalcisomes, whereas in AP3-deficient cells it was not detectable, suggesting that the protein is mislocalised and likely degraded. The V-H+-PPase also contains several tyrosine motifs that may interact with AP3. The two most prominent of these were disrupted by site-directed mutagenesis, but this did not affect the localisation of the V-H+-PPase, suggesting that these two sites are not, or not solely, important for AP3 binding or that the V-H+-PPase is not bound by AP3 directly.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Leishmania, parasite, secretion, peptidase, protein trafficking, bem46, acidocalcisome, AP3 adaptor complex
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QR Microbiology > QR180 Immunology
Q Science > QL Zoology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: Mottram, Prof Jeremy C.
Date of Award: 2010
Depositing User: Miss Daniela Tonn
Unique ID: glathesis:2010-1649
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Apr 2010
Last Modified: 07 Jun 2013 12:23
URI: http://theses.gla.ac.uk/id/eprint/1649

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