Étude des éléments régulateurs « cis » et « trans » impliqués dans la stabilité du transcrit de l'amastine au stade intracellulaire chez « Leishmania »

Le genre Leishmania regroupe des parasites protozoaires transmis par piqûre d’un insecte vecteur et qui sont responsables des leishmanioses. Le cycle de Leishmania alterne entre promastigotes dans l’appareil digestif de l’insecte et amastigotes dans les phagolysosomes des macrophages d’un hôte mammifère. Les delta-amastines sont une famille de protéines membranaires qui jouent potentiellement un rôle dans la virulence. L’expression exclusivement au stade intracellulaire de l’un de ces gènes est permise par une accumulation préférentielle de l’ARNm et la stimulation de la traduction, toutes deux chez les amastigotes. L’objectif de cette thèse est de caractériser les mécanismes permettant l’expression différentielle de l’ARNm de la delta-amastine. Ces organismes ont divergé rapidement des autres eucaryotes, ce qui engendre plusieurs différences fonctionnelles, dont notamment l’absence de régulation transcriptionnelle. Notre hypothèse est que la présence d’une région riche en uridines (URE) dans l’extrémité 3’ non traduite (3’UTR) du transcrit peut être impliquée dans la dégradation de l’ARNm. Nous démontrons que le URE est responsable d’une dégradation du transcrit au stade promastigote, par un phénomène indépendant de la déadénylation. Nous avons identifié une protéine à domaine Alba, LiAlba20, liant l’ARNm de la delta-amastine dans une région proche de l’URE. La suppression de cette protéine réduit l’accumulation du transcrit au stade amastigote. Ainsi, deux mécanismes complémentaires sont responsables de l’expression différentielle de ce transcrit. Le génome de Leishmania code pour une seconde protéine à domaine Alba, LiAlba13. Ces protéines interagissent ensemble, mais LiAlba13 n’affecte pas l’abondance de l’ARNm de la delta-amastine. Les protéines Alba ont une évolution exceptionnelle puisqu’elles stabilisent l’ADN chez les Archaea, et sont retrouvées dans les complexes RNase P/MRP chez les eucaryotes supérieurs. Nos résultats montrent qu’elles régulent l’expression de protéines spécifiques du stade amastigote, ce qui concorde avec les récents travaux chez d’autres parasites protozoaires. Ces protéines sont cytoplasmiques dans les deux stades de développement. Cependant, pendant la différenciation, elles s’accumulent dans le flagelle et le nucléole, respectivement décrits comme senseur et coordinateur de la réponse au stress. Nos travaux suggèrent donc l’implication du flagelle et du nucléole dans la coordination de la régulation de facteurs de virulence pendant la différenciation du parasite.The Leishmania genus encompasses protozoan parasites which are transmitted through the bite of an insect vector and are responsible for leishmaniasis. The Leishmania life cycle alternates between promastigote forms within the gut of the insect vector and amastigotes which multiply in the phagolysosomal vacuoles of the mammalian host’s macrophages. Delta-amastins are part of a multigenic family of membrane proteins that potentially act in parasite virulence. One of the delta-amastin's exclusive expression in the intracellular stage is mediated by mRNA accumulation and translation stimulation, both taking place in the amastigote stage. The aim of this thesis is to characterize the mechanisms implicated in the differential expression of delta-amastin mRNA. Leishmania splits early in evolution from other eukaryotes and this split correlates with many functional differences, including the absence of transcriptional control of gene expression. Our hypothesis is that the presence of a uridine-rich element (URE) within the 3’ untranslated region (3’UTR) of the transcript might be implicated in an mRNA decay mechanism. We reveal that the URE is responsible for a fast mRNA decay only in the promastigote stage, performed by an unusual deadenylation-independent pathway. We next identified an Alba domain protein, LiAlba20, which binds to the delta-amastin mRNA in a region flanking the URE. Depletion of this protein leads to a reduced mRNA accumulation in the amastigote stage specifically. Therefore, we identified two complementary mechanisms taking part in the transcript’s differential expression. The Leishmania genome encodes a second Alba domain protein, LiAlba13. These proteins interact together, but LiAlba13 does not affect the delta-amastin mRNA level during the parasite life cycle. Alba domain proteins have a remarkable evolution, being involved in DNA stabilization in Archaea and subunits of the RNAses P/MRP complexes in higher eukaryotes. In addition, our data show that these proteins regulate stage-specific protein expression, which is in agreement with recent works in other protozoan parasites. Alba domain proteins are constitutively expressed in the cytoplasm of both parasite life cycle stages. Nevertheless, during the differentiation, those proteins accumulate in flagellar and nucleolus compartments, respectively described as sensor and stress response coordinators in higher eukaryotes. Our work suggests that the flagellum is implicated in the coordination of stage-specific transcript expression in response to stress in Leishmania

Differential Subcellular Localization of <i>Leishmania</i> Alba-Domain Proteins throughout the Parasite Development

<div><p>Alba-domain proteins are RNA-binding proteins found in <i>archaea</i> and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in <i>Leishmania infantum</i>. We show that in contrast to other protozoa, <i>Leishmania</i> have two Alba-domain proteins, <i>Li</i>Alba1 and <i>Li</i>Alba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. <i>Li</i>Alba1 and <i>Li</i>Alba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The <i>Leishmania</i> Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the <i>Leishmania</i> flagellum confirmed <i>Li</i>Alba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the <i>Leishmania</i> flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in <i>Leishmania</i>.</p></div

Sequence alignment and phylogeny of Alba-domain proteins in <i>Leishmania</i>, <i>Trypanosoma brucei</i> and <i>T</i>. <i>cruzi</i> species.

<p>(A) Neighbor-joining tree showing the phylogenetic relationship between the Alba-domain proteins of TriTryps. Evolutional distances (scale) were estimated as the number of amino acid substitutions per site, considering Poisson correction. The two subgroups Rpp20-like and Rpp25-like are marked. (B) ClustalW alignment of Rpp20-like Alba-domain proteins merged with the in silico structure prediction of <i>Li</i>Alba1 (LinJ.13.0270) using the Phyre algorithm. The best score was obtained with the Alba protein from <i>Sulfolobus solfataricus</i> (<i>Ss</i>)(NCBI WP_010923153.1). ss: secondary structure <i>in silico</i> prediction; C: coil; H: helix; E: Sheet. Red squares indicate amino acids known to be phosphorylated on these specific genes (<i>T</i>. <i>cruzi</i> and <i>T</i>. <i>brucei</i>). The black star shows the expected position for Sir2 acetylation and the red stars underline the signature motif of the subgroup. Sequence variations in coiled regions between <i>Trypanosoma</i> spp. and <i>Leishmania</i> spp. are underlined with a black bar. <i>Li</i>Alba1 was used for Phyre structure prediction. (C) As in B for the Rpp25-like Alba-domain proteins. Structure prediction of <i>Li</i>Alba3 using Phyre outputs Alba2 from <i>Aeropyrum pernix</i> (<i>Ap</i>) K1 (NCBI WP_010866616.1) as the best match. <i>Li</i>Alba3 (LinJ.34.2410) was used for Phyre structure prediction. LinJ: <i>L</i>. <i>infantum</i>; LmjF: <i>L</i>. <i>major</i>; LtaP: <i>L</i>. <i>tarentolae</i>; LbrM: <i>L</i>. <i>braziliensis</i>; Tb: <i>T</i>. <i>brucei</i>; Tc: <i>T</i>. <i>cruzi</i>.</p

Alba-domain proteins are associated with ribosomal subunits.

<p>Polysome fractionation of <i>L</i>. <i>infantum</i> expressing HA-tagged Alba1 and Alba3 proteins by 15–45% sucrose gradient was carried out using logarithmic phase promastigotes (26°C) (A) or heat-stressed parasites grown O/N at 37°C (B). Graphical representations present the RNA content of each collected fraction after ultracentrifugation on 15–45% sucrose gradient. F: Free RNA; 40S, 60S and 80S: ribosomal subunits and monosomes, respectively. Each fraction was loaded on 12% SDS-PAGE and transferred on a nylon membrane for Western blot analysis to detect HA-<i>Li</i>Alba3 and <i>Li</i>Alba1-HA proteins using an anti-HA antibody. As a control, half of the protein extracts were incubated with EDTA before ultracentrifugation to disrupt association of the polyribosomes with mRNAs.</p

<i>L</i>. <i>infantum</i> Alba-domain protein putative partners identified by co-immunoprecipitation and LC-MS/MS analysis.

<p><i>L</i>. <i>infantum</i> Alba-domain protein putative partners identified by co-immunoprecipitation and LC-MS/MS analysis.</p

Alba-domain proteins translocate from the cytoplasm to the flagellum and the nucleolus upon <i>Leishmania</i> amastigote differentiation.

<p>Subcellular localization of <i>Li</i>Alba1-HA and HA-<i>Li</i>Alba3 proteins in promastigotes (A) and upon amastigote differentiation (8 h in MAA medium pH 5.8 at 37°C) (B) was assessed by indirect immunofluorescence studies using an anti-HA antibody as described in Materials and Methods. DAPI staining (red) allows detection of the nucleus (N) and kinetoplastid DNA (K). C) Immunofluorescence images of wild type <i>L</i>. <i>infantum</i> episomally co-expressing pSP-NEOalphaIR-eYPF-<i>Li</i>Alba1 and pSP-HYGalphaIR-mCh-<i>Li</i>NOP10 grown as promastigotes (Pro), differentiating amastigotes (Diff) and amastigotes (Ama). <i>Li</i>NOP10 was used as a nucleolar (Nu) control.</p

Heat stress triggers differential localization of Alba-domain proteins in <i>Leishmania</i>.

<p>Immunofluorescence images for eYPF-<i>Li</i>Alba1 (green) and mCh-<i>Li</i>NOP10 (red) in <i>L</i>. <i>infantum</i> promastigotes co-expressing pSP-NEOalphaIR-eYPF-<i>Li</i>Alba1 and pSP-HYGalphaIR-mCh-<i>Li</i>NOP10 submitted to heat stress (from 25°C to 37°C) or to acidic pH (pH 5.8) O/N. Nu: nucleolus.</p

Flagellum purification demonstrates an enrichment of <i>Li</i>Alba3 in the <i>Leishmania</i> flagellum during heat stress.

<p>Phase contrast images of intact <i>L</i>. <i>infantum</i> promastigotes (A) and of purified flagella after sucrose gradient isolation (B) as described in Materials and Methods. (C) Summary of MS/MS identified proteins from four independent experiments of flagellum purification (two from promastigote cell extracts and two from 8 h-differentiating amastigotes). Identified genes were classified according to their gene ontology and to characterized orthologs in <i>Trypanosoma</i> spp. based on GeneDB and TriTrypDB gene annotations. Only genes identified at least twice with a minimum of 2 peptides are shown here (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137243#pone.0137243.t002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137243#pone.0137243.s012" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137243#pone.0137243.s013" target="_blank">S4</a> for the complete list of the identified <i>L</i>. <i>infantum</i> flagellum proteins). (D) Confirmation of flagellar localization of PFR2C-HA in recombinant <i>L</i>. <i>infantum</i> expressing pSP-alphaIRNEOalphaIR-PFR2C-HA. (E) Western blot analysis and quantification of endogenous <i>Li</i>Alba3 in purified flagellum fractions upon promastigote conditions of growth (Pro) or following 8 h of temperature stress (37°C). After flagellum purification, flagella from promastigotes and heat-stressed parasites were counted on a Malassey (hemocytometer) to load an equivalent number of flagella on the gel. Total proteins of promastigote cells were loaded as a control. Relative quantification was performed using ImageJ blot.</p

Alba-domain proteins co-localize to the cytoplasm of promastigote and amastigote <i>Leishmania</i> life stages.

<p>Direct fluorescence images of recombinant <i>L</i>. <i>infantum</i> promastigotes (Pro) and axenic amastigotes (Ama) at passage 4 co-expressing eYPF-<i>Li</i>Alba1 (green) and mCh-<i>Li</i>Alba3 (red) proteins. Green and red pixels overlapped in the digital images yielding yellow/orange signals. The nucleus (N) and kinetoplastid DNA (K) were stained with DAPI (blue).</p