No description available.

En 1977, Carl Woese sépare les procaryotes en deux grands groupes en proposant une nouvelle classification basée sur des critères phylogénétiques. Les Archaea deviennent ainsi un domaine à part entière aux cotés des Bacteria et des Eucarya. Depuis, la compréhension de ce nouveau groupe et de ses relations avec les deux autres domaines, essentielles pour comprendre l'évolution ancienne du vivant, est largement passée par l'étude de leur phylogénie. Presque 40 ans de recherche sur les archées ont permis de faire évoluer leur image : de bactéries vivant dans des milieux spécialisés, souvent extrêmes, on est passé à un domaine indépendant, très diversifié aussi bien génétiquement, métaboliquement ou encore écologiquement. Ces dernières années la barre symbolique de cent génomes complets d'archées séquencés a été franchie et, parallèlement, les projets génomiques et métagénomiques sur des groupes peu caractérisés ou de nouvelles lignées de haut rang taxonomique (e.g. Nanohaloarchaea, Thaumarchaeota, ARMAN, Aigarchaeota, groupe MGC, groupe II des Euryarchaeota, etc.) se sont multipliés. Tout ceci apporte un matériel sans précédent pour l'étude de l'histoire évolutive et de la diversité des Archaea. Les protéines ribosomiques ont été utilisées de façon courante pour inférer la position phylogénétique des nouvelles lignées d'Archaea. Néanmoins, les phylogénies résultantes ne sont pas complètement résolues, laissant des interrogations concernant d'importantes relations de parenté. La recherche de nouveaux marqueurs est donc cruciale et c'est dans ce contexte que mon projet de thèse s'inscrit. À partir de l'analyse des génomes de deux Thaumarchaeota et d'une Aigarchaeota, nous avons identifié 200 protéines conservées et bien représentées dans les différents phyla d'archées. Ces protéines sont impliquées dans de nombreux processus cellulaires, ce qui peut apporter un signal phylogénétique complémentaire à celui des marqueurs de type informationnel utilisés par le passé. En plus de confirmer la plupart des relations phylogénétiques inférées à partir de ces derniers (i.e., protéines ribosomiques et sous unités de l'ARN polymérase), l'analyse phylogénétique de ces nouveaux marqueurs apporte un signal permettant une meilleure résolution de la phylogénie des archées et la clarification de certaines relations jusqu'ici confuses. Un certain nombre de ces nouveaux marqueurs sont aussi présents chez les bactéries. Les relations entre les grands phyla d'archées restant encore non résolues, nous avons utilisé ces protéines pour essayer de placer la racine de l'arbre des Archaea en utilisant comme groupe extérieur les bactéries. Nous avons ainsi pu identifier 38 protéines, parmi les 200 sélectionnées précédemment, ayant un signal phylogénétique suffisamment fiable pour cette étude, auxquelles nous avons ajouté 32 protéines ribosomiques universelles. L'utilisation conjointe de ces données nous a permis de placer la racine entre les Euryarchaeota, d'une part, et un groupe rassemblant les Thaumarchaeota, les Aigarchaeota, les Korarchaeota et les Crenarchaeota, d'autre part. Ce nouvel éclairage sur l'évolution ancienne des archées nous a amené à proposer une révision de leur taxonomie avec, principalement, la création du nouveau phylum "Proteoarchaeota" contenant les quatre phyla actuels que nous proposons de rétrograder en classes : Thaumarchaea, Aigarchaea, Korarchaea et Crenarchaea.Finalement, l'analyse des protéines codées dans les trois génomes qui ont servi de point de départ de ma thèse nous a permis de générer une masse considérable de données qui ont révélé des traits particuliers ou encore des histoires évolutives inattendues. Un exemple est l'histoire du complexe formé par la chaperonne DnaK et de ses co-chaperonnes GrpE, DnaJ, et DnaJ-Fer chez les Thaumarchaeota, impliquant plusieurs transferts horizontaux entre les trois domaines du vivant.

No description available.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.