Mecanismos intraflagelares de transporte e trânsito de proteínas: uma abordagem pós-genômica e computacional de caracterização da virulência flagelar em Leishmania spp. / Intraflagelares mechanisms of transport and transit proteins: a post-genomics and computational characterization of virulence flagellar Leishmania spp.

Michely Correia Diniz

Leishmania are flagellated protozoa that cause a main neglected diseasease, collectively known as leishmaniases, posing a constant challenge for the control and prevention of the disease due to the wide-world distribution and prevalence of these parasites. As an insinuating pathogen, Leishmania has a specialized organelle for motility, the flagellum, which is essential for parasite migration, invasion and persistence on host tissues. Here we study the flagellum in Leishmania chagasi, L. amazonensis and L. major, whereas the process by which specific proteins (either actinassociated or not) are targeted to the flagellar environment is highlighted towards better understanding their causing diseases. The overall aim of this study is to provide a postgenomic analysis (proteomics and bioinformatics) on the flagellum of Leishmania spp., since it is a key organelle concerning their intraflagellar transport of proteins, including actin-interacting proteins, AIPs, and relevant motif-containing proteins. Therefore, we have applied the following methods: flagellum isolation and further purified fractions from Leishmania promastigotes; proteome analysis by means of two-dimensional electrophoresis [2D-E] in order to identify significant proteins; data interpretation to infer functions and implications through bioinformatics tools. A set of L. amazonensis and L. major flagellar proteins was obtained from 2D gels in which the detected spots were of good quality, allowing the prediction of their respective PI (isoelectric point) and MW (molecular weight). Coronin and Arp2/3 were two of the identified proteins in such proteome map, and since they are well known as AIPs, we have chosen them to build in silico models used to putatively ascertain their functional roles in trypanosomatid virulence. Moreover, we have applied an assorted computer approach to discover protein motifs from searches in Leishmania genomes databases that combined multi-relational data mining (MRDM) techniques performed after implementation of pattern recognition methods through hidden Markov models (HMM) customized with the Viterbi algorithm (VA). Such exacting approach, named MRDM/HMM/VA, was able to identify Leishmania proteins containg tetratricopeptide-repeat (TPR) motifs and their related motifs (pentatricopeptide - PPR e half-a-tetratricopeptide HAT), allowing the classification of protein families comprising the so-called TPR-like superfamily. Due to the fact that they are important structural modules in protein-protein interactions thought to play several molecular functions in numerous cell mechanisms, TPR-like motifs and TPR-containing proteins (TPRPs) were analysed in terms of intraflagellar apparatus. In comparison with currently standard methods, our MRDM/HMM/VA approach was more accurate in identifying TPRPs. Taken together, our results provide insights on the pathways through which coronins and Arp2/3 might be involved in the phagosome formation during polymerization and epolymerization of parasite actin and actin motor-associated proteins towards counting for the parasite survival within phagosomes as related to the flagellar apparatus. The arrangement of these two AIPs (coronins and Arp 2/3) seem to play a role in endosome delayed maturation and endosome-phagosome fusion in such a way that promastigotes differentiate in more resistant amastigotes, meaning a flagellar putative function on Leishmania virulence. AIPs and TPRPs might be potential targets for rational drug design concerning Leishmania differentiation, which is, indeed, one critical event on behalf of its infectivity.

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