A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

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Bacterial elongation factor P (EF-P) is a poorly understood soluble protein that has been shown to enhance the first step of peptide bond formation through an interaction with the ribosome and initiator tRNA. The crystal structure of EF‐P shows that EF‐P mimics the tRNA shape. Orthologous proteins have been found in both archaeal and eukaryotic systems, known as aIF5A and eIF5A, respectively. eIF5A, which was recently shown to increase translation elongation rates, is post-translationally modified at a highly conserved lysine residue (K50) through the addition of the rare amino acid hypusine. A similar pathway was recently elucidated for EF-P, in which EF-P is post-translationally modified by the enzymes YjeA and YjeK at lysine 34, corresponding to a homologous site of hypusylation in a/eIF5A. As a paralog of class II LysRS, YjeA catalyzes the addition of lysine onto EF-P, but is incapable of modifying tRNA. YjeK is a 2,3-(β)-lysine aminomutase and is responsible for converting lysine to β-lysine, which YjeA was recently shown to recognize as a preferred substrate for EF-P modification. However, fully modified EF-P requires a third enzyme, YfcM, which acts as a hydroxylase and hydroxylates the C4 or C5 position of K34 of EF-P, but not the added β-lysine. Based on a complete description of the EF-P modification and pathway, in this project we focused on further studies to address the mechanism of action of EF-P and especially to investigate how the different stages of EF-P’s modifications can affect E. coli cells. Using E. coli Keio knockout collection (Δefp, ΔyjeK, ΔyjeA, ΔyfcM) and E. coli Keio parental strain (wild-type) as reference, we checked the effect of the deletion strains on the cells under different environmental stress conditions (varying growth temperatures and nutrition conditions, susceptibility to antibiotics), showing that Δefp strain has growth defects and that E. coli efp mutants show sensitivity to non-ribosomal inhibitors, such as ampicillin and rifampicin, suggesting a possible secondary role of EF-P related to the cell envelope. Moreover, we tested the ability of deletion strains to restore viability in the presence of the appropriate plasmid and showed that EF-P is important for cell viability under certain conditions in E. coli. As reported previously, YjeA and YjeK are important in bacteria virulence. In addition, EF‐P is recognized as one of the proteins important for bacteria motility in Bacillus subtilis. However, motility and virulence are often linked together. Here, we tested deletion strains for their ability to produce flagella. Further, using external fluorescence staining and confocal microscopy we revealed differences in morphology of the E. coli deletion strains, and we performed Histidine tag protein purification with Ni-NTA agarose beads and gel filtration, in order to purify YfcM, an uncharacterized protein, and set initial screens for crystallization. Finally, our future goal is to clone the following polycistronic construct, “- yjeK - yjeA - yfcM - his-efp -“, overexpress and crystallize it, so as to see the crystal structure of the whole modification pathway of EF-P and study better the function of EF-P in translation extracts from different mutants / Ο βακτηριακός παράγοντας επιμήκυνσης EF-P, είναι μια διαλυτή πρωτεΐνη που βοηθά στο σχηματισμό του πρώτου πεπτιδικού δεσμού, αλληλεπιδρώντας με το ριβόσωμα και το εναρκτήριο tRNA. Η κρυσταλλική δομή του EF-P δείχνει ότι μιμείται στη μορφή το tRNA. Ορθόλογες πρωτεΐνες έχουν βρεθεί και στα αρχαία και τα ευκαρυωτικά κύτταρα, γνωστές ως aIF5A και eIF5A, αντίστοιχα. Ο eIF5A, για τον οποίο αποδείχθηκε πρόσφατα ότι συμμετέχει και στο στάδιο της επιμήκυνσης της μετάφρασης, υφίσταται μια μοναδική μετα-μεταφραστική τροποποίηση στη λυσίνη 50 (Κ50), μέσω της προσθήκης σε αυτή ενός σπάνιου αμινοξέος, της υπουσίνης (hypusine). Μια παρόμοια τροποποίηση αποδείχθηκε ότι υφίσταται ωστόσο και ο EF-P της Escherichia coli (E. coli), ο οποίος τροποποιείται μετα-μεταφραστικά στη λυσίνη 34 (Κ34) με τη βοήθεια των ενζύμων YjeA και YjeK. Το YjeA αποτελεί παράλογο της δεύτερης κλάσης των tRNA συνθετασών της λυσίνης (LysRSs: Lysyl-tRNA synthetases), και καταλύει την προσθήκη της λυσίνης επάνω στον EF-P. Το YjeK είναι μια 2,3 αμινομουτάση της λυσινης (LAM: Lysine-2-3-aminomutase) και είναι αρμόδια για τη μετατροπή της α-λυσίνης σε β-λυσίνη. Εντούτοις, πρόσφατες έρευνες έδειξαν ότι ο πλήρως τροποποιημένος EF-P απαιτεί ένα επιπλέον ένζυμο, το YfcM, το οποίο ενεργεί ως υδροξυλάση και υδροξυλιώνει τον C4 ή C5 άνθρακα της K34 του EF-P. Στη συγκεκριμένη μελέτη εστιάσαμε στην εξέταση του μηχανισμού δράσης του EF-P και ειδικότερα στις επιπτώσεις που μπορεί να έχουν τα διαφορετικά στάδια των τροποποιήσεών του σε κύτταρα E. coli. Χρησιμοποιώντας τα knockout E. coli στελέχη της Keio (Δefp, ΔyjeK, ΔyjeA, ΔyfcM), ελέγξαμε την επίδραση διαφόρων περιβαλλοντικών συνθηκών στα στελέχη (διαφορετικές θερμοκρασίες, συνθήκες διατροφής, ευαισθησία σε αντιβιοτικά), δείχνοντας ότι το Δefp στέλεχος έχει μειωμένη αύξηση και ότι τα μεταλλαγμένα στελέχη παρουσιάζουν ευαισθησία σε μη-ριβοσωματικούς αναστολείς, όπως για παράδειγμα την αμπικιλίνη και τη ριφαμπικίνη. Επιπλέον, εξετάσαμε τη δυνατότητα των μεταλλαγμένων στελεχών να ανακάμπτουν στην ανάπτυξή τους παρουσία του κατάλληλου πλασμιδίου και είδαμε ότι ο EFP είναι σημαντικός για την in vivo ανάπτυξη της E. coli σε στρεσσογόνες καταστάσεις. Όπως αναφέρθηκε πρόσφατα, οι YjeA, YjeK και EF-P πρωτεΐνες συμβάλουν στη μείωσης της τοξικότητας στη Salmonella. Επιπλέον, έχει δειχθεί πως ο EF-P είναι μια από τις πρωτεΐνες, που παίζουν σημαντικό ρόλο στην κινητικότητα των βακτηρίων στο Bacillus subtilis. Ωστόσο, έρευνα των Josenhans και Suerbaum το 2002, έδειξε πως η κινιτηκότητα και η τοξικότητα συχνά συνδέονται μεταξύ τους. Έτσι εξετάσαμε, τα μεταλλαγμένα στελέχη για την ικανότητά τους να κινούνται, δημιουργώντας μαστίγια, σε semi-solid θρεπτικό υλικό. Περαιτέρω έρευνες χρησιμοποιώντας external fluorescence staining και confocal microscopy, αποκαλύψε διαφορές στη μορφολογία των μεταλλαγμένων στελεχών της E. coli. Επίσης, με στόχο τη μελέτη της πρωτεΐνης YfcM, η λειτουργία της οποίας δεν είναι ακόμη γνωστή, απομονώσαμε και καθαρίσαμε την YfcM, με Νi-NTA agarose beads και gel filtration για τη μελλοντική κρυσταλοποίησή της,. Τέλος, μελλοντικός στόχος μας είναι η κλωνοποίηση του ακόλουθου πολυκιστρονικού γονιδίου “- yjeK - yjeA - yfcM - Ηis-efp - “, με σκοπό την υπερέκφραση και την κρυσταλλοποίησή του, ώστε να λάβουμε μια εικόνα του πως μοιάζει ολοκληρωμένο το μονοπάτι της τροποποίησης του EF-P, και επιπλέον, να μελετήσουμε καλύτερα τη λειτουργία του EF-P σε εκχυλίσματα της μετάφρασης από διαφορετικές μεταλλάξεις..

Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4. / Dissertation (MSc)--University of Pretoria, 2013. / Biochemistry / unrestricted

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Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. / Russian Foundation for Basic Research / Revisión por pares

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