Patogenia da leishmaniose cutânea experimental: a importância da necrose na eliminação dos parasitos das lesões

Um estudo histopatológico e ultraestrutural das lesões da leishmaniose cutânea causada pela Leishmania mexicana amazonensis em duas cepas isogênicas de camundongo, uma susceptível (Balb/c) e outra resistente (A/J), demonstrou que os amastigotas ficavam bem preservados nos vacúolos parasitóforos dos macrófagos, igualmente em ambas as cepas. A reação de imunofluorescência revelou antigenos parasitários no interior e na membrana dos macrófagos de maneira idêntica para ambas as cepas. A diferença ocorria quando os macrófagos apareciam destruídos e as leishmanias ficavam livres ou fagocitadas por polimorfonucleares, neutrófilos e eosinófilos. Estes parasitos exibiam então graus variáveis de nítidas alterações degenerativas. No camundongo resistence, a necrose, de tipo caseoso ou fibrinóide, era mais disseminada e mais freqüente que no animal susceptível. Os achados observados indicaram que as leishmanias não são destruídas no interior dos macrófagos e sim fora deles, especialmente quando fagocitadas por leucócitos polimorfonucleares. A necrose apareceu como o mecanismo mais saliente através do qual o hospedeiro elimina os parasitos das lesões, sendo a mesma um aspecto importante da reação de hipersensibilidade tardia que ocorre nos animais resistentes.

The use of different concentrations of leishmanial antigen in skin testing to evaluate delayed hypersensitivity in american cutaneous leishmaniasis

Three concentrations of Leishmania mexicana amazonensis sonicated whole promastigote antigen (30, 9.6 and 3 ug N in 0.1 ml) wereprepared and 0.1 ml of each inoculated intradermally intopatients who live in one endemic leishmaniasis region in Brazil. Patients were divided into groups with active cutaneous leishmaniasis (ACL), healed cutaneous leishmaniasis (HCL), mucosal leishmaniasis (ML), and Controls (C). Skin reactions were recorded by measuring induration 48 hours after inoculation. Skin tests using 9.6 ugN/0.1 mlyielded the best diagnostic resultssince 97% of 30 patients with active lesions (cutaneous or mucosal) and 83% with HCL showed reactions of 5 mm orgreater as compared with 4% Controls. Tests using 30ug N/O. 1 ml causedan unacceptable levei of skin reactions with necrosis (10% of ACL patients tested and 17% of HCL, respectively). Tests using 3 ug N/O. 1 ml were less sensitive since only 87% of patients with active lesions and 68% with HCL had reactions of 5mm orgreater. The 3 ug N/O. 1 ml dose was utilized to ask the questions whether skin delayed hypersensitivity decreased with time after the initial lesion and whether mucosal involvement is associated with enhaced hypersensitivity to leishmanial antigen. Decreased delayed hypersensitivity was noted only in those patients who had an initial lesion more than 30 years ago. The mean induration of the reaction in 10 patients with ML was 11.3 mm ± 7.15, in 41 patients with HCL, 9.27 mm ± 6.78 and in 20patients with ACL 10. 7 mm ± 6.10 mm. The percent of patients with 5 mm orgreater induration was ML 80%, HCL 71%, ACL 90%. Thus, we could not confirm an association between enhanced delayed hypersensitivity and mucosal involvement in leishmaniasis.

Observações sobre calazar em Jacobina, Bahia. VI - Investigações sobre reservatórios silvestres e comensais

Durante os anos de 1982 a 1986, a investigação sobre mamíferos comensais e silvestres, da periferia da cidade de Jacobina, Bahia, mostrou, ao lado do escasso número de exemplares, uma reduzida variedade específica dessa fauna. Capturou-se apenas 11 espécies, entre as quais, predominou o Didelphis albiventris, que abrangeu 44% dos 213 espécimens capturados. Entre os 193 com exames já concluídos, 84 eram exemplares de D. albiventris e 2 estavam infectados pela Leishmania donovani senso lato, 1 por L. mexicana amazonensis, 1 por L. braziliensis, subespécie e 3 por Trypanosoma cruzi Também foram observadas formas suspeitas de serem amastigotas de leishmanics, nos esfregaços de órgãos de 3 exemplares de Dasyprocta aguti, 1 Cercomys cunicularius - e 1 Oryzomys eliurus. 0 restante dos exemplares, inclusive 14 de Lycalopex vetulus, estava negativo para flagelados. Apesar de reforçado por outros indicadores epidemiológicos, como a predominância específica, a freqüência domiciliar, a atratividade para a vetora Lutzomyia longipalpis, e a concomitância com casos humanos nos mesmos locais, o índice de 2,3% de infecção natural do Didelphis albiventris, não autoriza a conclusão definitiva de ser o marsupial o mais importante reservatório natural da leishmaniose visceral em Jacobina.

American cutaneous leishmaniasis: presentation and problems of patient management

We report our experience with the diagnosis and treatment of 60 patients with American cutaneous leishmaniasis. They were infected in Panama (55), Brazil (4) or Colombia (I). Among 35 patients with a 3 week exposure in Panama, the mean maximum incubation period was 33 days (range 4-81 days). Diagnosis was delayed an average of 93 days after onset of skin lesions, due to the patient's delay in seeking medical attention (31 days), medical personnel's delay in considering the diagnosis (45 days), and the laboratory's delay in confirming the diagnosis (17 days). Forty-four patients (73%) developed ulcers typical of cutaneous leishmaniasis. Sixteen additional patients (27%) had atypical macular, papular, squamous, verrucous or acneiform skin lesions that were diagnosed only because leishmanial cultures were obtained. Of the 59 patients treated with pentavalent antimonial drugs, only 34 (58%) were cured after the first course of treatment. Lesions which were at least 2 cm in diameter, ulcerated, or caused by Leishmania braziliensis were less likely to be cured after a single course of treatment than were lesions smaller than 2 cm, nonulcerated or caused by Leishmania mexicana or Leishmania donovani.

Características epidemiológicas da leishmaniose tegumentar americana em uma região endêmica do Estado da Bahia: III. Fauna flebotomínica

A fauna flebotomínica da região de Três Braços, uma área endêmica de leishmaniose cutânea-mucosa localizada no sudeste do Estado da Bahia, na região cacaueira, é muito variada. Foram identificadas 30 espécies de Lutzomyia em 13.535 exemplares coletados entre os anos de 1976 e 1984. Lu. withmani foi a espécie altamente predominante no ambiente peridoméstico e no interior das residências, com percentuais de 99,0 e 97,5, respectivamente. Na floresta, as espécies predominantes foram Lu. ayrozai e Lu. yuilli, aparecendo Lu. whitmani com apenas 1,0% do total de exemplares examinados. Lu. flaviscutellata, vetor comprovado da Leishmania mexicana amazonensis, foi também coletada em baixos índices. Lu. wellcomei, vetor da L. braziliensis braziliensis na Serra dos Carajás, Pará, Brasil, não foi encontrada na região de Três Braços onde o parasito causando infecções humanas é predominantemente L. b. braziliensis. Embora não se tenha encontrado infecção natural por promastigotas em 1.832 fêmeas de diversas espécies examinadas, discute-se a possibilidade de Lu. whitmani ser um vetor da L.b. braziliensis na região, mantendo, provavelmente, a transmissão entre o cão e o homem.

Seroepidemiological Studies of Cutaneous Leishmaniasis in the Campeche State of México

Seroepidemiological studies of cutaneous leishmaniasis were carried out on 169 individuals in a rural area of the Campeche state of México. Fifty showed cutaneous lesions suggestive of leishmaniasis, 70% were parasite positive and 96% skin test positive. An overall 40% positivity to skin test with Montenegro's antigen was found. Most of the affected individuals were males from 11 to 30 years-old. Antibodies were determined by immunofluorescent antibody test (IFA) and by Western blot. Two antigen preparations were used, one from a Leishmania mexicana strain which produced localized cutaneous leishmaniasis (LCL) and the other from a diffuse cutaneous leishmaniasis (DCL). In the general population from the area of study 19% gave positive IFA tests with DCL antigen and 20% with LCL antigen while for the patients 67% gave positive IFA tests with DCL and 71% with LCL. By Western blot analysis most of the patients recognized more antigens in the DCL than in the LCL strain. In the DCL strain 78% of patients recognized a 105 kDa, 34% a 139 kDa, 28% a 117 kDa and 26% a 205 kDa MW antigen. In the LCL strain 40% of patients recognized a 205 kDa and 22% a 175 kDa antigens

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87% of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 μg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Descripción del ARN de transferencia mitocondrial para Serina (UCN) de Lutzomyia columbiana (Diptera, Psychodidae)

Lutzomyia columbiana es un flebotomíneo considerado como vector sospechoso de Leishmania mexicana y Leishmania braziliensis en Colombia. Este insecto pertenece al grupo verrucarum, que incluye algunos taxones isomórficos, lo que ha estimulado la búsqueda de marcadores moleculares que permitan, además de diferenciar las especies, estudiar sus relaciones de parentesco. En este artículo se describe por primera vez la estructura putativa del ARN de transferencia mitocondrial para serina que reconoce el codón UCN (ARNtSer) de Lu. columbiana. El ADN genómico fue extraído, amplificado y secuenciado a partir de seis especímenes colectados con cebo humano. La estructura secundaria del ARNtSer fue inferida con el programa tRNAscan-SE 1.21. El gen ARNts consistió de 67 pares de bases (pb), encontrándose un solo haplotipo en los seis individuos secuenciados. El ARNtSer de Lu. columbiana mostró 7 apareamientos intracatenarios en el brazo aceptor del aminoácido, 3 en el brazo dihidrouridina (DHU), 5 en el brazo del anticodón y 5 en el brazo ribotimidina-pseudouridina-citosina (TøC). El tamaño de las lupas correspondió a 5 nucleótidos en la DHU, 7 en la anticodón, 4 en la variable y 7 en la TøC. Lu. columbiana se distingue del resto de especies de Lutzomyia y Phlebotomus secuenciadas a la fecha por la presencia de una guanina en la posición nucleotídica 64, que produce un apareamiento no canónico tipo uracilo-guanina en el brazo aceptor. Se necesitan más estudios para confirmar la utilidad del ARNtSer como marcador molecular para la discriminación de especies de flebotomíneos.

The role of Notch receptors in T helper differentiation

After encountering antigens, naïve CD4+ Τ cells can differentiate into various effector Τ helper (Th) cell subsets, including CD4+ Thi, Th2, Thi7, regulatory Τ cells and the recently described follicular Τ helper cells (TFH cells). To date, most of the studies used either gain-of-function approaches that do not reflect the physiological Notch signaling intensity or loss-of-function models that block the entire Notch pathway. The contribution of single Notch receptors during Th differentiation occurring upon infection has not been investigated yet. In the present thesis, we wanted to assess the individual role of Notchi and Notch2 in Th differentiation, by using mice with Τ cell-specific deletion of Notchi, Notch2 or both (NiN2/iCD4Cre) in different models of infection/immunization.¦In the first part, we characterized the role of Notchi and Notch2 in Thi differentiation. We used experimental infection with the protozoan parasite Leishmania major, known to induce a protective Thi immune response in mice on the C57BL/6 background. Mice deficient for both Notchi and Notch2 developed unhealing lesions and were unable to control the parasite burden in their footpad. A profound defect in IFNy secretion by CD4+ Τ cells was shown to be responsible for the susceptibility of these mice. Although CD4+ Τ cells did not secrete IFNy following L. major infection, they exhibited higher IFNymRNA expression as well as higher frequency of CD4+IFNy+Τ cells in dLN. Altogether, these data indicate that Notch is dispensable for the differentiation of Thi cells expressing IFNy but controls, directly or not, the secretion of IFNy, allowing the development of a fully functional Thi immune response.¦In the second part of this thesis, we determined whether Notch is involved in differentiation of follicular Τ helper (TFH) cells. Using different models of immunization (NP-CGG, Schistosoma mansoni eggs) or infection (Leishmania mexicana), we showed that NiN2ACD4Cre mice were unable to generate TFH cells, displayed impaired germinal center (GC) formation as well as a profound defect in high affinity specific-antibodies secretion. We demonstrated an essential and previously unknown role of Notch in TFH cell development, the consequent GC formation and high affinity antibodies secretion, although the mechanisms by which Notch affects TFH development remain to be clearly demonstrated.¦-¦Lors d'une réponse immune, les lymphocytes Τ CD4+ se différencient en différentes sous- populations de lymphocytes Τ auxiliaires (T helper ou Th en anglais) incluant les populations de cellules Thi, Th2, Thn.7, Τ régulatrices ou Τ folliculaires. De nombreuses études ont montré un rôle de la voie de signalisation Notch dans la différentiation des lymphocytes Τ auxiliaires, bien que les résultats soient controversés. A ce jour, la majorité de ces études sont basées sur des modèles de gain de fonction qui ne reflètent pas le niveau physiologique du signal ou des modèles de perte de fonction pour lesquels toute la voie de signalisation est bloquée. De ce fait, nous avons voulu établir le rôle individuel de Notchi et Notch2 dans la réponse immune de type Thi et dans la différentiation des lymphocytes Τ auxiliaires folliculaires avec l'aide de souris déficientes pour Notchi, Notch2 ou les 2 (NiN2ACD4Cre) à la surface de leurs cellules T.¦Dans la première partie de cette thèse, nous avons analysé le rôle de Notch dans la différentiation de type Thi suite à infection avec le parasite Leishmania major, connu pour induire une forte réponse Thi dans des souris de souche C57BL/6. Les souris déficientes pour Notchi et Notch2 développent une importante lésion et sont incapables de contrôler la prolifération du parasite au site d'infection. Le profond défaut de la sécrétion d'IFNy par les cellules Τ des ganglions drainants est probablement responsable de la susceptibilité de ces souris à L. major. Bien que les cellules Τ ne sécrètent pas d'IFNy, nous avons observé des niveaux plus importants d'expression au niveau de l'ARN messager, et une proportion plus élevée de cellules positives pour CD4 et IFNy. Ces résultats indiquent que Notch est nécessaire pour la sécrétion d'IFNy mais pas pour la différentiation de cellules compétentes pour l'IFNy.¦Dans un second temps, nous avons voulu déterminer si Notch est impliqué dans la différentiation des cellules Τ folliculaires. En utilisant divers modèles d'immunisation (avec NP-CGG ou des oeufs de Schistosoma mansoni) ou d'infection (avec L. mexicana), nous avons montré que les souris NlN2ACD4Cre sont incapables de générer des cellules Τ folliculaires. En conséquence, la formation des centres germinatifs et la sécrétion d'anticorps de haute affinité sont profondément affectés. Nous avons démontré dans cette seconde partie un rôle crucial et inconnu à ce jour de Notch dans la différentiation des cellules Τ et en conséquence dans la formation des centres germinatifs et la sécrétion des anticorps de haute affinité, bien que les mécanismes par lesquels Notch contrôle cette différentiation restent à identifier.¦-¦Lors d'une réponse immune, les lymphocytes Τ CD// se différencient en différentes sous- populations de lymphocytes Τ auxiliaires de types Thi, Th2, Thi7, régulatrices ou folliculaires, définies selon la sécrétion de cytokines spécifiques. Le rôle de ces sous-populations dans le contrôle de diverses infections ou leur association avec de nombreuses maladies rend la compréhension des mécanismes de différentiation de ces cellules particulièrement importante. De nombreux facteurs sont impliqués dans ce processus, tels que la présence de diverses cytokines dans l'environnement, la nature de l'antigène ou encore la force de la stimulation. Par ailleurs, de nombreuses études ont montré un rôle de la voie de signalisation Notch dans la différentiation des lymphocytes T, bien que les résultats soient controversés. Dans cette thèse, nous avons voulu évaluer le rôle individuel des récepteurs Notch dans la différentiation des cellules Τ auxiliaires de type Thi et folliculaires à l'aide de souris dont les récepteurs Notch sont spécifiquement absents à la surface des lymphocytes T.¦Dans la première partie, nous avons utilisé le modèle d'infection au parasite Leishmania major, connu pour induire une forte réponse protectrice de type Thi dans la majorité des souches de souris. Suite à l'infection, les souris déficientes pour les récepteurs Notch sont incapables de contrôler la prolifération du parasite et développent une importante lésion au site d'infection. Cette susceptibilité est due à l'incapacité des cellules Τ auxiliaires à sécréter une cytokine spécifique des cellules de type Thi et nécessaire à l'éradication du parasite, l'IFNy. Ces résultats indiquent que les récepteurs Notch sont indispensables au développement d'une réponse Thi fonctionnelle, permettant la guérison suite à l'infection avec L. major.¦Dans la deuxième partie de cette thèse, nous avons voulu déterminer si Notch est impliqué dans la différentiation des lymphocytes Τ folliculaires. Ces cellules ont la particularité d'aider les lymphocytes Β à former des centres germinatifs au sein desquels les lymphocytes Β prolifèrent et sécrètent des anticorps, un processus nécessaire à la protection contre les pathogènes. Actuellement, l'efficacité de la majorité des vaccins repose sur la sécrétion d'anticorps par les lymphocytes B, aidés par les cellules Τ folliculaires. En raison du rôle important de ces cellules dans l'éradication des pathogènes et lors d'un processus de vaccination, il est important de connaître les facteurs et les mécanismes permettant la différentiation de ces cellules. Dans cette étude, nous montrons que la formation des cellules Τ folliculaires dépend de la voie de signalisation Notch, impliquant un rôle essentiel de cette molécule dans l'induction de la sécrétion d'anticorps par les lymphocytes B.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Structural basis for selective inhibition of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase: Molecular docking and 3D QSAR studies

The glycolytic enzyme glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) is as an attractive target for the development of novel antitrypanosomatid agents. In the present work, comparative molecular field analysis and comparative molecular similarity index analysis were conducted on a large series of selective inhibitors of trypanosomatid GAPDH. Four statistically significant models were obtained (r(2) > 0.90 and q(2) > 0.70), indicating their predictive ability for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results. Molecular modeling studies provided further insight into the structural basis for selective inhibition of trypanosomatid GAPDH.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.