Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. RESULTS: In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. CONCLUSIONS: The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis.

BACKGROUND: RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight) known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether Leishmania PUF proteins result antigenic during visceral leishmaniasis (VL). FINDINGS: Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in Leishmania infantum. Additionally, the 10 genes of the L. infantum PUF family were cloned and used to express the Leishmania PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The Leishmania PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients. CONCLUSION: Our results suggest that Leishmania PUFs are targets of the humoral response during L. infantum infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as Trypanosoma cruzi.

Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes.

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship.

BACKGROUND: Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. RESULTS: By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. CONCLUSION: SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in post-transcriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions (AU)

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Immunization strategies against visceral leishmaniosis with the nucleosomal histones of Leishmania infantum encoded in DNA vaccine or pulsed in dendritic cells.

Immunization of BALB/c mice with a DNA vaccine encoding the nucleosomal histones from Leishmania infantum resulted in a complete failure of protection against visceral leishmaniosis (VL), whereas the adoptive transfer of bone marrow-derived dendritic cells pulsed with the same pathoantigens plays an essential role in controlling parasite growth in half of the cases. Reduction of the visceral parasite burden seems to be related to low persistence of regulatory T-cells in the spleen from vaccinated mice. These results provide clues for the optimization of this vaccine strategy with the four Leishmania nucleosomal histones against L. infantum infection.

The Leishmania HSP20 is antigenic during natural infections, but, as DNA vaccine, it does not protect BALB/c mice against experimental L. amazonensis infection.

Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis.

Transitory or long-lasting immunity to Leishmania major infection: the result of immunogenicity and multicomponent properties of histone DNA vaccines.

The present studies were designed to analyze the immunization against cutaneous leishmaniosis with plasmids encoding Leishmania histones either individually or genetically linked in tandem, or with cocktails encoding the four nucleosomal histones (H2A, H2B, H3 and H4). Genetic immunization of BALB/c mice with the individual histones only resulted in a delay in lesion development, whereas the immunization with any one of the plasmids encoding a pair of histones provided stronger, though still partial protection against Leishmania major infection compared to the combination of the four histones. These results provide direct evidence that all four nucleosomal histones of Leishmania are necessary to maintain complete protection against L. major reinfection.

The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L: amazonensis Infection

Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis(AU)

Protozoos del género Leishmania son agentes causantes de la leishmaniasis, que es un importante problema de salud tanto en medicina humana y veterinaria. Aquí se describe una nueva proteína de choque térmico (HSP) de Leishmania, que pertenecen a la pequeña HSP (sHSP) en kinetoplastids familia. La proteína está altamente conservada en diferentes especies de Leishmania, mostrando importantes diferencias en lugar sHSP con la de otros organismos. La respuesta humoral contra esta proteína suscitado durante la infección por Leishmania se ha investigado en seres humanos infectados naturales y perros, y en hámsters infectados experimentalmente. Leishmania HSP20 es un antígeno para canina anfitriones, por el contrario, la proteína parece ser un pobre de antígenos para el sistema inmunitario humano. Curso de análisis de tiempo de aparición de anticuerpos anti-HSP20 en hamsters dorados indicó que estos anticuerpos se producen en etapas tardías de la infección, cuando los síntomas clínicos de la enfermedad son patentes. Por último, la eficacia protectora de HSP20 se evaluó en ratones utilizando una vacuna de ADN antes de enfoque desafío con Leishmania amazonensis

Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania.

Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.

A postgenomic view of the heat shock proteins in kinetoplastids.

The kinetoplastids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi are causative agents of a diverse spectrum of human diseases: leishmaniasis, sleeping sickness and Chagas' disease, respectively. These protozoa possess digenetic life cycles that involve development in mammalian and insect hosts. It is generally accepted that temperature is a triggering factor of the developmental programme allowing the adaptation of the parasite to the mammalian conditions. The heat shock response is a general homeostatic mechanism that protects cells from the deleterious effects of environmental stresses, such as heat. This response is universal and includes the synthesis of the heat-shock proteins (HSPs). In this review, we summarize the salient features of the different HSP families and describe their main cellular functions. In parallel, we analyse the composition of these families in kinetoplastids according to literature data and our understanding of genome sequence data. The genome sequences of these parasites have been recently completed. The HSP families described here are: HSP110, HSP104, group I chaperonins, HSP90, HSP70, HSP40 and small HSPs. All these families are widely represented in these parasites. In particular, kinetoplastids possess an unprecedented number of members of the HSP70, HSP60 and HSP40 families, suggesting key roles for these HSPs in their biology.

The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3'-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 degrees C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 degrees C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 degrees C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 degrees C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 degrees C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 degrees C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the deltaHSP70 mutant synthesizes HSP70 at a lower rate than the wild-type parasites. Overall, our data suggest that the biological function of the HSP70-II gene is to top up HSP70 levels under conditions of stress.

Changes in Haemoproteus sex ratios: fertility insurance or differential sex lifespan?

There is little direct evidence of the fitness effects of changes in malaria gametocyte sex ratio. Gametocyte sex ratios in haemospororin parasites (phylum Apicomplexa) are usually female skewed. However, in some cases and especially in Haemoproteus parasites, less female-biased and even male-biased sex ratios are encountered. The 'fertility insurance hypothesis' tries to explain these biases as an evolutionary strategy to facilitate gamete encounter. Thus, the hypothesis predicts that, if there is a reduction in gametocyte density (intensity of infection) or other factors preventing gametes from meeting, a change to a higher proportion of male gametocytes may be favoured. By contrast, a change in sex ratio may be caused by other non-adaptive mechanisms, for example differential survival of the gametocytes of each sex. We study within-host changes in Haemoproteus majoris sex ratios following an experimental reduction in the density of the parasites in the blood in a breeding population of blue tits (Parus caeruleus). Medication with the antimalarial drug primaquine induced a significant reduction in Haemoproteus gametocyte infection intensity in two different breeding seasons and under two different doses of medication. Sex ratios became male skewed following the experimental treatment in agreement with the predictions of the 'fertility insurance' hypothesis. Also in support of the hypothesis, a significant change towards male-biased sex ratios emerged for non-medicated birds in one year, probably owing to the natural immune reduction of the density of the parasites in the blood. The alternative possibility that changes are caused by different lifespans of gametocytes is not supported by changes in sex ratios in control hosts, where new production and release of gametocytes occur.

The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation.

BACKGROUND: Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation. RESULTS: Here we report the studies on the expression of the heat shock protein 83 (HSP83) genes of Leishmania infantum. Confirming previous observations for other Leishmania species, we found that the L. infantum HSP83 transcripts also show a temperature-dependent accumulation that is controlled by a post-transcriptional mechanism involving sequences located in the 3'-untranslated region (3'-UTR). However, contrary to that described for L. amazonensis, the accumulation of the HSP83 transcripts in L. infantum is dependent on active protein synthesis. The translation of HSP83 transcripts is enhanced during heat shock and, as first described in L. amazonensis, we show that the 3'-UTR of the L. infantum HSP83 gene is essential for this translational control. Measurement of the steady-state levels of HSP83 transcripts along the promastigote-to-amastigote differentiation evidenced a specific profile of HSP83 RNAs: after an initial accumulation of HSP83 transcripts observed short after (2 h) incubation in the differentiation conditions, the amount of HSP83 RNA decreased to a steady-state level lower than in undifferentiated promastigotes. We show that this transient accumulation is linked to the presence of the 3'-UTR and flanking regions. Again, an 8-fold increase in translation of the HSP83 transcripts is observed short after the initiation of the axenic differentiation, but it is not sustained after 9 h. CONCLUSIONS: This transient expression of HSP83 genes could be relevant for the differentiation of Leishmania, and the underlying regulatory mechanism may be part of the developmental program of this parasite.

Cell-cycle-dependent translation of histone mRNAs is the key control point for regulation of histone biosynthesis in Leishmania infantum.

The cell-cycle-dependent expression of the four core histones (H2A, H2B, H3 and H4) has been studied in the protozoan parasite Leishmania infantum. For that purpose, the cell cycle was arrested by incubation of promastigotes with the DNA synthesis inhibitor hydroxyurea, which induced an accumulation of cells stalled in G1 phase. Hydroxyurea release resulted in a semi-synchronous entry into the cell cycle, as determined by flow cytometry. The steady-state levels of histone mRNAs in the G1, S and G2/M phases were found to be constant along the cell cycle. However, the levels of histone synthesis increased when parasites enter the S phase, in agreement with previous results showing that histone synthesis in Leishmania is tightly coupled with DNA replication. In addition, we analysed the distribution of histone mRNAs on polyribosomes at different stages of the cell cycle by separation of cytoplasmic RNAs in sucrose gradients. Remarkably, a drastic change in the polysome profiles of histone mRNAs was observed during the progression from G1 to S phase. Thus, in the S phase, histone mRNAs are present in ribosome-bound fractions, but in the G1 phase, the histone transcripts are exclusively found in the ribosome-free fractions. These results support a regulatory model in which the cell-cycle-regulated synthesis of histones in Leishmania is controlled through a reversible interaction between translational repressors and histone mRNAs.