Pedro J. Alcolea

Transcriptomics throughout the life cycle of Leishmania infantum: high down-regulation rate in the amastigote stage.

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin. The promastigote and amastigote stages alternate in the life cycle of the parasite, developing inside the sand-fly gut and inside mammalian phagocytic cells, respectively. High-throughput genomic and proteomic analyses have not focused their attention on promastigote development, although partial approaches have been made in Leishmania major and Leishmania braziliensis. For this reason we have studied the expression modulation of an etiological agent of visceral leishmaniasis throughout the life cycle, which has been performed by means of complete genomic microarrays. In the context of constitutive genome expression in Leishmania spp. described elsewhere and confirmed here (5.7%), we found a down-regulation rate of 68% in the amastigote stage, which has been contrasted by binomial tests and includes the down-regulation of genes involved in translation and ribosome biogenesis. These findings are consistent with the hypothesis of pre-adaptation of the parasite to intracellular survival at this stage.

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

BackgroundThe extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.ResultsImmunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.ConclusionsThe specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.

Genome-wide analysis reveals increased levels of transcripts related with infectivity in peanut lectin non-agglutinated promastigotes of Leishmania infantum

Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA(-) promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.

Proteome profiling of Leishmania infantum promastigotes.

ABSTRACT. A proteome analysis of the promastigote stage of the trypanosomatid parasite Leishmania infantum (MON‐1 zymodeme) is described here for the first time. Total protein extracts were prepared at early logarithmic and stationary phases of replicate axenic cultures and processed by 2D electrophoresis (pH 3–10). A total of 28 differentially regulated proteins were identified by matrix‐assisted laser desorption/ionization‐tandem time of flight mass spectrometry. This approach has revealed that the electron transfer flavoprotein (ETF) and the eukaryotic elongation factor 1α (eEF1α) subunit have the same differential expression pattern at the protein and mRNA levels, up‐regulation in the stationary phase. A low‐molecular‐weight isoform and an alternatively processed form of the eEF1α subunit have been detected. A 51 kDa subunit of replication factor A is up‐regulated in dividing logarithmic promastigotes. None of the proteins described here shows opposite differential regulation values with the corresponding mRNA levels. Taken together with previous approaches to the proteome and the transcriptome, this report contributes to the elucidation of the differential regulation patterns of the ETF, the eEF1α subunit, the 40S ribosomal protein S12, α‐tubulin and the T‐complex protein 1 subunit γ throughout the life cycle of the parasites from the genus Leishmania.

Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection

Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte

BackgroundLeishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes.ResultsA total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations.ConclusionsThe up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.

Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

Graphical abstract

Structure of tyrosine aminotransferase from Leishmania infantum

The structure of the tyrosine aminotransferase from the parasitic protozoa L. infantum was solved to 2.35 Å resolution. The difference in substrate specificity and enzymatic activity between leishmanial and mammalian TAT is explained based on the presence of two residues (Gln55 and Asn58).

Genome-wide gene expression profile induced by exposure to cadmium acetate in Leishmania infantum promastigotes

Leishmania infantum is the etiological agent of visceral leishmaniasis in Mediterranean areas. The life cycle of the protist is dimorphic and heteroxene, as promastigotes develop inside the gut of sand-fly vectors and amastigotes multiply inside mammalian phagocytic cells. In previous studies, we analyzed the expression profiles of these stages and the modulation of gene expression triggered by temperature increase and acidification, both of which are crucial in the differentiation of promastigotes to amastigotes. Differential expression profiles of translation initiation and elongation factors were detected. Here we report that the presence of 1 mM cadmium acetate in the culture medium leads to a shock response consisting of growth arrest, morphological changes, the absence of motility, and the up-regulation of genes that code for: a heavy metal transporter, trypanothione reductase, a haloacid-dehalogenase-like hydrolase, and a metalloexopeptidase from the M20 family, among others. This response is probably controlled by the differential expression of regulatory genes such as those encoding initiation factors 4E, eukaryotic translation initiation factor 3 subunits 8 and 2α, and elongation factor 1β. The initiation factor 2α gene is induced in anomalous environments, i.e., those outside of the protists normal life-cycle progression, for example, in response to the presence of cadmium ions, acidification without temperature increase, and vice versa. Our results suggest that the regulation of gene expression is a key component of the shock response.