Molecular crosstalks in Leishmania-sandfly-host relationships

Sandflies (Diptera: Phlebotominael are vectors of Leishmania parasites, causative agents of important human and animal diseases with diverse manifestations. This review summarizes present knowledge about the vectorial part of Leishmania life cycle and parasite transmission to the vertebrate host. Particularly, it focuses on molecules that determine the establishment of parasite infection in sandfly midgut. It describes the concept of specific versus permissive sandfly vectors, explains the epidemiological consequences of broad susceptibility of permissive sandflies and demonstrates that genetic exchange may positively affect Leishmania fitness in the vector. Last but not least, the review describes recent knowledge about circulating antibodies produced by hosts in response to sandfly bites. Studies on specificity and kinetics of antibody response revealed that anti-saliva IgG could be used as a marker of host exposure to sandflies, i.e. as a useful tool for evaluation of vector control.     

Anti-leishmanial and anti-trypanosomal activities of 1,4-dihydropyridines: In vitro evaluation and structure-activity relationship study

Leishmaniasis and Chagas' disease constitute a relevant health and socio-economic problem in Latin America, Africa, and Asia. The therapeutic interventions rely on inefficient and highly toxic drugs with systemic side effects in patients. Considering the multiple biological activities of the calcium channel blockers and the high versatility of 1,4-dihydropyridines, eight clinically used 1,4-dihydropyridines (azelnidipine, amlodipine, cilnidipine, lercanidipine, nicardipine, nifedipine, nimodipine and nitrendipine) were in vitro tested against Leishmania and Trypanosoma cruzi parasites, and their cytotoxicity was tested against mammalian cells. In addition, a QSAR study was performed in order to delineate further structural requirements for the anti-protozoan activity and to predict the biological potency of 1,4-dihydropyridines. The tested compounds were effective against Leishmania (L.) amazonensis, Leishmania (V.)braziliensis, Leishmania (L.) chagasi, and Leishmania (L.) major promastigotes, L. (L.) chagasi intracellular amastigotes and T. cruzi trypomastigotes with 50% inhibitory concentration (IC(50)) values in the range of 2.6-181μM. The QSAR provided useful information about the structural features of the anti-protozoan activities, including diphenylpropyl and diphenylmethylazetidin groups at position 4 of the 1,4-dihydropyridine ring, allowing the prediction of two novel potential anti-protozoan analogs.     

Consequences of the natural propensity of Leishmania and HIV-1 to target dendritic cells

Recent studies have shown that both Leishmania and HIV type-1 (HIV-1) hijack dendritic cell (DC) functions to escape immune surveillance using an array of elaborate strategies. Leishmania has developed a variety of adaptations to disrupt cellular defense mechanisms, whereas HIV-1 targets DCs to achieve a more efficient dissemination. The capacity of Leishmania and HIV-1 to target DCs through a common cell-surface molecule, namely DC-SIGN (dendritic cell specific ICAM-3-grabbing non-integrin), points to a possible dangerous liaison between these two pathogens. This review explores our knowledge of how Leishmania and HIV-1 interact dynamically with DCs, and how they exploit this cell type for their reciprocal benefit.     

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) Complex and the Lizard Leishmania: Their Classification and Evidence for a Neotropical Origin of the Leishmania-Endotrypanum Clade

ABSTRACT. The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania . The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.     

ABC transporters in the protozoan parasite Leishmania

ATP-binding cassette (ABC) transporters constitute one of the biggest and most conserved protein families in the evolutionary scale. Many of them are of enormous clinical relevance, due to their relationship with genetic diseases and drug resistance during the treatment of cancer and infectious diseases. Leishmaniasis is a major and globally widespread group of parasitic diseases, whose treatment has been complicated by the expansion of resistance to conventional drugs. Here, we review the current knowledge about ABC transporters in Leishmania spp, with special attention to their relationship with the drug-resistance phenotype.     

Folylpolyglutamates in Leishmania major

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.     

Effect of natural L- to D-amino acid conversion on the organization, membrane binding, and biological function of the antimicrobial peptides bombinins H

Antimicrobial peptides (AMPs) are evolutionarily old components of innate immunity found in all living pluricellular organisms. Interestingly, some organisms express families of AMPs with only a slight variation among their members, possibly to increase their spectrum of activity. Despite the growing body of knowledge about their biological activity and mode of action on bacteria, only a few of them have been tested on Leishmania, a worldwide spread protozoan pathogen, and the parameters contributing to this activity are yet to be determined. We report on the anti-Leishmania activity and mode of action of bombinins H2 and H4 isolated from the skin secretion of the frog Bombina variegata. H4, the most active, is the first natural AMP of animal origin with a single L- to D-amino acid isomerization. Membrane depolarization and membrane permeation assays, as well as electron microscopy, suggest that the lethal mechanism involves plasma membrane permeation and/or disruption. To better understand the enhanced activity of H4, we determined the peptide's structure in membranes mimicking those of mammals, bacteria, and Leishmania by using ATR-FTIR and CD spectroscopies and assessed their membrane binding by using surface plasmon resonance. The data reveal that (i) H2 but not H4 partially aggregates in membranes mimicking those of Leishmania, (ii) H2 is slightly more helical than H4 in all membranes, and (iii) H4 binds the Leishmania model membrane approximately 5-fold better than H2. This study highlights the importance of a single alpha-amino acid epimerization as a tool used by nature to modulate the activity of AMPs. In addition, our findings suggest bombinins H as potential templates for the development of new drugs with a new mode of action against Leishmania.     

Characterisation of a developmentally regulated amino acid transporter gene from Leishmania amazonensis

The metabolism of protozoan parasites of the Leishmania genus is strongly based on amino acid consumption, but little is known about amino acid uptake in these organisms. In the present work, we identified a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid transporter that belongs to the amino acid/auxin permease family, a group of H(+)/amino acid symporters. This single copy gene is upregulated in amastigotes, the life cycle stage found in the mammalian host. La-PAT1 putative orthologous sequences were identified in Leishmania infantum, Leishmania donovani, Leishmania major and Trypanosoma.     

Deciphering the Leishmania exoproteome: what we know and what we can learn

Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis. Survival and transmission of these parasites in their different hosts require membrane-bound or extracellular factors to interact with and modify their host environments. Over the last decade, several approaches have been applied to study all the extracellular proteins exported by an organism at a particular time or stage in its life cycle and under defined conditions, collectively termed the secretome or the exoproteome. In this review, we focus on emerging data shedding light on the secretion mechanisms involved in the production of the Leishmania exoproteome. We also describe other methodologies currently available that could be used to analyse the Leishmania exoproteome. Understanding the complexity of the Leishmania exoproteome is a key component to elucidating the mechanisms used by these parasites for exporting proteins to the extracellular space during its life cycle. Given the importance of extracellular factors, a detailed knowledge of the Leishmania exoproteome may provide novel targets for rational drug design and/or a source of antigens for vaccine development.     

Water soluble cationic trans-platinum complexes which induce programmed cell death in the protozoan parasite Leishmania infantum

We have evaluated the cytotoxic properties against the protozoan Leishmania infantum of four water soluble cationic trans-Pt(II)Cl(2) compounds containing as inert groups NH3 and piperazine (1), 4-picoline and piperazine (2), n-butylamine and piperazine (3), and NH3 and 4-piperidino-piperidine (4). The leishmanicidal activity of compounds 3 and 4 against promastigotes of the parasite Leishmania infantum was 2.5- and 1.6-times higher than that of the cytotoxic drug cis-diamminedichloroplatinum(II), respectively. Interestingly, compounds 3 and 4 produce in Leishmania infantum promastigotes a higher amount of programmed cell death than cisplatin, which is associated with cell cycle arrest in G2/M. In contrast to cis-diamminedichloroplatinum(II), binding of compounds 3 and 4 to calf thymus DNA induces conformational changes more similar to those of trans-diamminedichloroplatinum(II) that may be attributed to denaturation of the double helix. Similarly to cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II), the interaction of compounds 3 and 4 with ubiquitin results in an increase of the alpha-helix content of the protein as observed by circular dichroism spectroscopy. However, fluorescence studies indicate that compounds 3 and 4 produce a decrease in the fluorescence of the tyrosine 59 residue of ubiquitin higher than both cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II). Altogether, our results suggest that the biochemical mechanism of cytotoxic activity of compounds 3 and 4 against Leishmania infantum must be different from that of cis-diamminedichloroplatinum(II). To the best of our knowledge, compounds 3 and 4 are the first reported trans-platinum complexes that show antiparasitic activity.     

LD1 amplifications in Leishmania

The genome of Leishmania is quite plastic. Chromosomal rearrangements and DNA amplifications are common events in all the species of the genus. Gene amplification occurs both as a mechanism of drug resistance and in the absence of drug pressure. The best known spontaneous amplification in Leishmania is the so-called LD1 family of amplicons. In the past few years there have been great advances in our knowledge of LD1 elements; here, Manuel Segovia and Ginés Ortiz review all the available data.     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

Protein S-myristoylation in Leishmania revealed with a heterologous reporter

Reversible esterification of myristic acid to cysteine residue(s) (S-myristoylation) was documented recently in the protozoan Trypanosoma brucei. Unlike N-myristoylation, S-myristoylation appears to be rare (or non-existent) in animal cells and has not been documented in any other trypanosome. Reasoning that a lack of knowledge of appropriate substrates may have contributed to this state of affairs, we devised an assay to test for protein S-myristoylation in the ancient eukaryote Leishmania. A cDNA encoding a glycosylphosphatidylinositol-phospholipase C (GPI-PLC) from T. brucei was transfected into Leishmania and the expressed protein analyzed for covalent lipid modifications. Leishmania modified the reporter with myristate in a thio-ester linkage. From these observations, we infer that (i) GPI-PLC may be used as a reporter of this lipid modification in eukaryotes, and (ii) protein S-myristoylation might have ancient origins.     

Molecular trees of trypanosomes incongruent with fossil records of hosts

Molecular trees of trypanosomes have confirmed conventionally accepted genera, but often produce topologies that are incongruent with knowledge of the evolution, systematics, and biogeography of hosts and vectors. These distorted topologies result largely from incorrect assumptions about molecular clocks. A host-based phylogenetic tree could serve as a broad outline against which the reasonability of molecular phylogenies could be evaluated. The host-based tree of trypanosomes presented here supports the " invertebrate first " hypothesis of trypanosome evolution, supports the monophyly of Trypanosomatidae, and indicates the digenetic lifestyle arose three times. An area cladogram of Leishmania supports origination in the Palaearctic during the Palaeocene.     

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.     

Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA

A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of Leishmania spp. in areas of endemicity.     

Ticks as vectors of Leishmania parasites

Sand flies are the only accepted biological vectors of Leishmania parasites. However, secondary modes of transmission have been extensively discussed and speculated about in recent years. In particular, the hypothesis of ticks as vectors of Leishmania infantum was studied in the 20th century and today is being revisited using modern molecular biology techniques. Recent studies have shed new light on the discussion, but have also led to misleading conclusions on the role of ticks as Leishmania vectors. In this article, the role of brown dog ticks, Rhipicephalus sanguineus, as vectors of L. infantum is discussed, and the need for further research to better understand their participation in the epidemiology of leishmaniasis is advocated.     

Identification of substrates of an S-phase cell cycle kinase from Leishmania donovani

Despite the importance of cyclin-Cdk related kinases (CRK) in regulation of cell and life cycle of kinetoplastida parasites, only limited knowledge about their substrates are presently available. Here, the potential substrates were searched for an S-phase LdCyc1-CRK3 complex from Leishmania donovani based on the presence of Cdk target phosphorylation site together with the cyclin interacting Cy-motif in genome-derived putative protein sequences. Three substrates could be identified with one of them being a unique protein with no known homologues. Another identified substrate is similar to MYST family of histone acetyl transferase and the third one contains Ku-70 related conserved domains. All the substrates interact directly with LdCyc1 and are phosphorylated in a Cy-motif dependent manner suggesting the importance of Cy-motif for their functions.