Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneider's Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneider's Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneider's Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection.     

Developmental Life Cycle of Leishmania—Cultivation and Characterization of Cultured Extracellular Amastigotes1

ABSTRACT. The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host-macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease-maintaining stage of this parasite.     

Dissecting Leishmania infantum Energy Metabolism - A Systems Perspective

Leishmania infantum, causative agent of visceral leishmaniasis in humans, illustrates a complex lifecycle pertaining to two extreme environments, namely, the gut of the sandfly vector and human macrophages. Leishmania is capable of dynamically adapting and tactically switching between these critically hostile situations. The possible metabolic routes ventured by the parasite to achieve this exceptional adaptation to its varying environments are still poorly understood. In this study, we present an extensively reconstructed energy metabolism network of Leishmania infantum as an attempt to identify certain strategic metabolic routes preferred by the parasite to optimize its survival in such dynamic environments. The reconstructed network consists of 142 genes encoding for enzymes performing 237 reactions distributed across five distinct model compartments. We annotated the subcellular locations of different enzymes and their reactions on the basis of strong literature evidence and sequence-based detection of cellular localization signal within a protein sequence. To explore the diverse features of parasite metabolism the metabolic network was implemented and analyzed as a constraint-based model. Using a systems-based approach, we also put forth an extensive set of lethal reaction knockouts; some of which were validated using published data on Leishmania species. Performing a robustness analysis, the model was rigorously validated and tested for the secretion of overflow metabolites specific to Leishmania under varying extracellular oxygen uptake rate. Further, the fate of important non-essential amino acids in L. infantum metabolism was investigated. Stage-specific scenarios of L. infantum energy metabolism were incorporated in the model and key metabolic differences were outlined. Analysis of the model revealed the essentiality of glucose uptake, succinate fermentation, glutamate biosynthesis and an active TCA cycle as driving forces for parasite energy metabolism and its optimal growth. Finally, through our in silico knockout analysis, we could identify possible therapeutic targets that provide experimentally testable hypotheses.     

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background

Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Methodology/Principal Findings

We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain.

Conclusions/Significance

The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.     

Evolutionary Processes Acting on Candidate cis-Regulatory Regions in Humans Inferred from Patterns of Polymorphism and Divergence

Analysis of polymorphism and divergence in the non-coding portion of the human genome yields crucial information about factors driving the evolution of gene regulation. Candidate cis-regulatory regions spanning more than 15,000 genes in 15 African Americans and 20 European Americans were re-sequenced and aligned to the chimpanzee genome in order to identify potentially functional polymorphism and to characterize and quantify departures from neutral evolution. Distortions of the site frequency spectra suggest a general pattern of selective constraint on conserved non-coding sites in the flanking regions of genes (CNCs). Moreover, there is an excess of fixed differences that cannot be explained by a Gamma model of deleterious fitness effects, suggesting the presence of positive selection on CNCs. Extensions of the McDonald-Kreitman test identified candidate cis-regulatory regions with high probabilities of positive and negative selection near many known human genes, the biological characteristics of which exhibit genome-wide trends that differ from patterns observed in protein-coding regions. Notably, there is a higher probability of positive selection in candidate cis-regulatory regions near genes expressed in the fetal brain, suggesting that a larger portion of adaptive regulatory changes has occurred in genes expressed during brain development. Overall we find that natural selection has played an important role in the evolution of candidate cis-regulatory regions throughout hominid evolution.     

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.     

GIP: an open-source computational pipeline for mapping genomic instability from protists to cancer cells

Genome instability has been recognized as a key driver for microbial and cancer adaptation and thus plays a central role in many diseases. Genome instability encompasses different types of genomic alterations, yet most available genome analysis software are limited to just one type of mutation. To overcome this limitation and better understand the role of genetic changes in enhancing pathogenicity we established GIP, a novel, powerful bioinformatic pipeline for comparative genome analysis. Here, we show its application to whole genome sequencing datasets of Leishmania, Plasmodium, Candida and cancer. Applying GIP on available data sets validated our pipeline and demonstrated the power of our tool to drive biological discovery. Applied to Plasmodium vivax genomes, our pipeline uncovered the convergent amplification of erythrocyte binding proteins and identified a nullisomic strain. Re-analyzing genomes of drug adapted Candida albicans strains revealed correlated copy number variations of functionally related genes, strongly supporting a mechanism of epistatic adaptation through interacting gene-dosage changes. Our results illustrate how GIP can be used for the identification of aneuploidy, gene copy number variations, changes in nucleic acid sequences, and chromosomal rearrangements. Altogether, GIP can shed light on the genetic bases of cell adaptation and drive disease biomarker discovery.     

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms.     

Leishmania donovani Infection Causes Distinct Epigenetic DNA Methylation Changes in Host Macrophages

Infection of macrophages by the intracellular protozoan Leishmania leads to down-regulation of a number of macrophage innate host defense mechanisms, thereby allowing parasite survival and replication. The underlying molecular mechanisms involved remain largely unknown. In this study, we assessed epigenetic changes in macrophage DNA methylation in response to infection with L. donovani as a possible mechanism for Leishmania driven deactivation of host defense. We quantified and detected genome-wide changes of cytosine methylation status in the macrophage genome resulting from L. donovani infection. A high confidence set of 443 CpG sites was identified with changes in methylation that correlated with live L. donovani infection. These epigenetic changes affected genes that play a critical role in host defense such as the JAK/STAT signaling pathway and the MAPK signaling pathway. These results provide strong support for a new paradigm in host-pathogen responses, where upon infection the pathogen induces epigenetic changes in the host cell genome resulting in downregulation of innate immunity thereby enabling pathogen survival and replication. We therefore propose a model whereby Leishmania induced epigenetic changes result in permanent down regulation of host defense mechanisms to protect intracellular replication and survival of parasitic cells.     

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.