Leishmania: Responding to environmental signals and challenges without regulated transcription.

Here we describe the non-canonical control of gene expression in Leishmania, a single-cell parasite that is responsible for one of the major neglected tropical diseases. We discuss the lack of regulated RNA synthesis, the post-transcriptional gene regulation including RNA stability and regulated translation. We also show that genetic adaptations such as mosaic aneuploidy, gene copy number variations and DNA sequence polymorphisms are important means for overcoming drug challenge and environmental diversity. These mechanisms are discussed in the context of the unique flow of genetic information found in Leishmania and related protists.

Introduction

Infections by parasites of the genus Leishmania manifest in three main forms: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL), which together are counted among the most important neglected tropical diseases [1]. Leishmaniasis is endemic in large parts of the tropical and subtropical regions of the World and poverty-related [2]. The genus Leishmania is part of the order Trypanosomatida and of the early-branching phylum Euglenozoa [3], [4]. Leishmania spp. are closely related to other important pathogens, such as the causative agents of human sleeping sickness, Trypanosoma brucei ssp, and T. cruzi, the aetiologic agent of Chagas disease.

There are over 20 human-pathogenic species of Leishmania, divided into sub-genera, of which Leishmania and Viannia [5], [6] are the most prominent. All leishmaniae undergo a biphasic life cycle with two main morphologically distinct stages. In their vectors, sandflies such as Phlebotomus spp. and Lutzomyia spp., leishmaniae proliferate rapidly as elongated, flagellated promastigotes within the midgut of females, which require mammalian blood for fertility. These promastigotes undergo small morphological changes during the insect stage, emerging as infective, highly motile forms that upon injection into the mammalian skin during the next sandfly blood meal end up in phagocytic, antigen-presenting cells (APCs). Within those, they undergo a critical differentiation into ovoid, aflagellated, non-motile amastigotes that reside and proliferate inside the phagosomes of macrophages and other APCs. By shedding protein-loaded exosomes [7], they can modulate macrophage activity and thus ensure their survival.

Proliferating amastigotes will destroy the host cell, escape and infect new APCs. The ensuing immune reaction involves influx of various immune cells, inflammatory responses, tissue swelling and destruction, either restricted to the infection site (CL), spreading through the lymphatic system (diffuse CL, MCL), or afflicting the major lymphatic organs (spleen, liver, bone marrow) in VL. The latter is almost invariably lethal in untreated cases or when therapeutic options fail. The manifestation of Leishmania infections in CL, MCL or VL is mostly preordained by the infecting species, but the host immune status has also a considerable influence on the outcome. In recent years, it has become increasingly clear that not all Leishmania infections cause a symptomatic disease and that parasites can persist in humans in subclinical infections for years [8]. These subclinical cases are thought to constitute an important reservoir for anthroponotic Leishmania species but may exacerbate upon permanent or temporary immune suppression.

Conversion from the insect stage to the mammalian stage requires Leishmania to change its gene expression. While gene expression in most eukaryotes is controlled mainly at the level of transcription initiation, by cis-acting promoters and trans-acting transcription factors [9], Leishmania spp. and the entire order Trypanosomatida lack gene-specific, regulated transcription by RNA Polymerase II, missing both canonical promoter elements and genes for cognate transcription factors [10]. Rather, Leishmania chromosomes comprise of large polycistronic transcription units (PTUs) of functionally unrelated genes [11], [12]. The regions between the PTUs are called strand switch regions where transcription is initiated and terminated [13], [14] (Fig. 1). However, episomal DNA, e.g. plasmid- or cosmid-based transgenes, are efficiently transcribed [15], [16], without harbouring strand switch region DNA. This argues for additional, unspecific initiation of RNA synthesis. The polycistronic pre-mRNAs are processed into mature mRNAs by coupled trans-splicing of a leader RNA and polyadenylation [17]. With very few exceptions, Leishmania genes lack introns and cis-splicing [18], eliminating alternative splicing as a means of gene regulation.

Fig. 1

Polycistronic transcription and RNA processing in Leishmania. The figure shows the distribution of coding sequences (CDSs) on the L. donovani chromosome 2. Arrows in red signify upper strand CDSs, green arrows indicate lower strand CDSs. Polycistronic transcripts are generated and processed into mature, monocistronic mRNA by trans-splicing of a 39-nucleotide spliced leader RNA (SL) and coupled poly-adenylation (poly A). The SL also contributes the CAP structure (yellow diamond) to the mRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The differentiation from the non-pathogenic promastigote form of the insect host to the pathogenic amastigote stage is induced by factors that are not yet fully understood. It is known, however, that for a range of Leishmania spp., an acidic pH of 5.5 and a temperature increase to 33–37 °C are the major triggers for differentiation even under axenic culture conditions [19], [20]. Conversion into amastigotes is crucial for survival within the mammalian host and thus plays an important role in the pathogenicity of Leishmania spp.. The pivotal role of heat shock proteins in differentiation and adaption was proposed early and has been extensively studied in Leishmania [21].

In the following, we shall discuss the ways by which Leishmania can respond to environmental stimuli using post-transcriptional gene regulation and adapt to environmental challenges by constitutive genetic diversity and selection.

Post-transcriptional responses to environmental signals

Post transcriptional regulation

The response to environmental stimuli is mediated by post-transcriptional regulation of gene expression in Leishmania spp., a fact that was demonstrated by nuclear run-on experiments and using the universal cellular heat shock response as model [22], [23]. Nuclear run-on and microarray studies showed that Leishmania genes are constitutively transcribed, consistent with the lack of gene-specific transcription regulation. However, a global down regulation of RNA synthesis was observed for axenic amastigotes when compared with promastigotes [24], [25]. The stage differentiation process itself is associated with a globally increased protein synthesis [26], but also with specific increases for amastigote stage-specific proteins [27].

The role of mRNA stability and translation efficiency

While early research focused mainly on mRNA levels of selected genes, later proteome analysis and system-wide RNA abundance studies revealed that the regulation of Leishmania gene expression occurs on levels other than RNA synthesis, i.e. RNA processing and stability [28]. Even then, changes of mRNA abundance do not necessarily translate into corresponding protein synthesis rates or abundance, hinting at translation efficiency and protein half life as additional targets of regulation [29], [30], [26], [31], [32], [33].

RNA half life as a post-transcriptional regulatory mechanism was shown for resistance to antimony, which differs in various Leishmania species and correlates with the stability of the mRNA coding for aquaglyceroporin (AQP1) and is mediated by the 3′-untranslated region (3′ UTR) [34]. RNA stability is regulated by non-long terminal repeat retrotransposons in the 3′ UTR. In L. major, two families of these Short Interspersed DEgenerated Retroposons (SIDER) could be identified, LmSIDER1 and LmSIDER2. For SIDER2 it was demonstrated that it destabilises the mRNA and even leads to an mRNA decay [35], [36], while the SIDER1 family can regulate mRNA translation in a stage-specific manner [37].

Ribosome profiling shows a regulated translation in Leishmania

RNA-Seq analysis is a powerful tool to obtain a genome-wide view of mRNA abundance patterns and is often seen as the equivalent to genome-wide gene expression analysis. However, in Leishmania and the related Trypanosoma spp., inducible mRNA abundance and corresponding protein synthesis/abundance show only a limited correlation [26], [33], [38], [39], [40]. To study the effect of environmental triggers on gene expression in the parasite, the steady-state level of proteins or translation rates can be measured. The former can be done by mass spectrometry-based proteomics [26], [27], [41] and the latter is achieved by ribosome profiling analysis [42] or a combined metabolic labeling/mass-spectrometry strategy [43]. The combination of ribosome profiling and RNA-Seq facilitates not only correlation studies of mRNA abundance and translation, but also gives a measure of the relative translation efficiencies of mRNAs in response to environmental triggers. Ribosome profiling analyses in L. donovani, combined with RNA-Seq, showed that inhibition of HSP90, while having no global impact on gene expression, changes steady state levels for many mRNAs and causes increased or decreased protein synthesis rates for <10% of the proteome. RNA abundance variations correlate poorly with changes to translation rates [33]. Among the proteins that show increased synthesis upon HSP90 inhibition are many that are known as markers of early amastigote differentiation, while induced morphological changes are reminiscent of in vitro stage conversion [16], [26], [33], [44].

Translation factors

While no canonical transcription factors are involved in controlling gene expression in Leishmania, translation factors may play important roles. Axenically induced amastigote differentiation coincided with reduced overall translation and phosphorylation of translation factor eIF2α [45]. Reduction of protein synthesis in amastigotes may reflect the adaptation to the reduced proliferation rate observed for the intracellular stage [46]. Furthermore, it is known for eukaryotes that stress causes a switch from cap-dependent to cap-independent translation. Leishmania spp. can adapt their translation machinery to environmental stress by a special cap structure (cap-4) and associated cap-binding protein eIF4E [47]. There are several factors, e.g. Leish4E-IP, that bind to eIF4E and thus mediate the switch from cap-dependent to alternative translation initiation mechanisms [48]. Apart from these findings, very little is known about translation factors and their role in the regulation of inducible gene expression in Leishmania.

Post-translational modifications

Post-translational modifications (PTMs) are also involved in the regulation of gene expression patterns by influencing the function and stability of proteins through phosphorylation, acetylation, methylation, and glycosylation. High throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodologies were used to study the prevalence of PTMs during axenic differentiation into amastigotes and showed altered abundance of modified proteins [49], [50], [51]. MAP kinases (MAPK), for instance, play a major role in altering gene expression profiles. They are highly conserved serine/threonine-specific protein kinases in all eukaryotes and are important in signal transduction cascades. By phosphorylating their substrates, MAPKs regulate critical cellular functions in Leishmania affecting cell viability, parasitic life cycle control, morphology and drug resistance [52], [53], [54], [55], [56], [57], [58], [59]. Phosphorylation of HSP70 and HSP90 by the L. donovani MAP kinase 1 is thought to affect the stability of heat shock proteins and their functions during the life cycle [60].

Casein kinases also play crucial roles in overcoming environmental adversity. They are exported to the host cell cytoplasm via exosomes and interact with parasite chaperones and host proteins [61], [62], [63], [64], [94].

Genetic diversity and selection under environmental challenge

The lack of gene regulation at the transcription level and the absence of cis-splicing in Leishmania, the Trypanosomatida and possibly all Euglenozoa sets these organisms apart from other eukaryotic phyla [10], [28], [65], [66]. Although Leishmania chromosomes are divided into gene arrays that are transcribed in a polycistronic mode [11], [13], [67], those arrays do not constitute operons of jointly regulated, functionally related genes. Nevertheless, Leishmania populations are able to adapt to environmental adversity.

Starting in the 1980s, researchers discovered that certain Leishmania genes were present in more than one copy per haploid set of chromosomes [68], [69], [70], [71], [72], and the copy numbers often varied between species and parasite isolates. This already hinted at gene copy number variation (CNV) as a mechanism of genetic adaption.

The advent of reverse genetics in Leishmania research [73], [74] provided more puzzles. Double-allelic gene replacement by homologous recombination using different selection marker genes for both alleles often left detectable copies of the genes of interest (GOI), in spite of a successful and verified gene replacement and marker gene insertion [74], [75].

The availability of deep sequencing technology in recent years has greatly expanded our knowledge of the structure and dynamics of Leishmania genomes. Parasite populations selected under virtually any sort of pressure, e.g. drug challenge, temperature stress, pH milieu, oxidative stress, but also loss-of-function mutations with growth defects, can be subjected to whole genome sequencing (WGS) alongside the pre-selection parental strain. The resulting sequence read densities can then be analysed for ploidy changes, gene CNVs, and single nucleotide polymorphisms (SNPs) [76].

Aneuploidy and mosaic aneuploidy

Individual chromosome ploidy in Leishmania is highly variable within populations [77], [78], [79]. Between one and up to six sister chromosomes may occur. Chromosome ploidy can be assessed from the relative NGS read alignment density, normalised to a reference strain [76] (Fig. 2A). Intermediate fold values can indicate mosaic aneuploidy, e.g. variances within the population, or partial chromosome duplication events. Distinction between these possibilities is done by plotting read alignment density against the length of the chromosome: even read alignment density indicates whole chromosome/partial population changes (Fig. 2B), while localised read alignment density increases may hint at partial chromosome/whole population events.

Fig. 2

Constitutive and selected aneuploidy in Leishmania donovani selected under IC50 of a pteridin reductase 1 inhibitor (PTR1-i) or an equivalent dose of solvent (DMSO) for 40 days. (A) NGS read alignment densities were established for each chromosome using the Bowtie2 algorithm, normalised against the overall average read density set at 2, and displayed as heat map. Note that i) chromosome 31 is constitutively tetrasomic, ii)chromosomes 8, 12, and 24 are constitutively trisomic, and chromosome 23 shows trisomy after PTR1-i selection. In addition, chromosome 5 shows an intermediate ploidy indicating mosaic aneuploidy or partial chromosome amplification. (B) Sequence read alignment over chromosome 5 for DMSO- (upper panel) or PTR1-i-selected (lower panel) shows equal relative read distribution, indicating mosaic aneuploidy for chromosome 5 in the PTR1-i-selected population.

Aneuploidy has been studied extensively in the context of drug resistance. Decades of use of pentavalent antimony-based drugs in endemic regions have led to wide-spread resistance, rendering these drugs almost obsolete in certain areas [80], [81], [82], [83]. The advances of systems biology strategies in Leishmania spp. confirmed initial proposals [84] linking antimony resistance with aneuploidy patterns specific for resistant parasites [85], [86], [87]. However, mosaic aneuploidy also plays a role in the adaption to vector and host as distinct patterns of ploidy are observed for vector- and host-derived leishmaniae [86], but also for in vitro cultivated cells [86], [88], [89].

Even more telling is a recent finding comparing somy patterns of identical L. donovani strains obtained from cultivated parasites or directly from clinical biopsies. This approach skips the in vitro cultivation of parasites and the concomitant karyotypic diversification prior to whole genome sequencing. While cultured parasites showed a wide variety of somy patterns, ploidies were remarkably similar for bone-marrow-derived parasite genomes [90], in keeping with comparable selective pressures within the human hosts.

In multicellular organisms, aneuploidy is known to have severe consequences, including tumour formation and chromosome instability [91], [92], [93]. Yet, the leishmaniae seem to cope well with genome plasticity. Firstly, aneuploidy is reversed quickly once the benefits of a supernumerary chromosome expire. Secondly, over expression due to additional gene copies appears to be limited at the protein level [86], [94]. Regulated translation may therefore play a role in ameliorating the effects of aneuploidy.

Gene CNVs

As mentioned before, gene CNVs were observed for Leishmania species and strains, often affecting heat shock gene arrays [12], [44], [68], [69], [70], [71], [72], which can be assumed to mediate tolerance against various physical and chemical stresses. Indeed, increased abundances of HSP90 or HSP70 appear to protect Leishmania spp. against antimony-containing drugs [95], [96], [97]. Drug selection pressure can also lead to increased copy numbers for genes encoding ABC transporter proteins [98]. The prevalent mechanism for gene amplification was shown to depend on the SIDERs that are distributed over the Leishmania genomes. In addition to their roles in mRNA stability, they can be found flanking amplified intra-chromosomal and episomal genes and gene clusters, suggesting a role of such repeats in the formation of linear and circular episomes [94], [99]. Such amplifications occur frequently and stochastically within a given population and, like mosaic aneuploidy, contribute to the constitutive genetic diversity within Leishmania isolates [85], [86], [87]. In fact, these fluctuations of gene copy numbers are known to account for a majority of genetic adaptations [100]. However, while gene dosage affects the abundance of the corresponding RNA(s) proportionally, their effect on protein abundance during natural gene copy number variation [86] and targeted over expression [94], [101] is extenuated, likely due to modulated translation. Still, increased gene copy numbers lead to increased expression.

SNPs

SNPs in non-coding or protein-coding sequences may affect expression or function, respectively, of genes and their products. They are usually associated with a loss-of-function phenotype. In the context of drug resistance, SNPs were found in Leishmania selected for resistance to the antileishmanial drug miltefosine. Selected parasites carry a plethora of SNPs in the miltefosine transporter gene causing amino acid exchanges or open reading frame shifts [102], [103].

Another example for the selection of SNPs involves the L. major P46 virulence enhancing protein, which, depending on the geographic origin of L. major isolates, shows distinct patterns of SNPs. Defined SNP patterns were indeed selected in vitro and in murine hosts, suggesting a role of P46 sequence polymorphism in the adaption to diverse reservoir hosts found in various L. major-endemic regions [104].

Mimicking CNV in the laboratory

Adaptive, extrachromosomal gene amplifications can be mimicked in a laboratory setting by complementation genetics, more specifically by functional cloning from a cosmid library representing a Leishmania genome [15], [105], [106], [107], [108], [109]. Since Leishmania genes do not contain introns but need their flanking sequences for proper RNA processing via trans-splicing and polyadenylation [110], unaltered, genomic DNA-bearing cosmids contain all the information needed for proportional over expression of several neighbouring gene loci. The compactness of the Leishmania genomes (~32–34 Mb) contributes to this, with less than 5000 individual cosmid clones covering >99% of the genome [107], and allowing for a genome-wide search for dominant genetic markers using a manageable number of individual transfectants. Using shuttle cosmid vectors, propagation in Escherichia coli and Leishmania spp. is feasible. Lately, the technology was greatly improved by the use of next generation sequencing (NGS) to map the origin of selected cosmids and to establish the relative preference within a number of selected gene loci [111], [112], [113]. This cos-seq strategy can now be employed to identify dominant drug resistance markers for established drugs and for drug leads, improving sustainable drug development [111], [114].

Conclusions

By dispensing with individually regulated RNA polymerase II transcription, Leishmania spp., like all Trypanosomatida, lost an important route to up-regulate the expression of genes needed for overcoming environmental adversity, while allowing them to dispense with cis- and trans-acting transcription regulators, thus reducing genome size. The constitutive transcription of most of their genome and the resulting waste of biochemical energy equivalents is compensated in part by the compact arrangement of coding sequences, the lack of cis-splicing and introns, and the parasitic life style, utilising the host's energy resources. Generally speaking, obligate parasites such as Leishmania spp. live in the stable environments of either sandflies or mammals and are thus shielded against acute environmental changes. Nevertheless, their extreme and constitutive genetic variability as expressed in mosaic aneuploidy, episomal and intrachromosomal gene amplification, a short generation time, and an effective post-transcriptional stress response allows them to adapt — as a population — to environmental challenges in an exemplary fashion.

Authors statement

J.G. and J.C. contributed equally to the creation of this manuscript, including conceptualisation, literature research, drafting and finalising of the manuscript. J.C. contributed the Figures.