The chemotherapy against leishmaniasis remains the main strategy to manage the disease control, but several implications regarding the treatment should be considered [11-17]. Pentavalent antimonials are considered one of the main options of treatment, however these drugs have several toxic side effects and high resistance rates [9,11-13,16,17]. Thus, the comprehension of resistance molecular mechanisms in Leishmania spp. is very crucial to identify potential drug targets to prevent or reverse such mechanisms. In this study, RNA Seq has been used successfully to quantify transcript levels of SbIII-resistant and wild-type L. infantum lines. These data allowed us to identify several potential candidates for biochemical pathways associated with antimony resistance phenotype in L. infantum. Differentially expressed (DE) transcripts between wild-type and SbIII-resistant L. infantum lines were grouped into functional classes according to Gene Ontology database and results are discussed below.
Protein phosphorylation
Protein phosphorylation is one of the most important post-translational modifications regulating various signaling processes. GO enrichment analysis showed that 37 transcripts belonging to protein phosphorylation category are 2.04 to 8.93-fold upregulated in the LiSbR line (Table 1 and Additional file 1). Protein kinases are important regulators of many different cellular processes, such as transcriptional control, cell cycle progression, differentiation and response to stress [46–48]. They represent promising drug targets for trypanosomes and Leishmania, since some of them are essential for viability of parasites and have significant sequence differences from mammalian homologous [46,49].
Five transcripts encoding phosphatidylinositol kinase (PIK) were 2.52 to 3.97-fold upregulated in the LiSbR line. According Velásquez (2015), the phosphatidylinositol signaling pathway is interesting to explore since that it is involved in the survival of the parasite by controlling osmoregulation, transport through membranes, and activation of transcription factors [50]. Target of rapamycin, (TOR) kinases are key regulators of cell growth proliferation. Our data showed that TOR1 and TOR3 are 2.5 and 2.74-fold upregulated in the LiSbR line, respectively. Interestingly, TOR1 and TOR2 appear to be essential for Leishmania, and TOR3 is required for acidocalcisome biogenesis and animal infectivity [51].
RAB GTPases, 2-fold upregulated in the LiSbR, play a key role in regulation of exocytic and endocytic pathways in eukaryotic cells. This protein was also more abundant in the LiSbR line [22,52]. It has been shown that RAB GTPases of L. major are highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis [53]. L. donovani overexpressing RAB6 showed a resistant phenotype, by allowing trans-dibenzalacetone treated parasites to both increase internal thiol levels and enhance MRP pump activity [53].
Dual specificity protein phosphatase (DUSP) was 8.93-fold upregulated in the LiSbR line. DUSPs are the negative regulator of the mitogen-activated protein (MAP) kinase pathway. This enzyme is able to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues on a single substrate simultaneously. DUSP1 protein is overexpressed by metacyclic forms and its knockout in L. mexicana causes both reduction of growth ratios in promastigotes and virulence attenuation in mouse macrophages [54].
Other transcripts associated with dephosphorylation, as protein phosphatase and protein phosphatase 2C, were 17.52 to 2.28-fold upregulated in the LiSbR line, respectively (Additional files 1 and 2). Proteomic analysis using these same L. infantum lines showed that both enzymes were also more abundant in SbIII-resistant line [22].
Some transcripts encoding cyclins 10, 11 and CYC2-like were 2.27 to 5.56-fold upregulated in LiSbR line. Cyclins play important roles in cell cycle regulation in eukaryotes. The cell cycle progression in eukaryotes is regulated through the phosphorylation of many proteins involved in the process by cyclin-dependent kinases (Cdks) [55]. CYC2 knockout attempts in T. brucei suggest that this is an essential gene for this parasite [56].
Elongation factor 2 (EF2), a relevant factor for production of proteins, can be regulated through inhibitory phosphorylation at threonine 56 by EF2 Kinase [57]. This enzyme was 3.23-fold upregulated in the LiSbR line. Our group showed that the EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of SbIII, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy [22,58].
A comparative phosphoproteomic analysis of SbIII resistant and susceptible lines of L. braziliensis identified several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite [22,58]. In the antimony-resistant L. infantum line, we also observed that different kinases and phosphatases are differentially expressed in this parasite (Additional files 1 and 2).
Microtubule-based movement
In this category, 20 transcripts were found to be 2.0 to 5.9-fold upregulated in the LiSbR line (Table 2 and Additional file 1). The microtubules play important roles in diverse cellular activities, as mitosis, intracellular transport and for the parasites motility in free living stages [59].
Dyneins were 2.0 to 5.9-fold upregulated in the LiSbR line. They are high molecular weight microtubule based motor proteins responsible for beating of the flagellum [60–62]. Eukaryotic organisms with flagella also express two cytoplasmic dyneins (dynein-1 and dynein-2) containing at least one dynein heavy chain (DHC) and a variable number of dynein light chains [63]. Interestingly, it has been shown that the genomes of L. mexicana, L. major, L. infantum and T. brucei actually have two cytoplasmic dynein-2 heavy chain genes, with different functions, and only one of them is essential for L. mexicana [63]. Cytoplasmic dyneins, upregulated in the LiSbR line, are related with nuclear migration, organization of the mitotic process, chromosome separation, and the positioning and function of intracellular organelles [64].
Ten transcripts encoding kinesins were 2.02 to 5.87-fold upregulated in the LiSbR (Table 2 and Additional file1). They are responsible for ATP hydrolysis and microtubule binding. In L. donovani, kinesin plays an important role in cell division, cytoskeleton filament formation, flagellar beating and intracellular transport of vesicles and organelles [65]. The kinesin motor domain region of L. donovani induces a strong Th1-type immune response, important for protection, being a potential vaccine candidate against visceral leishmaniasis [66–69].
Others transcripts upregulated in the LiSbR encodes tryptophan-aspartic acid (WD) protein and tetratricopeptide repeat domain (TPR). The WD protein mediates protein-protein interactions and shows protective immunity in L. amazonensis infected mice [70]. The TPR repeat domain is a degenerate 34-amino acid sequence that has been recruited by a significant number of co-chaperones that interact especially with Hsp90 and Hsp70 [71]. Dores-Silva et al. (2012) investigated the structural features of Hip (Hsp70-interacting protein) from L. braziliensis (LbHip) [72]. This protein is a TPR that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation [22]. TPR repeat domain can have several different functions, including: control of mitosis and apoptosis, cell cycle control, endocytosis, interaction with beta-amyloid proteins and growth hormone receptors, protein folding, protein transport and transcriptional repression [73]. It has also been shown that the TPR repeat domain of L. infantum is a potential candidate for a diagnostic marker and also for vaccine development [73].
Protein ubiquitination
Ubiquitination is a crucial process in all eukaryotic organisms. It is involved in several essential functions, such as degradation of denatured proteins, DNA repair, endocytosis, regulation of protein levels, transcription, and apoptosis [74,75]. Twenty transcripts that are 2.03 to 9.14-fold upregulated in the LiSbR line were assigned to this category, described as: ubiquitin, ubiquitin-transferase (HECT domain - homologous to the E6-AP carboxyl terminus and SPRY domain - SPla and the RYanodine Receptor), cullin protein (involved in ubiquitination through participation in multisubunit ubiquitin ligase complexes), Zinc finger containing proteins and others (Table 2 and Additional file 1). Similar to our results, antimony-resistant L. tropica isolate also showed overexpression of ubiquitin [76]. These data suggest that increased levels of protein ubiquitination may contribute to degradation of oxidized proteins, protecting the parasite against oxidative stress from antimony.
Cellular process
Zinc finger proteins are RNA binding proteins involved in many biological processes by binding nucleic acids or participating in transcriptional or translational processes by mediating protein-protein interactions and membrane association [77]. Zinc finger domains in proteins were recently proposed as potential targets for SbIII, due to the ability of SbIII to compete with ZnII. A previous study suggested that the interaction of SbIII with zinc finger proteins may modulate the pharmacological action of antimonial drugs [10,14,78]. In our study, four transcripts encoding different zinc finger proteins (C3HC4 type - RING finger and FYVE) were 2.15 to 3.77-fold upregulated in the LiSbR line (Table 1 and Additional file 1). The FYVE domain is a small zinc binding module that recognizes phosphatidylinositol 3-phosphate and the majority of these proteins are implicated in membrane trafficking, protein sorting and signaling transduction [79].
Glycosomal transporter (GAT3) also was 3.39-fold upregulated in the LiSbR line. It is homologous to peroxisomal ABC transporters with association to glycosomal membrane. It has been shown that GAT3 is not an essential gene for T. brucei [80]. While on the other hand glycosomal transporters GAT1 and GAT2 seem to be responsible for transporting fatty-acids, GAT3 knockdown in T. brucei does not change parasites fatty-acid profile. In addition, GAT3 also seems to be unrelated to glucose transport in T. brucei [80]. It was also demonstrated that GAT3 sequences are less conserved, which can result in changes in its function [81].
Biosynthetic process
Serine palmitoyltransferase catalyzes the first rate-limiting step in the synthetic pathway of de novo sphingolipid biosynthesis [82]. This enzyme was 2.99-fold upregulated in the LiSbR line (Table 1 and Additional file 1). Metabolomic analysis revealed differences in the phospholipid and sphingolipid contents between -susceptible and antimony-resistant L. donovani isolates [83]. According to Zhang and Beverley (2010), these two lipid classes are both abundant and critical to virulence and viability in Leishmania [84].
Ribonucleoprotein complex assembly
Transcripts encoding ATP-dependent RNA helicase were 2.38 to 3.34-fold upregulated in the LiSbR line (Table 1, Additional files 1 and 2). It plays an essential function in RNA metabolism, including RNA degradation, translation, regulation and RNA editing [85,86]. It has been shown that a member of RNA helicases “DDX3 DEAD-Box” of Leishmania plays a central role in preventing reactive oxygen species-mediated damage and in maintaining mitochondrial protein quality control [85].
Stress granule assembly
One transcript assigned as pumilio protein was 5.59-fold upregulated in the LibSbR line. Pumilio are RNA-binding proteins that regulate mRNA stability and translation by enhancing the deadenylation and subsequent degradation of mRNAs or repressing translation initiation [87]. Previous study showed that Pumilio-domain protein PUF6 interacts with SIDER2 (short interspersed degenerate retroposon) regulatory sequences and to accelerate mRNA decay in Leishmania [88].
Regulation of membrane lipid distribution; phospholipid-translocating
Four transcripts related to Phospholipid-transporting ATPase/P-type ATPase proteins were upregulated in the LiSbR line. ATPases are membrane proteins that perform active ion transport across biological membranes, which are found in bacteria and all eukaryotic cells, including Leishmania [89]. Fernandez-Prada et al. (2016) demonstrated that different point mutations in a P-type ATPase transporter in L. infantum are implicated with cross-resistance to miltefosine and amphotericin B [90].
Cellular metabolic process
The HSPs have important functions in folding, secretion, assembly, intracellular localization, regulation and degradation of other proteins [91]. In general, the heat shock response is a homeostatic mechanism that protects cells from the deleterious effects of environmental stress, such as heat and drug exposure [92]. Several authors reported the overexpression of HSPs in antimony-resistant isolates of L. donovani [93–95], L. braziliensis and L. infantum lines [22,96]. Here, a transcript encoding a 100 KDa heat shock protein was 2.86-fold upregulated in LiSbR line. HSP100 has the unique capability of recognizing misfolded proteins within an aggregate and actively unfolding them, ultimately disassembling the insoluble structure and delivering substrates into refolding pathways [97].
Primary metabolic process; cellular macromolecule biosynthetic process; cellular nitrogen compound metabolic process
DNAJ proteins, also known as HSP40s, are crucial partners for HSP70 chaperones, and much of the functional diversity of the HSP70s is driven by this diverse class of cofactors [97]. Here, DNAJ was 2.13-fold upregulated in the LiSbR line. This protein plays a relevant role in the differentiation process from promastigote to amastigote stage in L. infantum, since it suffers a dramatic increase in phosphorylation [98]. Interestingly, HSP40 was also found increased in artemisinin-resistant L. donovani [99].
Regulation of membrane lipid distribution; Phospholipid translocation
In our study, many transcripts belonging to ATP-binding cassette (ABC) transporters were upregulated in the LiSbR line. ABC transporters comprise a superfamily of integral membrane proteins involved in the ATP-dependent transport of a variety of molecules across biological membranes, including amino acids, sugars, peptides, lipids, ions and chemotherapeutic drugs [100]. They have been associated with drug resistance in various diseases. In Leishmania, the first ABC protein identified was MRPA (multidrug-resistance protein - PgpA) [101] which is a member of the ABCC subfamily, able to confer resistance to antimonials by sequestering thiol-metal conjugates into an intracellular vesicle [101,102]. Our previous data showed an association of chromosomal amplification of MRPA gene with the drug resistance phenotype in SbIII-resistant Leishmania spp. lines [22,102]. Similarly, it has been shown that L. infantum knockout for the MRPA gene is more sensitive to Sb [103]. As ABC transporters are important regulators of drug susceptibility, they are excellent candidates for inhibitor design [104].
Quorum sensing involved in interaction with host, multi-organism cellular process
Since the regulation of gene expression in trypanosomatids occurs largely at post-transcriptional levels, the main control points in gene expression are mRNA degradation and translation [105]. The RNA-binding proteins (RBP) play essential roles in regulating RNA processing, transport, localization, translation and degradation. RBPs contain various structural motifs, such as RNA recognition motif (RRM), dsRNA binding domain, zinc finger and others [106]. RRM is a region of around eighty amino acids containing several conserved residues [107]. Four transcripts of RRM were 2.87 to 14.4-fold upregulated in the LiSbR line. Literature data show that one or more RRMs are found in a variety of RNA binding proteins, including heterogeneous nuclear ribonucleoproteins (hnRNP), translation factors, small nuclear ribonucleoprotein (snRNP) polypeptides, proteins involved in pre-mRNA and pre-rRNA processing, and poly(A)-binding proteins [108].
Ribonucleoprotein complex subunit organization, rRNA processing, ribosome biogenesis, translation
According to GO enrichment analysis, the transcripts encoding ribosomal proteins such as 40S and 60S ribosomal proteins, nucleolar and nuclear proteins are downregulated in the LiSbR line. The ribonucleoprotein complex subunit organization, rRNA processing, ribosome biogenesis and translation are essential for many biological processes of parasites, including cell development, differentiation and proliferation, and any positive and/or negative regulation of this cellular mechanism could direct affect the proteins synthesis. Transcriptomic analysis of L. infantum revealed high down-regulation of genes involved in translation and ribosome biogenesis rate in the amastigote stage [109].
In our study, 14 transcripts encoding ribosomal proteins of the small ribosomal 40S subunit and 11 transcripts encoding ribosomal protein components of 60S subunit were downregulated in the LiSbR line (Table 2). Data showed that the recombinant 40S ribosomal S3a protein of L major plays a dual role in regulation of T- and B-cell activation [110].
Nucleosome assembly
Modifications of core histones are involved in many biological processes such as DNA packing, transcription, DNA repair and gene regulation. In our study, transcripts encoding the histones H2A, H2B, H3 and H4 were found 2.0 to 2.56-fold downregulated in antimony resistant L. infantum line (Table 2, Additional files 1 and 3). It has been described that histone proteins are potent vaccine candidates against leishmaniasis [111]. A combination of recombinant H2A, H2B, H3 and H4 proteins from L. donovani were highly immunogenic and confer considerable experimental protection against visceral leishmaniasis [112]. It was demonstrated that the knockout of histone H3.V does not affect L. major viability, while the variants H2A.Z and H2B.V are essential in these parasites and also in T. brucei [113,114]. In our study, downregulation of histones could be associated with gene transcription regulation, thus favoring the antimony-resistance phenotype.
Proteins without GO enrichment for biological process
Ribosomal proteins play an important role in the translation and they also regulate cell growth and apoptosis. In our study, the 60S ribosomal L23a, a component of 60S subunit of ribosome large subunit, was found 2.07-fold upregulated in the LiSbR line (Additional file 2). In agreement with our results, proteomic analysis showed that this protein also was overexpressed in antimony-resistant L. donovani isolates [115]. Interestingly, 60S ribosomal L23a-overexpressing L. donovani line was more resistant to sodium antimony gluconate (SbV), miltefosine and paromomycin [116].
Cytochrome b5 and cytochrome P450 reductase that are involved in oxidoreductase activity were 6.37 and 4.33-fold upregulated in the LiSbR line, respectively (Additional file 2). Cytochrome b5 is a flavohemoprotein associated with oxidative reactions such as catabolism of xenobiotics and compounds of endogenous metabolism [117]. Mukherjee et al. (2012) observed that L. major deficient in cytochrome b5 oxidoreductase domain present decreasing of linoleate synthesis followed by increased oxidative stress and cell death by apoptosis [118]. Cytochrome P450 reductase is located on the endoplasmic reticulum in many types of cells and it is also related to drug metabolism [118].
Gamma-glutamylcysteine synthetase (γ-GCS) is the first enzyme of the glutathione pathway that produces γ-glutamylcysteine, a direct precursor of glutathione [119]. Our results showed that one transcript encoding this enzyme was found 2.6-fold upregulated in the LiSbR line (Additional file 2). γ-GCS has been shown to be essential for L. infantum, where it confers protection against oxidative stress and SbV [119]. According to our results, an increase of GSH1 mRNA levels also have been reported in some L. tarentolae samples with in vitro-induced resistance to antimony [120] and some SbV-resistant L. donovani field isolates [121]. Overexpression of γ-GCS is associated with antimony-resistance phenotype in L. guyanensis [122].
Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. Our data showed that one transcript encoding this enzyme is 2.54-fold upregulated in the LiSbR line (Additional file 2). Interestingly, our group overexpressed the GPI-14 gene in L. braziliensis and observed the involvement of the GPI-14 enzyme in the SbIII-resistance phenotype of L. braziliensis [123].
We have also identified two transcripts encoding proteins classified as amastins that were 3.33 and 2.97-fold upregulated in the LiSbR (Additional file 2). Amastins are glycoproteins related to parasite virulence [124]. They are encoded by a multigene family present in trypanosomatid genomes, comprising more than 40 genes in L. infantum [123,124]. Amastin transcripts share common 3’UTR elements that are implicated in stage-specific regulation of gene expression [125,126]. De Paiva et al. (2015) showed that amastin knockdown in L. braziliensis affects parasite-macrophage interaction and impaired viability of this parasite [124].