This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

The identification of RNAs that are not translated into proteins was an important breakthrough, defining the diversity of molecules involved in eukaryotic regulation of gene expression. These non-coding RNAs can be divided into two main classes according to their length: short non-coding RNAs, such as microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). The lncRNAs in association with other molecules can coordinate several physiological processes and their dysfunction may impact in several pathologies, including cancer and infectious diseases. They can control the flux of genetic information, such as chromosome structure modulation, transcription, splicing, messenger RNA (mRNA) stability, mRNA availability, and post-translational modifications. Long non-coding RNAs present interaction domains for DNA, mRNAs, miRNAs, and proteins, depending on both sequence and secondary structure. The advent of new generation sequencing has provided evidences of putative lncRNAs existence; however, the analysis of transcriptomes for their functional characterization remains a challenge. Here, we review some important aspects of lncRNA biology, focusing on their role as regulatory elements in gene expression modulation during physiological and disease processes, with implications in host and pathogens physiology, and their role in immune response modulation.

Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg-). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection.

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis-infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat2B and Cat1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr6, Tlr9, Ly96, Tirap, Traf 6, Ticam1, Tollip, Casp8, Map3k1, Mapk8, Nfkbib, Nfkbil1, Ppara, Mapk8ip3, Hspd1, and Ube2n, as well as immunomodulators, such as Ptgs2/Cox2, Csf 2, Csf 3, Ifnb1, Il6ra, and Ilr1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses.

BackgroundLeishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes.Methodology/Principal findingsA total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis.Conclusions/SignificanceAll data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.

BackgroundLeishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability.Methodology/Principal findingsRNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes.Conclusions/SignificanceIn this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.

Background: Leishmaniases are neglected tropical diseases that are caused by Leishmania, being endemic worldwide. L-arginine is an essential amino acid that is required for polyamines production on mammal cells. During Leishmania infection of macrophages, L-arginine is used by host and parasite arginase to produce polyamines, leading to parasite survival; or, by nitric oxide synthase 2 to produce nitric oxide leading to parasite killing. Here, we determined the metabolomic profile of BALB/c macrophages that were infected with L. amazonensis wild type or with L. amazonensis arginase knockout, correlating the regulation of L-arginine metabolism from both host and parasite. Methods: The metabolites of infected macrophages were analyzed by capillary electrophoresis coupled with mass spectrometry (CE-MS). The metabolic fingerprints analysis provided the dual profile from the host and parasite. Results: We observed increased levels of proline, glutamic acid, glutamine, L-arginine, ornithine, and putrescine in infected-L. amazonensis wild type macrophages, which indicated that this infection induces the polyamine production. Despite this, we observed reduced levels of ornithine, proline, and trypanothione in infected-L. amazonensis arginase knockout macrophages, indicating that this infection reduces the polyamine production. Conclusions: The metabolome fingerprint indicated that Leishmania infection alters the L-arginine/polyamines/trypanothione metabolism inside the host cell and the parasite arginase impacts on L-arginine metabolism and polyamine production, defining the infection fate.Int. J. Mol. Sci. 2019, 20, 6248 2 of 20 forms found in the proboscis of the phlebotomine sand fly host, to amastigote forms in the interior of macrophage phagolysosomes in the mammalian host [3]. Leishmania infection results in the regulation of pathways that are involved in the inflammatory response and leishmanicidal mechanisms, such as nitric oxide (NO) production via nitric oxide synthase 2 (NOS2), which is induced by Th1-cytokines [4][5][6][7][8][9]. However, Leishmania can subvert these mechanisms, which impacts on Th1/Th2 cytokine balance and induces macrophage arginase 1 (ARG1) activity to produce polyamines, putrescine, spermidine, and spermine, leading to Leishmania survival [6,[10][11][12].L-arginine is an essential amino acid and a precursor in the synthesis of proteins, urea, ornithine, citrulline, NO, creatinine, agmatine, glutamate, proline, putrescine, spermidine, and spermine, supporting the proline, glutamate, and polyamine metabolism at the whole organism level or cellular level in mammals. Its availability and metabolization can modulate inflammation and immune response regulation during infections and also recover the physiological steady-state [13,14].In mammalian cells, the endogenous synthesis of L-arginine from proline or glutamate via ornithine is not sufficient for supplying several pathways that need this amino acid as a precursor. L-arginine uptakes occur via cationic amino acid transporters (CAT1, CAT2A, ...

Leishmaniases are neglected diseases that cause a large spectrum of clinical manifestations, from cutaneous to visceral lesions. The initial steps of the inflammatory response involve the phagocytosis of Leishmania and the parasite replication inside the macrophage phagolysosome. Melatonin, the darkness-signaling hormone, is involved in modulation of macrophage activation during infectious diseases, controlling the inflammatory response against parasites. In this work, we showed that exogenous melatonin treatment of BALB/c macrophages reduced Leishmania amazonensis infection and modulated host microRNA (miRNA) expression profile, as well as cytokine production such as IL-6, MCP-1/CCL2, and, RANTES/CCL9. The role of one of the regulated miRNA (miR-294-3p) in L. amazonensis BALB/c infection was confirmed with miRNA inhibition assays, which led to increased expression levels of Tnf and Mcp-1/Ccl2 and diminished infectivity. Additionally, melatonin treatment or miR-30e-5p and miR-302d-3p inhibition increased nitric oxide synthase 2 (Nos2) mRNA expression levels and nitric oxide (NO) production, altering the macrophage activation state and reducing infection. Altogether, these data demonstrated the impact of melatonin treatment on the miRNA profile of BALB/c macrophage infected with L. amazonensis defining the infection outcome.