Transcriptomic profiling of gene expression and RNA processing during Leishmania major differentiation.

Protozoan parasites of the genus Leishmania are the etiological agents of leishmaniasis, a group of diseases with a worldwide incidence of 0.9-1.6 million cases per year. We used RNA-seq to conduct a high-resolution transcriptomic analysis of the global changes in gene expression and RNA processing events that occur as L. major transforms from non-infective procyclic promastigotes to infective metacyclic promastigotes. Careful statistical analysis across multiple biological replicates and the removal of batch effects provided a high quality framework for comprehensively analyzing differential gene expression and transcriptome remodeling in this pathogen as it acquires its infectivity. We also identified precise 5' and 3' UTR boundaries for a majority of Leishmania genes and detected widespread alternative trans-splicing and polyadenylation. An investigation of possible correlations between stage-specific preferential trans-splicing or polyadenylation sites and differentially expressed genes revealed a lack of systematic association, establishing that differences in expression levels cannot be attributed to stage-regulated alternative RNA processing. Our findings build on and improve existing expression datasets and provide a substantially more detailed view of L. major biology that will inform the field and potentially provide a stronger basis for drug discovery and vaccine development efforts.

Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions.

BACKGROUND: Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate. RESULTS: We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure. CONCLUSIONS: The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.

Early Leukocyte Responses in Ex-Vivo Models of Healing and Non-Healing Human Leishmania (Viannia) panamensis Infections.

Early host-pathogen interactions drive the host response and shape the outcome of natural infections caused by intracellular microorganisms. These interactions involve a number of immune and non-immune cells and tissues, along with an assortment of host and pathogen-derived molecules. Our current knowledge has been predominantly derived from research on the relationships between the pathogens and the invaded host cell(s), limiting our understanding of how microbes elicit and modulate immunological responses at the organismal level. In this study, we explored the early host determinants of healing and non-healing responses in human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) panamensis. We performed a comparative transcriptomic profiling of peripheral blood mononuclear cells from healthy donors (PBMCs, n=3) exposed to promastigotes isolated from patients with chronic (CHR, n=3) or self-healing (SH, n=3) CL, and compared these to human macrophage responses. Transcriptomes of L. V. panamensis-infected PBMCs showed enrichment of functional gene categories derived from innate as well as adaptive immune cells signatures, demonstrating that Leishmania modulates adaptive immune cell functions as early as after 24h post interaction with PBMCs from previously unexposed healthy individuals. Among differentially expressed PBMC genes, four broad categories were commonly modulated by SH and CHR strains: cell cycle/proliferation/differentiation, metabolism of macromolecules, immune signaling and vesicle trafficking/transport; the first two were predominantly downregulated, and the latter upregulated in SH and CHR as compared to uninfected samples. Type I IFN signaling genes were uniquely up-regulated in PBMCs infected with CHR strains, while genes involved in the immunological synapse were uniquely downregulated in SH infections. Similarly, pro-inflammatory response genes were upregulated in isolated macrophages infected with CHR strains. Our data demonstrate that early responses during Leishmania infection extend beyond innate cell and/or phagocytic host cell functions, opening new frontiers in our understanding of the triggers and drivers of human CL.

The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector.

The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of Leishmania insect stages during their cyclical development in vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.

Correction: Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion.

[This corrects the article DOI: 10.1371/journal.pntd.0004992.].

Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

UNLABELLED: Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. IMPORTANCE: Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in infection that was reduced at later time points. A similar expression pattern was observed in the parasites. Our analyses provide specific insights into the interplay between human macrophages and Leishmania parasites and constitute an important general resource for the study of how pathogens evade host defenses and modulate the functions of the cell to survive intracellularly.

Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion.

Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes.

The generation of macrophages with anti-inflammatory activity in the absence of STAT6 signaling.

Macrophages readily change their phenotype in response to exogenous stimuli. In this work, macrophages were stimulated under a variety of experimental conditions, and phenotypic alterations were correlated with changes in gene expression. We identified 3 transcriptionally related populations of macrophages with immunoregulatory activity. They were generated by stimulating cells with TLR ligands in the presence of 3 different "reprogramming" signals: high-density ICs, PGE2, or Ado. All 3 of these cell populations produced high levels of transcripts for IL-10 and growth and angiogenic factors. They also secreted reduced levels of inflammatory cytokines IL-1ß, IL-6, and IL-12. All 3 macrophage phenotypes could partially rescue mice from lethal endotoxemia, and therefore, we consider each to have anti-inflammatory activity. This ability to regulate innate-immune responses occurred equally well in macrophages from STAT6-deficient mice. The lack of STAT6 did not affect the ability of macrophages to change cytokine production reciprocally or to rescue mice from lethal endotoxemia. Furthermore, treatment of macrophages with IL-4 failed to induce similar phenotypic or transcriptional alterations. This work demonstrates that there are multiple ways to generate macrophages with immunoregulatory activity. These anti-inflammatory macrophages are transcriptionally and functionally related to each other and are quite distinct from macrophages treated with IL-4.