We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL), inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection.

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.