The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.

Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF-kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. We describe the effect of Leishmania amazonensis infection on NF-kappaB transcriptional factor activation in macrophages and the subsequent reduction in inducible nitric oxide synthase (iNOS) expression. L. amazonensis promastigote infection activates the p50/p50 NF-kappaB complex, a classic transcriptional repressor. Interestingly, L. amazonensis promotes the change of the classical p65/p50 NF-kappaB dimer induced by LPS, leading to the p50/p50 NF-kappaB complex activation in macrophages stimulated with LPS. Moreover, this parasite promotes the reduction of p65 total levels in infected macrophages. All these effects contribute to the observation that this parasite is able to restrain the NF-kappaB-dependent transcriptional activity induced by LPS. Strikingly, L. amazonensis reduces the mRNA levels of the iNOS in addition to protein expression and the production of nitric oxide in LPS-stimulated macrophages. Accordingly, as revealed by reporter-gene assays, L. amazonensis-induced iNOS repression requires NF-kappaB sites in the iNOS promoter region. In summary, our results suggest that L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF-kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infection.

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The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in the protozoan of the genus Leishmania, which is associated with the immunopathogenesis and susceptibility of BALB/c mice to Leishmania major infection. We previously demonstrated that intranasal immunization with a plasmid DNA encoding the p36/LACK leishmanial antigen (pCI-neo-LACK) followed by challenge 7 days after a booster dose effectively protects BALB/c mice against both cutaneous and visceral leishmaniasis. In the present study, the correlation between systemic mRNA expression after nasal DNA uptake, and the duration of protective immunity was addressed. LACK mRNA transcripts were detected in the spleen, brain, cervical lymph nodes and popliteal lymph nodes as early as 7 days, lasting 3 months after vaccination with pCI-neo-LACK. The kinetics of transcript expression correlated with enhanced cutaneous hypersensitivity against parasite antigens. Leishmania chagasi infection at 7 days or 3 months, but not 6 months after vaccination resulted in significantly lower parasite loads as compared with non-vaccinated controls. Protection also correlated with enhanced spleen cell responsiveness to parasite antigens leading to increased IFN- γ and IL-4 and decreased IL-10 production. Together, these data demonstrate that the protection conferred by the intranasal DNA vaccine lasts at least 3 months and is associated with expression of vaccine mRNA in peripheral organs.

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.