CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes (original) (raw)
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PLoS ONE, 2013
Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780) is unique to Leishmania and contains a putative secretion signal peptide. The fulllength gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P,0.001). Analysis by flow cytometry showed a higher percentage, ,4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005). These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania. Citation: Dan-Goor M, Nasereddin A, Jaber H, Jaffe CL (2013) Identification of a Secreted Casein Kinase 1 in Leishmania donovani: Effect of Protein over Expression on Parasite Growth and Virulence. PLoS ONE 8(11): e79287.
Leishmania and the macrophage: a marriage of inconvenience
Immunology Today, 1989
The Leishmania protozoan successfully parasitizes the macrophage of its vertebrate host. Considerable interest now centres on the identification of the parasite ligands and the macrophage receptors to which they bind. In this article, David Russell and Patricia Talamas-Rohana discuss the attachment mechanisms of Leishmania promastigotes to macrophages, and the possible role of complement componenb in their opsonizalion.
Inhibitors of casein kinase 1 block the growth of Leishmania major promastigotes in vitro
International Journal for Parasitology, 2006
Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [ 3 H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites. Ó
European Journal of Immunology, 1999
We have previously demonstrated that murine macrophages (M ¤ ) infected with Leishmania promastigotes, in contrast to M ¤ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I-A d -restricted T cell hybrids and to the T cell clone 9.1-2. These T cells react with the LACK (158-173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK-specific T cell hybridoma OD12 by promastigote-infected M ¤ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log-phase or stationary-phase promastigotes of L. major or of L. amazonensis, M ¤ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by M ¤ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, M ¤ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected M ¤ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158-173)-I-A d complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158-173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by M ¤ infected with log-phase or stationary-phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4 + T lymphocytes. Abbreviations: BFA: Brefeldin A BMDM ¤ : Bone marrowderived M ¤ LACK: Leishmania homologue of receptors for activated C kinase Leu-oMe: L-Leucine methyl ester LPG: Lipophosphoglycan MOI: Multiplicity of infection PV: Parasitophorous vacuole 762 N. Courret et al.
Intracellular growth and pathogenesis of Leishmania parasites
Essays in biochemistry, 2011
Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in humans, as well as chronic long-term infections. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome compartment of host macrophages. Studies with defined Leishmania mutants in mouse models of infection have highlighted processes that are required for parasite survival in macrophages. Parasite mutants have been identified that (i) are poorly virulent when the insect (promastigote) stage is used to initiate infection, but retain wild-type virulence following transformation to the obligate intracellular amastigote stage, (ii) are highly attenuated when either promastigotes or amastigotes are used, and (iii) are unable to induce characteristic lesion granulomas, but can persist within macrophages in other tissues. From these analyses it can be concluded that promastigote stages of some species require the surface expression of lipophosphoglycan, ...
Leishmania Eukaryotic Initiation Factor (LeIF) Inhibits Parasite Growth in Murine Macrophages
PLoS ONE, 2014
The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-c, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/ IFN-c prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-c after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-c-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-c showed a clear upregulation in macrophage inflammatory protein 1a (MIP-1a) as well as tumor necrosis factor alpha (TNF-a) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-c combined with anti-TNF-a monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-a, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different. Overall, these results indicate that LieIF is a good candidate for use as an anti-leishmanial molecule.
Bioscience reports
In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related ...
Leishmania and its host cell: a complex relationship
Poster Abstract. Proceedings of 11th International Congress of Parasitology, ICOPA XI, Glasgow (Scotland, United Kingdom), 6-11 August 2006., 2016
Leishmania parasites invade host macrophages, causing infections that are either limited to skin (cutaneous), or spread" to internal organs (visceral). Macrophage-parasite interactions were investigated for three Leishmania species responsible for cutaneous leishmaniasis: L. major, L. aethiopica and L. tropica, using a monocyte cell line (THP-l). Fluorescence 2-D Difference Gel Electrophoresis (DIGE) was used and proteins differentially expressed during infection were identified. Over 100 proteins showed significant changes of expression following infection with each of the three species tested. Proteins of interest were then separated, digested with trypsin and the peptide masses measured using MALDI-TOF. The identities of the proteins were elucidated by PMF. The identities and the involvement of the DE proteins will be discussed.
The Leishmania-macrophage interaction: a metabolic perspective
Cellular Microbiology, 2007
Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon sources and growth-limiting nutrients, respectively, and their availability to intracellular amastigotes may be regulated by the activation state of host macrophages. Metabolic processes in both the parasite and host cell may thus be crucial determinants of disease outcome.