Features Partner Sites Information LinkXpress
Sign In
Advertise with Us


06 Jun 2016 - 09 Jun 2016
22 Jun 2016 - 24 Jun 2016
04 Jul 2016 - 06 Jul 2016

Unique Kinase Found Important for Parasite Survival and Virulence

By BiotechDaily International staff writers
Posted on 01 Jan 2014
Print article
Image: Phase contrast showing location of whole cells (Photo courtesy of Prof. C.L. Jaffe, Hebrew University, and of PLOS One).
Image: Phase contrast showing location of whole cells (Photo courtesy of Prof. C.L. Jaffe, Hebrew University, and of PLOS One).
Image: Presence of Casein Kinase 1 isoform 4 (CK1.4) in Leishmania donovani shown by immunoflourescent staining (red); control staining of nuclei is shown in blue (Photo courtesy of Prof. C.L. Jaffe, Hebrew University, and of PLOS One).
Image: Presence of Casein Kinase 1 isoform 4 (CK1.4) in Leishmania donovani shown by immunoflourescent staining (red); control staining of nuclei is shown in blue (Photo courtesy of Prof. C.L. Jaffe, Hebrew University, and of PLOS One).
A unique protein kinase (PK) secreted by the human parasite Leishmania was found to be important for the parasite's growth and virulence, making it a potential new chemotherapeutic target against leishmaniasis.

Enzymes of the casein kinase 1 (CK1) family are present in all known eukaryotes and are involved in a wide variety of cellular processes. Scientists at the Hebrew University - Hadassah Medical School (Jerusalem, Israel) led by Charles Jaffe, professor at the Department of Microbiology and Molecular Genetics, and Kuvin Center for the Study of Infectious and Tropical Diseases, investigated the role of CK1 isoform 4, unique to Leishmania (LCK1.4) and one of six putative CK1 isoforms encoded in the leishmanial genome.

In the study published in the journal PLOS One on November 15, 2013, Dan-Goor et al. investigated LCK1.4 in Leishmania donovani (LdCK1.4), a species that causes fatal, visceral disease. Full-length recombinant protein and three deletion constructs were cloned and expressed in E. coli. Only the full-length PK demonstrated significant activity, suggesting that both the N- and C-regions are required for LdCK1.4 PK activity.

Analysis of the LdCK1.4 amino acid sequence identified a putative secretion signal. To determine whether this PK is secreted, a C-terminus FLAG-tagged form, LdCK1.4-FLAG, was constructed and stably overexpressed in L. donovani promastigote-stage cells. LdCK1.4-FLAG or wild-type LdCK1.4 (native, untagged) release by mutant and/or wild-type parasites was monitored by Western blotting with polyclonal anti-LdCK1.4 and anti-FLAG upon transfer to induction buffer. The released PK was clearly observed in cell-free supernatants, peaking at 10 minutes. Differences in kinetics of release, and amounts of enzyme, were observed between mutant and wild type parasites.

LdCK1.4 was localized in fixed dividing-stage promastigotes using antibodies. Intracellular fluorescence was present both as weak diffuse cytoplasmic staining, as well as a strong punctate pattern adjacent to the nucleus and/or kinetoplast (no co-localization with a nuclear staining control was observed). This suggests that CK1.4 may have intracellular, as well as secreted extracellular roles.

Effect of LdCK1.4-FLAG overexpression on parasite growth and differentiation into virulent, metacyclic-stage promastigotes was examined daily. Parasites overexpressing the PK grew significantly faster, noted by day 3, and to higher densities than control cells. Interestingly, the LdCK1.4-FLAG overexpressing mutants differentiated more rapidly in culture, on day 3, into the metacyclic, virulent stage, than the wild-type parasites, as measured by flow cytometry. However, the final percentage of metacyclic parasites in stationary growth-phase cultures, on day 5, was similar in both types of parasites.

The researchers also examined the effect of LdCK1.4 overexpression on parasite virulence. CK1.4 expression increases rapidly during differentiation from the insect promastigote stage to intracellular amastigote stage in the mammalian host. Day 5 stationary phase promastigotes were used to infect mouse macrophages and intracellular amastigote infection was measured 72 hours later. The percentage of infected macrophages was significantly higher (~4.5–fold, p<0.0005) with the mutant, LdCK1.4-FLAG overexpressing parasites (~28%) compared to the wild-type parasites (~6%). Only a small, but significant difference in the number of parasites per infected macrophage was observed. Further characterization of LCK1.4 and its role in parasite survival and virulence will establish the potential of this unique leishmanial protein kinase as a putative drug target.

Related Links:

Hebrew University - Hadassah Medical School

Print article



view channel
Image: Follicular helper T-cells (TFH cells, shown in blue) play a crucial role in the maturation of antibody-producing B-cells (shown in green). Activated B-cells give rise germinal centers (shown in red), where mature B-cells proliferate and produce highly specific antibodies against pathogens. Top left: normal germinal center in a mouse tonsil. All others: Germinal centers fail to form when the interaction between ICOS and TBK1 is interrupted (Photo courtesy of Dr. Kok-Fai Kong, La Jolla Institute for Allergy and Immunology).

Molecular Pathway Controlling High-affinity Antibody Production Identified

A molecular pathway has been identified that controls formation of follicular helper T-cells (TFH cells) germinal centers and production of high-affinity antibodies through interaction with the inducible... Read more


view channel
Image: A space-filling model of the anticonvulsant drug carbamazepine (Photo courtesy of Wikimedia Commons).

Wastewater May Contaminate Crops with Potentially Dangerous Pharmaceuticals

Reclaimed wastewater used to irrigate crops is contaminated with pharmaceutical residues that can be detected in the urine of those who consumed such produce. Investigators at the Hebrew University... Read more

Lab Technologies

view channel

Huge Modifiable Biomedical Database to Be Available on the Wikidata Site

Genome researchers are exploiting the power of the open Internet community Wikipedia database to create a comprehensive resource for geneticists, molecular biologists, and other interested life scientists. While efficiency in generating scientific data improves almost daily, applying meaningful relationships between... Read more


view channel

European Biotech Agreement to Promote Antigen-Drug Conjugation Technology

Two European biotech companies have joined forces to exploit and commercialize an innovative, site-specific ADC (antigen-drug conjugate) conjugation technology. ProBioGen (Berlin, Germany), a company specializing in the development and manufacture of complex glycoproteins and Eucodis Bioscience (Vienna, Austria), a... Read more
Copyright © 2000-2016 Globetech Media. All rights reserved.