Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani
Cell Biology Section, Lab of Parasitic Diseases, NIAID/NIH, Bethesda, Maryland 20892-0425, USA. Cell Motility and the Cytoskeleton
(Impact Factor: 4.19).
04/2008; 65(4):269-80. DOI: 10.1002/cm.20259
Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units. Several GFP-LdK39B fusion constructs were generated and used for episomal-expression in these parasites. Results of confocal and immunoelectron microscopy indicated that the GFP-LdK39B-fusion proteins localized to a region adjacent to the flagellar pocket and the kinetoplast i.e. the mitochondrial-DNA containing organelle that is physically tethered to the flagellar basal bodies. Sub-cellular fractionation results showed that GFP-LdK39B proteins were insoluble in nature and remained tightly associated with purified flagella/kinetoplasts following their extraction with detergent and high salts. Our cumulative results suggest that the LdK39B may play a scaffold-like role in facilitating and maintaining the unique spatial/structural association between the flagellum-basal body-kinetoplast complex in these parasites.
Available from: Alicia Sánchez-Gorostiaga
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ABSTRACT: Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA(-) promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.
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ABSTRACT: Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania which affects an estimated 12 million people worldwide. The work presented herein describes characterization of several DNA aptamers which were developed against two different 39-amino acid synthetic
peptides derived from the highly conserved repetitive regions of a 230 kD flagellar Leishmania kinesin for their potential use in Leishmania diagnostics. Following ten rounds of aptamer affinity selection against the kinesin peptides and PCR amplification (i.e., SELEX), several
candidate aptamers demonstrated strong binding to the kinesin peptides in an enzyme-linked aptamer sorbent assay (ELASA). Confocal fluorescence microscopy using several aptamers with the highest affinities and the microtubule marker paclitaxel revealed localization patterns characterized by
diffuse, but often punctate, staining within the cytoplasm and in some cases along the flagella of L. major promastigotes. Aptamer association with microtubules was further evidenced by aptamer-colloidal gold staining and electron microscopy supporting the binding and specificity of
these aptamers for Leishmania kinesins. In addition, Southwestern blots confirmed that some of the top aptamers were specific for their respective ∼4 kD peptides. One aptamer in particular (K2-13R) also demonstrated clear detection of a 100 kD protein band and >250 kD bands in
L. major promastigote lysate. Given that the presence of antibodies recognizing the 39 kD kinesin (K39) or repetitive region of the 230 kD kinesin in patient serum is useful for the serodiagnosis of visceral leishmaniasis, but that few commercial antibodies exist for detection of K39
itself, these kinesin aptamers may have value as novel cellular and molecular probes for Leishmania diagnosis.
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