A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 106 copies per cell of this protein (Mr 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed

Ancient Leishmania coronin (CRN12) is involved in microtubule remodeling during cytokinesis

In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture

ADF/cofilin-driven actin dynamics in early events of Leishmania cell division

ADF/cofilin is an actin-dynamics-regulating protein that is required for several actin-based cellular processes such as cell motility and cytokinesis. A homologue of this protein has recently been identified in the protozoan parasite Leishmania, which has been shown to be essentially required in flagellum assembly and cell motility. However, the role of this protein in cytokinesis remains largely unknown. We show here that deletion of the gene encoding ADF/cofilin in these organisms results in several aberrations in the process of cell division. These aberrations include delay in basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division. In addition to these changes, the intracellular trafficking and actin dynamics are also adversely affected. All these abnormalities are, however, reversed by episomal complementation. Together, these results indicate that actin dynamics regulates early events in Leishmania cell division

Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum

Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum

An unconventional form of actin in protozoan hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirusinsect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs

Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani

Background: Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L. donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L. donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L. donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L. donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism

Leishmania actin binds and nicks kDNA as well as inhibits decatenation activity of type II topoisomerase

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling

Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania

Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids

Nuclear localization of an actin-related protein (ORF LmjF21.0230) in Leishmania

This article does not have an abstract

Overexpression of S4D Mutant of Leishmania donovani ADF/Cofilin Impairs Flagellum Assembly by Affecting Actin Dynamics

Leishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmania actin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum. R eorganization of actin cytoskeleton is central to several fundamental processes in eukaryotes, including cell division, cell shape regulation and, transmission of extracellular stimuli toward the cell interior. Such diverse functions of actin cytoskeleton have been attributed to the dynamic character of actin, which requires high turnover of actin monomers in its filamentous meshwork by a treadmilling process (11). This process is greatly facilitated by the actin-depolymerizing protein (ADF)/cofilin family of actin binding proteins (40). These proteins generally have three distinct biochemical activities, viz., F-actin depolymerization, actin filament severing, and nucleotide exchange (12). By virtue of these activities, ADF/cofilins play a key role in regulating the actin dynamics and associated functions in eukaryotes (7). Functions of the actin cytoskeleton have been considered important not only in higher eukaryotes but also in several parasites that cause lifethreatening human diseases, such as Plasmodium (5, 49), Acanthamoeba (10, 23), Trypanosoma (13, 21), Leishmania (30, 47), and others. Leishmania spp. constitute a group of medically important protozoan parasites that are responsible for a vast array of devastating human diseases, including kala-azar (visceral leishmaniasis). These organisms exist in two morphobiological forms, amastigotes (inside the human host) and promastigotes (in the insect vector), which undergo extensive cytoskeletal rearrangement during their transformation from one form to the other (25). The promastigote form possesses a single highly motile protruding flagellum, which drives the cell to move forward, whereas the rudimentary flagellum in amastigotes has been considered important to establish host-parasite interactions (22). Further, a direct involvement of the promastigote flagellum has been demonstrated in sandfly infection (16). Apart from being important for parasite biology, the Leishmania flagellum has also been considered a good model system to study the biology of flagella and cilia in connection with ciliopathies in humans The Leishmania flagellum is comprised of two main structural components, the axoneme and the paraflagellar rod (PFR). Whereas the axoneme powers beating in most eukaryotic flagella (44), the PFR has been implicated in flagellar motility and waveform generation (42). All eukaryotic flagella are microtubulebased dynamic structures, which utilize the microtubule-based motor proteins, kinesins and dyneins, for trafficking proteins from the base to the tip and vice versa in a process called intraflagellar transport (IFT) during their assembly and disassembly (recently reviewed in reference 28). Although there are several studies which have shown the presence of actin and actin binding proteins in the flagellar compartment Our previous studies have shown that besides containing actin (LdACT), Leishmania donovani parasites also contain a homolog of ADF/cofilin (LdCof), not only in their cell bodies but also in the flagella (47, 52). It has further been shown that knockout of the LdCof gene in Leishmania promastigotes results in short, stumpy, and nonmotile cells with shorter and paralyzed flagella (52). Additionally, it has been reported that in LdCof null mutants, most of the actin was present in the form of bundles, suggesting a possible role of LdCof-mediated actin dynamics in assembly of the flagellum. To further investigate this, we have now created LdCof mutants in which the serine-4 residue was replaced with aspartate (S4D) or alanine (S4A) and have analyzed the effects of their overexpression in wild-type cells. In addition, we expressed these mutant proteins in bacteria and, after their purification and characterization, analyzed their biochemical properties in terms of actin binding, actin depolymerization, and exchange of actin-bound nucleotides. Our results revealed that overexpression of the S4