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Originally published In Press as doi:10.1074/jbc.M106806200 on September 5, 2001

J. Biol. Chem., Vol. 276, Issue 46, 43253-43261, November 16, 2001
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Expression of a Mutant Form of Leishmania donovani Centrin Reduces the Growth of the Parasite*

Angamuthu SelvapandiyanDagger , Robert DuncanDagger , Alain DebrabantDagger , Sylvie Bertholet§, Gannavaram Sreenivas, Narender S. Negi||, Poonam Salotra, and Hira L. NakhasiDagger **

From the Dagger  Laboratory of Bacterial, Parasitic, and Unconventional Agents, Division of Emerging and Transfusion Transmitted Disease, and the § Laboratory of Parasitic Biology and Biochemistry, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and the  Institute of Pathology (Indian Council of Medical Research) and || Safdarjung Hospital, New Delhi, 110 029 India

Received for publication, July 19, 2001, and in revised form, August 28, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca2+ binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca2+ binding site suggest that Ca2+ binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G2/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leishmania donovani, a protozoan parasite and a member of the order Kinetoplastida, is the causative agent of visceral leishmaniasis in humans worldwide. The disease is also known as "kala-azar" in India and Nepal. Treatment for this disease involves chemotherapy using antimony-based drugs, which is less effective in immunocompromised individuals (1). To date no vaccine is available for this disease. L. donovani has a digenic life cycle. The flagellated form, called promastigote, resides extracellularly in the gut of a dipteran sand fly insect. The second form, amastigote, is found in the macrophages of the infected human host. The growth and differentiation of Leishmania involves both qualitative and quantitative changes in various biochemical parameters (2). In our previous studies, we have identified genes that may have a role in growth and differentiation of the parasite (3-5). Here we describe one such gene, centrin, and analyze its function with respect to growth of the parasite.

Centrins are cytoskeletal, calcium-binding (EF-hand) proteins that are localized in the microtubule-organizing center of eukaryotes (6). Centrins are one of the several regulatory proteins essential for duplication or segregation of the centrosome in higher eukaryotes and basal bodies in lower eukaryotes (7). In many organisms, more than one centrin isotype has been described e.g. three centrins in humans and mice. Though three centrin forms have been recognized in the unicellular algae Chlamydomonas, only one has so far been characterized (8). One subfamily of centrins, which includes human centrin 1 (HsCEN1), human centrin 2 (HsCEN2), and Chlamydomonas reinhardtii centrin (CrCEN1), is involved in centrosome segregation (9). The other subfamily, which includes human centrin 3 (HsCEN3) and yeast centrin (CDC31), is involved in centrosome duplication (10, 11). Results from diverse experimental systems, mostly in yeast, suggest that different types of proteins like PKic1p, protein kinase (11, 12), and Kar1p, a component of the half-bridge of the spindle pole body, (13, 14) bind to centrins. Though centrin has been characterized in a variety of eukaryotes, it has not been reported in the order Kinetoplastida.

The parasites of the Kinetoplastida are responsible for a wide variety of diseases affecting humans, animals, and plants (15). Members of this group have been considered to be one of the earliest eukaryotes, developing conventional organelles, but sometimes with extreme features rarely seen in other organisms (15). The role of such unique structural and functional features of these organelles, like the cytoskeleton and the flagellar apparatus, in infectivity is still obscure. Hence, the identification of genes that enable Leishmania to grow and differentiate within the harsh and diverse environments (sand fly gut and human macrophages) continues to be our objective (3, 16-18).

The nature and the role of either the microtubule-organizing center or the basal body apparatus in the primitive eukaryote Leishmania are still not known. None of the genes (e.g. centrin, calmodulin, and gamma -tubulin) that are associated with these organelles in higher eukaryotes has been characterized so far in this important human parasite. As a first step toward that, here we report our findings leading to cloning, sequencing, and characterization of a centrin gene from L. donovani (LdCEN). We have also undertaken a functional analysis of Leishmania centrin protein by performing deletion analysis of the centrin gene and expressing such truncated proteins in the parasites to test their potential role in the growth of the parasite.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Vitro Culture of Parasites-- L. donovani isolated from either the Indian kala-azar patient (K80) or the cloned line designated by the World Health Organization as MHOM/SD/62/1S-C12D (SD) (16) was used in all the experiments. Promastigotes and the axenic amastigotes were grown and harvested as described previously (16).

Cloning of Centrin and Sequence Analysis-- A cloned arbitrarily primed polymerase chain reaction fragment (AP-PCR-8), amplified specifically from an Indian kala-azar parasite DNA using AP-PCR primer 9 (5), hybridized differentially to 2.1 kb1 RNA from promastigotes compared with axenic amastigotes. This DNA was used as a probe to screen a genomic cosmid library of L. donovani (19). A positive cosmid clone was sequenced in the region of the AP-PCR8 fragment. Sequencing revealed that the 3'-end of AP-PCR8 fragment overlapped with the 5'-untranslated region of an ORF. The encoded protein of 149 amino acids was identified through BLAST search as a homolog of the centrin gene from other organisms. The authenticity of the start site of the centrin ORF was confirmed by performing a reverse transcription-PCR, using an internal reverse primer (5'-TTCGCAACCTCCTTCAAG) and a forward primer (5'-ACTAACGCTATATAAGTA) designed from the Leishmania-specific mRNA splice leader sequence (20). The reverse transcription-PCR-amplified products were cloned into pCRII-TOPO vector and sequenced with the M13 forward and reverse primers. Multiple sequence alignment of centrins from various organisms was conducted in MacVector 7.0 program and used to determine cluster relationships among the sequences and to construct dendrograms representing cluster relationships (21).

Isolation of Genomic DNA and Southern Blot Analysis-- Total Genomic DNA was isolated from either promastigotes or axenic amastigotes according to the methods described in the manual for GENOME DNA isolation kit from BIO 101 Inc. The DNA was digested with restriction endonucleases EcoRI, NcoI, and SalI and separated on 1% agarose gels. Southern blot analysis of digested DNA was done as described previously (22).

Isolation of RNA and Northern Blot Analysis-- Total RNA was isolated from promastigote and axenic amastigote cultures of L. donovani using RNA STAT-60 according to the manufacturer's instructions (Tel-Test, Inc. Friendswood, TX). Total RNA (10 µg) was analyzed by Northern blot as described (23). Both Northern and Southern blots were hybridized with a 32P-labeled centrin coding region (450 base pairs) probe. The membranes were exposed and scanned on the PhosphorImager system (Molecular Dynamics Amersham Pharmacia Biotech Piscataway, NJ). The intensity of the hybridized bands was quantitated using ImageQuant software version 1.1 (Molecular Dynamics).

Plasmid Constructs and Expression of Recombinant Wild Type and Mutant Centrin Forms-- Plasmid constructs to express either the wild type or the mutant forms of LdCEN in either Escherichia coli or L. donovani cells are described in Fig. 1. The full-length open reading frame of wild type centrin and its various mutant forms were PCR-amplified with primers as described in Fig. 1 and ligated individually in pCR-T7/CT TOPO vector (Invitrogen) and transformed into competent E. coli BL21 (DE3) PlysS (Invitrogen). The authenticity of each of the constructs and the PCR-amplified fragments was confirmed by DNA sequencing. Reverse primers that were used to amplify wild type or mutant centrin forms to be expressed in the Leishmania parasite contained a hemagglutinin (HA) tag sequence (24) added in frame with centrin at the 3'-end. All the amplified products were digested with SpeI and ligated at the same site of pKSNEO (25) and transfected into the parasite. To clone LdCEN in bacterial expression vector (pQE-70) the PCR-amplified fragment was digested with SphI and BglII and cloned into SphI and BamHI site of pQE-70 in frame with the histidine-tag (His6) at the 3'-end and transformed into E. coli M15 cells as described (26). The protein was expressed from E. coli, purified through Ni-agarose ion-exchange column chromatography according to the manufacturer protocol (Qiagen Inc., Valencia, CA), and the protein was used to generate polyclonal antibody in rabbits (26).


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  		Fig. 1.   Details of the primers, plasmids and host strains used in the study. A, list of oligos that were used and are described in 5' to 3' directions. Bold regions in the oligos denote HA tag (24). Underlined regions are the restriction sites for the enzyme SpeI in the sequences 1-4, SphI in the sequence 9, BglII in the sequence 10. F, forward primer; R, reverse primer. B, a schematic diagram of centrin (ORF), showing the regions from which the oligonucleotide primers (dark bars numbered) were designed. C, chart showing the combination of the oligonucleotide primers used in PCR, the nature of products, type of expression plasmids, and hosts used.

Immunoblot Analysis of Leishmania Centrin Protein (LdCenp)-- Antisera raised against two recombinant human centrins (HsCen2p and HsCen3p, both rabbit polyclonal; gifts from Dr. Michel Bornens, Institute Curie, Paris, France), C. reinhardtii centrin (CrCenp1 (20H5) mouse monoclonal; gift from Dr. J. L. Salisbury, Mayo Clinic Foundation, Rochester, MN), and LdCenp (rabbit polyclonal; prepared by Spring Valley Labs, Woodbine, MD) were used to determine immunogenic cross-reactivity of the recombinant LdCenp. 100-200 ng of recombinant centrin protein was separated in a 12 or 15% SDS-PAGE gel, transferred to nitrocellulose membranes, and analyzed by Western blot (27) using the centrin antibodies (dilutions: anti-LdCenp Ab 1/1000, anti-HsCen2p Ab 1/2000, anti-HsCen3p Ab 1/250, and anti-CrCen1p Ab 1/1000). The proteins were visualized using the SuperSignal Chemi-luminescent substrate system (Pierce). To analyze the endogenous centrin and determine its size, mid-log culture of either promastigotes or axenic amastigotes were resuspended in 20 mM HEPES buffer, protein concentration was determined by BCA (Pierce), and 20 µg of total cell protein was analyzed on SDS-PAGE by Western blot analysis using various centrin antibodies.

Calcium Binding of LdCenp-- The binding assay for 45Ca was performed as described (17, 28). Briefly, the various recombinant proteins were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with 1 µCi/ml 45Ca in buffer containing 60 mM KCl, 5 mM MgCl2, and 10 mM imidazole-HCl, pH 7.0, for 10 min at the room temperature, washed with distilled water, dried, and exposed to x-ray film. To confirm the calcium binding and conformational changes of LdCenp in vivo, metal chelator EGTA (100 mM) was added to the parasite's cell lysate in 10 µl of reaction volume, incubated for 5 min at room temperature, and processed for Western blot analysis on a non-denaturing gel according to NovexTM, San Diego, CA using anti-LdCenp antibody.

Immunofluorescence Analysis-- L. donovani promastigotes were fixed in suspension in 4% (w/v) paraformaldehyde in PBS (50 mM Na2HPO4, 150 mM NaCl, pH 7.4) for 20 min at room temperature, washed three times in PBS, and allowed to attach to glass slides. After air drying, the slides were first immersed in ice-cold methanol (-20 °C) for 5 min, blocked for 30 min in 1% (w/v) bovine serum albumin (United States Biochemical Co., Cleveland, OH) in PBS, and incubated 1 h with either the anti-LdCenp serum (1:200 dilution) or the anti-HA serum (1:30 dilution) diluted in 1% bovine serum albumin in PBS. After three washes in PBS, cells were incubated for 1 h with affinity-purified fluorescein-conjugated anti-rabbit IgG (H+L) antibody when probed with anti-LdCenp Ab and Texas red anti-mouse IgG (H+L) antibody when probed with anti-HA serum antibody. These secondary antibodies (Vector Laboratories Inc., Burlingame, CA), were diluted 1:200-fold in PBS containing 1% bovine serum albumin. Cells were subsequently washed three times with PBS and mounted in Vectashield containing 4'6-diamidino-2-phenylindole (DAPI) (Vector, Vector Lab. Inc.) to stain both nucleus and kinetoplast. Cells were examined for fluorescence under the microscope (Nikon (DIAPHOT-200), Tokoyo, Japan), with epi-fluorescence and images captured with Pixera (120ES) color digital camera. Confocal studies were conducted under 100× objective lens of Leica-DM IRBE (Leica Microsystem, Heidelberg, Germany) using krypton and argon/UV lasers. The focal plane chosen in all the cells was in the middle of the cells. The images were processed using Adobe Photoshop 5.5 (Adobe Systems Inc., Mountain View, CA).

Culture and Transfection of the Parasites-- Mid-log phase promastigotes (2-4 × 107 cells/ml) were harvested by centrifugation at 3000 g for 10 min at 4 °C. Cell pellets were washed in ice-cold PBS and electroporated with the DNA using conditions as described previously (29). Transfected promastigotes were selected with minimal doses of G418 (20 µg/ml). The drug-resistant cells were used in all subsequent experiments.

Flow Cytometry-- Promastigotes from early exponential cultures were collected, fixed in 70% ethanol, and washed with PBS. The fixed cells were treated with 100 µg/ml ribonuclease in PBS for 5 min at room temperature and stained with 50 µg/ml propidium iodide (Sigma) in PBS for 15 min on ice and analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and CELLQuest software. For each sample 20,000 fluorescent events were measured, and the data was analyzed using the Modfit Lt. Software was Verity Software House, Inc. (Topshan, ME).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Sequence Analysis of the L. donovani Centrin Gene-- We used an arbitrarily primed polymerase chain reaction (AP-PCR) approach, as previously described (5), to isolate genes from L. donovani that are differentially expressed during the growth and differentiation of this parasite. A 1-kb AP-PCR fragment was generated specifically using DNA isolated from L. donovani parasites collected from an Indian Kala-azar patient. The fragment was cloned and used as a probe to analyze total RNA from the promastigotes and axenic amastigotes of L. donovani. A 2.1-kb RNA hybridizing to the AP-PCR fragment was found to be expressed significantly more in promastigotes than in axenic amastigotes (data not shown). Based on such differential expression, the AP-PCR fragment was used to screen a L. donovani cosmid DNA library. Sequence analysis of a positive clone showed an ORF that overlapped with the 3'-end of the 1-kb AP-PCR sequence. Homology search of the open reading frame revealed that it has significant similarity with centrin proteins from many organisms. The complete nucleotide and deduced amino acid sequence of the L. donovani centrin (LdCEN) gene is shown in Fig. 2. The authenticity of the start site of the centrin's ORF was confirmed by performing a reverse transcription PCR, using an internal reverse primer and the splice leader forward primer. The nucleotide sequence of the reverse transcription PCR clones confirmed that the identified ORF occurs in a mature transcript of the LdCEN gene. The sequences of the amplified fragments also indicated the presence of at least two splice sites in the 5'-untranslated region of the gene (Fig. 2).


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  		Fig. 2.   Nucleotide and amino acid sequences of L. donovani centrin (GenBank accession number AF406767). The splice leader sequence is single underlined. Two putative splice sites are indicated by the arrows. The amino acid sequence of LdCenp is shown in single letter code. In each EF-hand, a 12-amino acid Ca2+ binding site (white box), is flanked on either side by a 9-amino acid alpha -helical stretch (gray box). Acidic amino acids in the Ca2+ binding site are shown in bold. Hydrophobic amino acids in the alpha -helical regions are shown as outlined characters. Asterisk indicates the stop codon.

Centrins and calmodulins, another closely related Ca2+-binding protein, have in general four EF-hand (Ca2+ binding) domains; however, the number of functional EF-hand domains vary among the centrins (30, 31). Sequence motif analysis of L. donovani centrin protein (LdCenp) predicted only two Ca2+ binding sites (EF-hand 1 and 4) (Fig. 2). In addition the LdCenp was also found to possess hydrophobic amino acids in their alpha -helices around both the EF-hands 1 and 4 (Fig. 2) as have been observed with other centrins. The amino acid sequence of LdCenp was analyzed by ClustalW alignment with centrins from different organisms, as shown in Fig. 3A. The calculated percent similarity shows that it is closer to HsCen2p (61%), HsCen1p (60%), or Trichomonas centrin (60%) than other centrins. The N'-terminal non-conserved domain of centrins, which is variable in length, is considered to be responsible for the functional diversity of centrins (32, 33). Interestingly, L. donovani centrin has a significantly small N-terminal region compared with centrins from other species (Fig. 3A).


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  		Fig. 3.   A, multiple alignment of centrin sequences of various eukaryotes: L. donovani (AAL01153); Trichomonas (CAB5 5607); Paramecium (Q27178); mouse (NP-031619); human isoforms 1-3 (NP-004057, P41208 and O15182); Giardia (AAB05594); Chlamydomons (P05434); yeast (NP-014 900). All the accession numbers are from GenPept data bank. A calmodulin gene sequence of yeast (NP-009667) is also included. Amino acids are listed in the standard one-letter code, and residues identical to Leishmania centrin are indicated by dashes. The gray boxes (EF-hands 1-4) are the putative Ca2+ binding domains. Acidic amino acids in the boxes are printed in bold. Bold numbers in parenthesis at the end of the last lines represent the percent similarity of each to L. donovani centrin. B, phylogenetic analysis of centrins from various eukaryotes. The dendrogram of complete protein sequences of centrins was generated in the ClustalW-alignment section of MacVector 7.0 program utilizing systematic, bootstrap, and neighbor-joining options. The numbers on the nodes indicate the proportion of times (%) the centrins (shown on the right) grouped together in 1000 bootstrap samples in the program. The branching order, rather than the actual distances on the tree, is shown.

A neighbor-joining systematic tree based on centrins of various eukaryotes was constructed, to study the phylogenetic relationship of LdCenp with centrins of other organisms. Two distinct clusters were seen in the tree (Fig. 3B). One cluster had CrCen1p, mouse centrin, HsCen2p, and HsCen1p, and the second cluster had Giardia centrin, CDC31p, and HsCen3p. However, centrins of Paramecium and Leishmania branched off independently from the common ancestor of all the centrins (Fig. 3B).

Northern and Southern Blot Analyses of LdCEN-- To analyze LdCEN mRNA levels in both promastigotes and axenic amastigotes, a Northern blot analysis was performed using total RNA obtained from the mid-log cultures of these two parasite stages. The 32P-labeled LdCEN probe recognized two bands (Fig. 4A): a major ~1.7-kb band that expressed equally in both promastigotes and axenic amastigotes and a 2.1-kb band expressed significantly more in promastigotes than in axenic amastigotes.


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  		Fig. 4.   Northern and Southern blots of Leishmania centrin. A, Northern blot: 12 µg of total RNA from each of the mid-log parasites (Pro, promastigote; Am, axenic amastigote) was separated in an agarose/formaldehyde gel. Arrows indicate the mRNAs hybridized. B, Southern blot: L. donovani genomic DNA (5 µg) was digested with the restriction enzymes indicated and separated on 1% agarose gel. Both the blots were hybridized with LdCEN probe. Molecular weight standards are mentioned on the right of both the panels.

To determine the copy number of LdCEN in the genome, Southern blot analysis of the total genomic DNA from L. donovani promastigotes was done using the centrin coding region as a probe (Fig. 4B). DNA digested individually with either SalI or EcoRI, whose sites are not present in the coding region of the centrin gene, gave a single band of ~4.1 or 4.3 kb, respectively. On the other hand, digestion of DNA with NcoI, which has two sites at positions 1 and 203 in the LdCEN coding region, resulted in three bands of ~0.2, 3.8, and 6.5 kb (Fig. 4B, lane 2). These results are consistent with LdCEN being a single copy gene.

Antigenic Similarity of LdCenp with Centrins from Other Organisms-- To test whether LdCenp has any antigenic similarity with centrins from other organisms, a Western blot containing the recombinant LdCenp expressed in E. coli was performed with antisera raised against human centrin 2, human centrin 3, C. reinhardtii centrin 1, and LdCenp (Fig. 5A). The recombinant LdCenp cross-reacted with anti-LdCenp Ab and anti-HsCen2p Ab, but did not react with either anti-HsCen3p Ab or anti-CrCen1p Ab (Fig. 5A). To further confirm that LdCenp is expressed in the parasite and to analyze whether there are more than one form of LdCenp that may cross-react with the various anti-centrin antibodies, we tested these antibodies in a separate Western blot using cell lysates from mid-log promastigotes and axenic amastigotes (Fig. 5B). Anti-LdCenp Ab and anti-HsCen2p Ab reacted with a 17-kDa protein in both the lysates (Fig. 5B, lanes 1-4). However, anti-Hs-Cen2p antibody reacted with an additional protein of ~25 kDa (Fig. 5B, lanes 3 and 4). On the other hand, anti-HsCen3p and anti-CrCen1p antibodies reacted with different size proteins (~19 kDa and ~18 kDa, respectively) in Leishmania cell lysates. (Fig. 5B, lanes 5-8). The intensity of cross-reacting centrin bands was similar in promastigotes and axenic amastigotes. These results suggest that additional centrin-like proteins may exist in the parasite.


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  		Fig. 5.   Western blot analysis using various centrin antibodies. A, 100 ng of nickel-purified recombinant LdCenp was run on a 15% SDS-PAGE and analyzed for its cross reactivity by Western blot using either polyclonal antibodies against LdCenp, HsCen3p, HsCen2p, or monoclonal antibody against CrCen1p. B, promastigote (Pro) and axenic amastigote (Am) cell lysates (25 µg of protein in each lane) were analyzed similarly by Western blot using all four antibodies. Molecular weight standards are shown in both the panels.

EF-Hand 4 Is the High Affinity Ca2+ Binding Site in LdCenp-- Amino acid sequence analysis of LdCenp and its comparison with known centrins revealed that it has two putative Ca2+ binding EF-hand domains (1 and 4). We tested whether LdCenp binds to Ca2+ in vitro and defined which of the predicted sites interact with Ca2+. To do so we constructed three LdCenp mutants that had either the first, the fourth, or both putative Ca2+ binding domains deleted (Fig. 6A), and expressed the full-length and mutant centrins in E. coli (Fig. 6B). The proteins were tested for their binding to 45Ca in vitro. Full-length and N-terminal-deleted (LdCenp Delta N) centrins bound a similar level of 45Ca (Fig. 6C, lanes 1 and 3). However, the C-terminal-deleted centrin (LdCenp Delta C) bound significantly less Ca2+ (Fig. 6C, lane 2), and the LdCenp that lacked both the C- and the N-terminal regions (LdCenp Delta NC) did not bind calcium at all (Fig. 6C, lane 4). The PhosphorImager quantitation of 45Ca-bound bands revealed that the LdCenp Delta N bound 16-fold more 45Ca than the LdCenp Delta C. The Ca2+ binding to centrin was specific, since bovine serum albumin, a non-Ca2+-binding protein, did not bind to 45Ca in this assay (data not shown). These results demonstrate that the LdCenp has two Ca2+ binding sites and that Ca2+ binds preferentially to the C-terminal site.


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  		Fig. 6.   Calcium binding analysis of recombinant LdCenp. A, schematic diagram showing the type of deletions made in the LdCEN gene. F, full-length centrin shows the EF-hands 1 and 4. Delta C, C-terminal-deleted centrin that removes the Ca2+ binding region of EF-hand 4 and the rest of the variable C-terminal region. Delta N, N-terminal end-deleted centrin that removes the first Ca2+ binding region, including the variable N-terminal region. Delta NC, centrin without both C- and N-terminal regions. B, Ponceau S-stained nitrocellulose membrane blotted from the SDS-PAGE of the various purified recombinant centrin proteins (500 ng in each lane) reported in panel A. Recombinant centrins confirmed through a separate Western blot analysis using the anti-LdCenp Ab (results not shown) are pointed out by arrows. The 22-kDa band seen in all the lanes could be a contaminant coming through the purification. C, autoradiograph showing 45Ca binding of the proteins shown in panel B.

Both N- and C-terminal Regions Are Necessary for the Ca2+-dependent Protein Conformation-- Recent reports using either NMR spectrum for C. reinhardtii centrin (34) or CD spectroscopy and size exclusion chromatography for Scherffelia dubia and human centrins (7) showed Ca2+ induced conformational changes in the proteins. Having shown that the LdCenp binds to Ca2+, we analyzed the Ca2+-induced folding conformation of LdCenp. Plasmid constructs containing either full-length centrin (pKSNEO LdCEN) or N-terminal-deleted centrin (pKSNEO LdCEN Delta N) and C-terminal-deleted centrin (pKSNEO LdCEN Delta C) along with vector control (pKSNEO) were transfected into wild type L. donovani promastigote cells. Proteins were extracted from all the transfected parasites at mid-log stage and analyzed by Western blot using anti-LdCenp Ab (Fig. 7A). Though the level of expression of each form of recombinant centrin differed, each corresponded to the predicted molecular weight and was sufficiently abundant for further analysis. These protein extracts were incubated in the presence or absence of the metal chelator EGTA, separated in a non-denaturing gel, and analyzed by Western blot using antibody against either LdCenp, which recognizes the endogenous centrin of the control parasite, or antibody against HA tag sequence, which recognizes the episomally expressed centrins in the transfected parasites (Fig. 7B). Endogenous centrin after treatment with EGTA migrated faster in the gel than untreated (Fig. 7B, lanes 1 and 2). The same EGTA-induced shift occurred with the transfected over-expressed full-length centrin (Fig. 7B, lanes 5 and 6). However, treatment with EGTA did not affect the mobility of either LdCenp Delta N or LdCenp Delta C (Fig. 7B, lanes 7-10). These results suggested that binding of Ca2+ to both binding sites induced a conformational change of this protein. Further, deletion of the N or C terminus resulted in a loss of the Ca2+ dependent conformational change.


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  		Fig. 7.   Demonstration of the effect of EGTA on the conformation of centrin. A, Western blot analysis of the episomally expressed centrins in the parasite. Total cell lysates of 20 µg of protein from control (C, transfected with pKS NEO), full (F, transfected with pKSNEO LdCEN), C-deleted (Delta C, transfected with pKSNEO LdCEN Delta C) and N-deleted (Delta N, transfected with pKSNEO LdCEN Delta N) were analyzed in Western blot using anti-LdCenp Ab. B, cell lysates of various mid-log promastigotes expressing either full-length centrin or its truncated mutants were treated with or without 100 mM EGTA. Samples separated in a 12% non-denaturing PAGE were transferred to nitrocellulose membrane and analyzed by Western blot using either anti-LdCenp or anti-HA tag antibodies. C, F, Delta N, and Delta C are same as mentioned in panel A.

Parasite Growth Regulated Expression of LdCenp-- Centrins have been implicated to have an essential function during the cell division cycle (35). As a first step toward understanding the role of LdCenp in Leishmania growth, we explored whether there is a correlation between the expression of LdCenp and the parasite growth. The level of expression of both centrin mRNA (the 1.7-kb) and protein was measured at different stages of promastigote and axenic amastigote growth. Quantitation of Northern (Fig. 8A) and Western blots (Fig. 8B) indicated that the level of LdCEN mRNA and protein, in both the promastigotes and axenic amastigotes, was maximal in the exponentially growing culture. The levels of both mRNA and protein steadily declined as the parasites progressed from late-log to stationary phase, thereby suggesting that the expression of centrin correlates with the growth rate of L. donovani.


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  		Fig. 8.   RNA and protein levels of LdCEN from growing and stationary phase parasites. Cells were collected at different time points during growth and stationary phase of both promastigote and axenic amastigote cultures (). These cells were then processed for isolation of either total RNA or total protein. The RNA samples were analyzed by Northern blot (panel A), and the protein was analyzed by Western blot (panel B) using either centrin DNA or centrin antibody as probes respectively. Fig. shows the quantitation of Northern (black-square) and Western (black-diamond ) blots at various time points. The data were the representative of two independent experiments.

LdCenp Localizes at the Basal Body Region of the Promastigotes and Axenic Amastigotes-- To ascertain the localization of centrin in L. donovani, we carried out immunofluorescence analysis. Paraformaldehyde-fixed mid-log phase promastigotes and axenic amastigotes were stained with anti-LdCenp Ab and DAPI and examined by confocal microscopy. The intensity of fluorescence by anti-LdCenp Ab was mostly concentrated in the anterior part of both the parasitic forms. In addition, a dense fluorescent spot was seen in the area close to the DAPI-stained kinetoplast, (Fig. 9). However, the stationary phase cells of either promastigotes or axenic amastigotes showed no significant staining by anti-LdCenp Ab (data not shown). The basal body has been shown to be localized at the flagellar root that remains tightly associated to the kinetoplast in Leishmania (15). The results showed that LdCenp is predominantly localized close to the kinetoplast of both growing promastigotes and axenic amastigotes and may be associated with the basal body of Leishmania.


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  		Fig. 9.   Immunolocalization of centrin in promastigotes and axenic amastigotes. Immunofluorescence analysis of both promastigotes (Pro) and axenic amastigotes (Am) using rabbit anti-LdCenp Ab (images 1 and 2). Nucleus (N) and kinetoplast (K) were stained with DAPI (images 3 and 4). Images were viewed under the confocal microscope. The stained images and phase were merged and shown (images 5 and 6).

Differential Localization of the Mutant Centrins-- To identify the domain of LdCenp, which is responsible for its targeting to the basal body, we analyzed log-phase Leishmania parasites that over-expressed either complete, N-deleted or C-deleted forms of centrin. Transfected parasites were stained with anti-HA tag Ab to reveal the over-expressed centrins. Parasites expressing full-length and carboxy-end-deleted centrins showed immunostaining throughout the parasite (Fig. 10, panels 4 and 8) possibly due to the high expression of both these centrin forms compared with control cells (Fig. 7A). On the contrary, in a significant number (~40%) of cells expressing LdCenp Delta N, the over-expressed protein was predominantly localized toward the posterior region (Fig. 10, panel 6). The redistribution of LdCenp Delta N toward the posterior region in these cells suggests that the N-terminal region may have the localization signal that could target the protein toward the basal body region.


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  		Fig. 10.   Immunolocalization of mutant centrin expression. Parasites were processed for immunofluorescence using mouse anti-HA tag Ab to stain the episomally expressing centrins. Texas red anti-mouse IgG as a secondary antibody stains the cells red (2, 4, 6, and 8). Cells were viewed by confocal microscopy. C, F, Delta N, and Delta C are same as mentioned in Fig. 7A.

LdCenp Lacking the N-terminal Region Has a Dominant Negative Effect on the Growth of the Parasite-- To determine the role of centrin in the growth of L. donovani, growth of the transfected parasites was analyzed in culture. The promastigotes that were transfected with either vector alone or the LdCenp Delta C construct grew almost at the same rate (Fig. 11A). The full-length centrin over-expressing line showed slightly slower growth rate compared with the control cells; however, reached the same stationary plateau. On the other hand, parasites expressing LdCEN Delta N displayed a 2-fold reduction in the growth rate compared with the other transfectants based on the slope of the curve during log phase (Fig. 11A). The maximum density of the LdCenp Delta N-expressing cells at the stationary stage was also low compared with the control cells. Similar suppression of growth due to LdCenp Delta N was also seen in the axenic amastigotes (data not shown). To determine the cause of slow growth of LdCenp Delta N-expressing cells, the mid-log stage promastigote cultures were subjected to cell cycle analysis using flow cytometry. Results showed a decrease of 23% in S-phase cells and an increase of 26% in G2-phase cells in the LdCenp Delta N-transfected line compared with the control line (Fig. 11B). There was no significant difference in the number of S-phase cells expressing either full-length centrin or LdCenp Delta C. However, ~10% more full-length centrin-expressing cells remained in G2-phase than in the control (Fig. 11B). These results suggest that the slower growth observed in LdCenp Delta N expressing cells could be due to a significant number of cells remaining for a longer period of time in the G2/M phase of the cell cycle compared with control cells.


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  		Fig. 11.   A, effect of the expression of various mutant centrin forms on the growth of the parasite. The graph shows the growth of the parasite (promastigotes) transfected with either full-length (F) or mutant centrins (Delta N and Delta C) or vector control constructs (C). The results were the mean of four independent growth experiments. B, flow cytometric analysis of the DNA content of L. donovani lines expressing various centrin mutants. Promastigotes transfected with either full-length (F) or mutant centrins (Delta N and Delta C) or vector control constructs were grown to early-exponential phase, fixed, stained with propidium iodide, and subjected to fluorescence-activated cell sorter analysis. For each sample 20,000 fluorescent events were measured. The difference in the number of cells between each centrin over-expressing sample and the control was expressed as % change and shown for each phase of the cell cycle. The data were averaged from three independent experiments and plotted with standard deviation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Centrin is a calcium-binding protein involved in the contractile function of the cytoskeletal structures in eukaryotes (32, 36). We report here the first cloning of such a gene among Kinetoplastids, whose members are considered to be primitive eukaryotes (15). The cloned gene, LdCEN, from the L. donovani parasite is more homologous to centrin than the other closely related Ca2+ binding EF-hand proteins such as calmodulins. Typically, centrin is distinguished structurally from calmodulin by a longer N-terminal variable region. This longer N terminus is thought to confer functional diversity to centrins (7, 32). However, this region is smaller in LdCEN than all other centrins and calmodulins. The short N terminus may reveal its ancestral position in the evolution of centrin. LdCenp cross-reacts with the antibody raised against human centrin protein (HsCen2p). Multiple centrins are localized at the centrosome and expressed in all the dividing cell types (36). Specifically HsCen2p expression level increases during ciliogenesis in the tracheal epithelial cells, and its involvement in cell division as well as cellular motility has been demonstrated (37, 38). Similarly, LdCenp was found to be localized at the basal body of the parasite, and its expression was high when cells were actively dividing and significantly reduced in the resting cells. This correlation of expression pattern suggests that Leishmania centrin may have a role in the growth of the parasite. However, it is also speculated that it may not have a role in the cellular movement, because this protein is equally expressed in the non-motile axenic amastigote stage and is present in lower concentration in the stationary stage promastigote cells, which are highly motile.

The actual number and position of the Ca2+ binding sites varies from centrin to centrin. In HsCen2 only the fourth EF-hand binds calcium (13), whereas, in LdCenp both EF-hand 1 and 4 bind to Ca2+. EF-hand 4 binds to calcium 16-fold more than EF-hand 1 as evidenced from our calcium binding study on the various recombinant mutant centrin proteins. At the molecular level, the calcium binding generates conformational changes in CrCen1p (7) and CDC31p (14). The Ca2+-dependent polymerization of the green algae S. dubia centrin protein results in a filamentous network (7). A similar Ca2+-induced protein conformational change of LdCenp was also demonstrated in the present study. The implications of this conformational change for the LdCenp remain to be studied.

At least three major types of centrin have been described by others. Type 1 (mouse centrin 1) is expressed during spermatogenesis suggesting a role in meiosis (39). The second type of centrin (HsCEN2) protein is up-regulated during ciliogenesis of mammalian cells (37) and has been shown to play a role in cellular motility and microtubule severing (38, 40). The third type of centrin, similar to yeast protein CDC31 and HsCEN3 appears to play a role in centrosomal duplication (41). The antisera against HsCen2p reacted with a protein equivalent in size to the recombinant LdCenp and with a larger size protein in the L. donovani lysate. Anti-HsCen3p Ab and anti-CrCen1 Ab reacted with proteins in the Leishmania lysates yet they differed in size from LdCenp. These results suggest the existence of more than one centrin isotype in L. donovani. The reason for the molecular heterogeneity in LdCenp protein and the functional analysis of the different putative forms of LdCenp remain to be analyzed, though each of them may serve a different function as described for human centrins (32, 35).

In our phylogenetic analysis, LdCenp branched off independently from a common centrin ancestor, while most other centrins can be grouped into two clusters. These clusters interestingly also correlate with their distinct biological functions as observed (9-11). HsCen1p, HsCen2p, and CrCen1p of one cluster have been involved in the segregation of centrosome, whereas CDC31p and HsCen3p of the other cluster have been involved in centrosome duplication during cell division (9-11). Despite Leishmania centrin's early divergence, the unique binding of the anti-HsCen2p Ab suggests structural relatedness of LdCenp and Hscen2p. This similarity to HsCen2p may not be reflected in the phylogenetic analysis due to amino acid differences in regions other than the common antigenic epitopes. Similar inconsistencies between phylogenetic trees and biochemical analysis have been observed by others (42, 43). Whether the structural similarity reflects a common function remains to be analyzed for LdCenp.

Centrin's association with growth in several eukaryotes has been studied (8). Injecting recombinant Chlamydomonas centrin or human centrin 2 in two cell stage frog embryos delayed cleavage and promoted the formation of abnormal blastomeres (35). Expression of both antisense and sense transcripts of centrin arrested spermiogenesis in Marsilea vestita (44). In the present study, we analyzed the role of LdCenp in the parasite growth by over-expressing the full-length and the mutant centrins. Though full-length and C-terminal-deleted centrins did not alter the growth of the parasite, N-deleted centrin did reduce the growth by 2-fold over the control in a dominant negative fashion. The slow growth of this culture was correlated with the significant number of cells remaining for a longer period in G2/M phase of the cell cycle, suggesting centrin function is crucial for completion of mitosis. The importance of the N-terminal sub-domain for centrin function was emphasized similarly by observing slower growth rate in the yeast cells expressing centrin (CDC31), which had the N-terminal region replaced with that of S. dubia centrin (7). Secondly, yeast centrin interacts with the cellular protein Kar1p at its C-terminal region (7, 14). In the present study in Leishmania, LdCenp could be similarly interacting with other cellular components after binding to Ca2+, and LdCenp Delta N could compete with the endogenous centrin for this interaction. Such an interaction, having no functional N-terminal region, would probably affect the role of centrin responsible for the growth of the parasite. The second abnormality noticed with such a slow growing parasite was the localization of the LdCenp Delta N in the cells. The protein was seen everywhere in the cell, though a significant number of cells (~40%) expressing LdCenp Delta N showed localization predominantly at the posterior region. This suggests that the basal body localization signal for LdCenp may be in its N-terminal region. Alternatively LdCenp may interact with another cellular protein through its N terminus for proper localization as observed for yeast centrin CDC31p. CDC31p interacts with Kar1p, a spindle pole body component that helps to localize CDC31p to the spindle pole body. No CDC31p was detected at this organelle in cells lacking Kar1p (45, 46). However, the exact mechanism by which LdCenp Delta N localizes to the posterior end of a significant number of cells remains to be investigated. Nonetheless, possible co-localization of LdCenp with the basal body, the correlation of centrin expression with Leishmania growth and alteration of growth rate, and alteration of the cell cycle upon expression of LdCenp Delta N may suggest a role for centrin in Leishmania cell division. In conclusion, understanding the role of centrin in Leishmania growth could provide ways to alter the growth of the parasite and clues for the development of attenuated Leishmania vaccine candidates.

    ACKNOWLEDGEMENTS

We acknowledge James McNally and Tatiana Karpova (Laboratory of Receptor Biology and Gene Expression, Fluorescence Imaging Facility, NCI, National Institutes of Health) for use of the Leica TCS confocal fluorescence microscope, Jeffrey L. Salisbury (Mayo Clinic, Rochester, MN) and Michel Bornens (Institut Curie, Paris, France) for providing certain anti-centrin antibodies, Dennis Dwyer (Cell Biology Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health) and Gerardo Kaplan (Laboratory of Hepatitis and Related Emerging Agents, DETTD, CBER, Food and Drug Administration) for valuable suggestions, and Mike Clutch (Laboratory of DNA Viruses, Division of Viral Products, CBER, and Food and Drug Administration) for technical help in DNA sequencing.

    FOOTNOTES

* This work was supported by INDO-US Vaccine Action Program Y3-AI-9319-01 through interagency agreement between NIAID, National Institutes of Health and CBER/Food and Drug Administration.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF406767 and to GenPept Data Bank with accession number(s) AAL01153.

** To whom correspondence should be addressed. Tel.: 301-496-2205; Fax: 301-480-7928; E-mail: nakhasi@cber.fda.gov.

Published, JBC Papers in Press, September 8, 2001, DOI 10.1074/jbc.M106806200

    ABBREVIATIONS

The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Ab, antibody; PBS, phosphate-buffered saline; HA, hemagglutinin; DAPI, 4'6-diamidino-2-phenylindole; AP-PCR, arbitrarily primed PCR; ORF, open reading frame; Delta N, N-terminal deletion; Delta C, C-terminal deletion; Delta NC, N- and C-terminal deletions.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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