Role of Centrins 2 and 3 in Organelle Segregation and Cytokinesis in <em>Trypanosoma brucei</em>

<div><p>Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in <em>Trypanosoma brucei</em> (<em>T. brucei</em>) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of <em>T. brucei</em> centrin2 (TbCen2) and <em>T. brucei</em> 3 (TbCen3) in the early events of <em>T. brucei</em> procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1–3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.</p> </div

Effect of ablation of centrins on cell shape and organelle number.

<p><b>A</b>: Effects on the duplication and segregation of basal bodies, kinetoplasts and nuclei in TbCen2 and 3-depleted cells. The cells were stained with DAPI for the nuclei and kinetoplasts, YL1/2 for the basal bodies and L8C4 for the flagella. Note the RNAi induced cells are large and pleomorphic in shape with multiple organelles with more than one flagellum and the new flagella are of detached type (middle and lower panels) compared to the control cell with organelles in single number with one attached flagellum (top panel). Scale bar, 5 µm. <b>B</b>: Electron microscopy of centrin-depleted cells. (<b>a</b>) A typical control cell in this particular section shows single flagellum, pair of basal bodies, kinetoplast and nucleus. (<b>b–d</b>) TbCen2-depleted cells with (<b>b</b>) multi basal bodies and the kinetoplast with enlarged size compared to the control in ‘<b>a</b>’, (<b>c</b>) multi nuclei and (<b>d</b>) abnormal kinetoplast. (<b>e–h</b>) TbCen3-depleted cells with (<b>e</b>) multi basal bodies, (<b>f</b>) multi flagella. Inset is the cross-section image of a detached flagellum displaying the normal axoneme (with 9+2 microtubule structure) and the paraflagellar rod, (<b>g</b>) multi nuclei and (<b>h</b>) an abnormal kinetoplast. Scale bars, 500 nm (<b>a</b>, <b>b</b>, <b>d–f</b> and <b>h</b>) and 2 µm (<b>c</b> and <b>g</b>). <b>C & D</b>: Bar graphs showing the percent of <i>T. brucei</i> procyclic cells with duplicated basal bodies (<b>C</b>) and flagella (<b>D</b>). The cells were analyzed on day 3 after induction for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells. Data represent the means ± SD of three independent experiments. For each TbCen2 and 3 RNAi studies, over 140 cells were manually counted and analyzed. F, flagellum; B, basal body; K, kinetoplast; N, nucleus; P, flagellar pocket, A, axoneme; R, paraflagellar rod; AF, attached flagellum; DF, detached flagellum; An, anterior; Po, posterior.</p

Images show direct effect of centrins' depletion on kinetoplast biology and overall organelle segregation in <i>T. brucei</i>.

<p><b>A</b>: Segregation patterns of the basal bodies along with the kinetoplasts during RNAi for TbCen2 and 3. The cells were stained with DAPI for nuclei and kinetoplasts and YL1/2 for basal bodies. Panels 1 and 2 display the typical segregation of basal bodies, kinetoplasts and nucleus in the control cells. Panels 3–5 and 6–8 display the 3 different segregation patterns of the organelles after RNAi of TbCen2 and 3 respectively, other than the typical pattern seen with the control cells (panel 2). Panel 1 is the ‘G’ stage of the cell cycle of the procyclic form with 1K1N, where both the basal bodies and kinetoplast are seen at the posterior region of the cell. Scale bar common for all the images, 5 µm. <b>B</b>: Histogram of cells with 2 basal body pairs displaying each of the 3 new segregation patterns of basal bodies and kinetoplasts in the RNAi cells. For each TbCen2 and 3 RNAi studies, over 100 bi-nucleated cells with abnormal organelle segregation were manually counted and analyzed. Data represent the means ± SD of three independent experiments. The time points at which the cells analyzed after induction was day 3 for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells. An, anterior end; B, basal body; K, kinetoplast; N, nucleus, Po, posterior end. Scale bars, 5 µm.</p

Centrins' RNAi and their effect on parasite growth.

<p><b>A</b>: Northern blots of RNA from induced (I) and uninduced (UN) TbCen2 and 3 cultures. Membranes containing 12 µg total RNA from day 2 uninduced and induced cells were used. Membranes, in addition to hybridization with DNA probes of either specific centrin or α-tubulin (loading control) were reprobed with DNA probes of other centrins (TbCen1-3). <b>B</b>: The effect of TbCen2 and 3 knockdown on the <i>in vitro</i> growth of <i>T. brucei</i> procyclics. The cells were grown with or without tetracycline. The data represent the means of ± SD of the three independent experiments. *p<0.02.</p

<i>T. brucei</i> procyclic cells after TbCen2 or 3 RNAi induction displaying the cell populations that contain undivided kinetoplasts.

<p>DAPI stained cells were used in the analysis. The cells were analyzed on day 3 after induction for TbCen2 RNAi cells and day 2 for TbCen3 RNAi cells.</p>*<p>Cells as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045288#pone-0045288-g004" target="_blank">Figure 4A</a>; panel 3.</p>**<p>Cells as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045288#pone-0045288-g004" target="_blank">Figure 4A</a>; panel 5.</p

Schematic diagram of the organelle segregation and the failure of segregation in the absence of TbCen2 or 3 in <i>T. brucei</i> procyclic form.

<p>Normal cell division and the three different types of abnormal segregation patterns of the organelles during centrin RNAi leading to the failure in cytokinesis and the generation of giant cells with multiple organelles are shown. Color coordination for organelles colored: red, basal body; green, kinetoplast; blue, nucleus.</p

Analysis of cell structure and kinetoplasts and nuclei number in the RNAi cells.

<p><b>A</b>: DAPI-stained images of the control and TbCen2 and 3-depleted cells, 4d after RNAi induction. DF, detached flagella; K, kinetoplast; N, nucleus. Scale bar, 5 µm. (<b>B & C</b>) Fluorescence-activated cell sorting analysis for relative DNA content of TbCen2 and 3 depleted cells. <b>B</b>: Histogram plots of cell count by DNA content obtained directly from the flow cytometry analysis are shown. <b>C</b>: The percentage of cells that contain two times the DNA content (2C), four times (4C), and more than 4 times (>4C) were measured at each time point and are shown. Data represent the means ± SD of three independent experiments. <b>D</b>: Bar graph showing increase in the proportion of multinucleated and multikinetoplast cells with time after RNAi knockdown of TbCen2 and 3. Cells after different days of RNAi induction were stained with PI and examined by fluorescence microscope for tabulation of the cells with different number of kinetoplasts and nuclei. The time points at which the cells were analyzed after induction was up to day 5 for TbCen2 RNAi cells and day 4 for TbCen3 RNAi cells. Data represent the means ± SD of three independent experiments. For each TbCen2 and 3 RNAi studies, over 140 cells were manually counted and analyzed.</p

Sequence of organelle biogenesis and segregation in normal and RNAi cells.

<p><b>A</b>: The control cells sequentially show duplication of DNA content from panel 1 to 2; division into two kinetoplasts from panel 2 to 3; segregation of the separated kinetoplasts to appropriate locations from panel 3 to 4. An, anterior end; K, kinetoplast; N, nucleus, Po, posterior end. Scale bar, 5 µm. <b>B</b>: Histogram showing the percent of TbCen2 and TbCen3 RNAi cells with undivided kinetoplasts in the two nucleated cells at different time points including day 3 for TbCen2 RNAi and day 2 for TbCen3 RNAi. For the TbCen2 and 3 RNAi, average 200 and 170 cells respectively were manually counted and analyzed during each time point. Data represent the means ± SD of three independent experiments.</p

Live Attenuated <i>Leishmania donovani</i> Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis

<div><p>Background</p><p>Visceral leishmaniasis (VL) caused by the protozoan parasite <i>Leishmania donovani</i> causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated <i>L</i>. <i>donovani</i> parasites (<i>LdCen-/-</i>) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed <i>LdCen-/-</i> parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice.</p><p>Methodology</p><p>Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with <i>LdCen-/-</i> parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4⁺ Th1 cell effector function compared to <i>LdWT</i> infected BMDCs <i>in vitro</i>. Similarly, parasitized splenic dendritic cells from <i>LdCen-/-</i> infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to <i>LdWT</i> infected mice. We also evaluated <i>in vivo</i> protection of the <i>LdCen-/-</i> immunized young and aged mice against virulent <i>L</i>. <i>donovani</i> challenge. Immunization with <i>LdCen-/-</i> induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent <i>L</i>. <i>donovani</i> challenge, <i>LdCen-/-</i> immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the <i>LdCen-/-</i> induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice.</p><p>Conclusions</p><p>Taken together, <i>LdCen-/-</i> immunization induced a significant but diminished host protective response in aged mice after challenge with virulent <i>L</i>. <i>donovani</i> parasites compared to young mice.</p></div

Ag-specific intracellular cytokine secretion analysis of CD4 and CD8 T cells from <i>LdCen-/-</i> immunized and non-immunized young and aged mice after virulent <i>L</i>. <i>donovani</i> challenge.

<p>The 8- wk post-immunized or non-immunized young and aged mice were challenged for 4-wk with virulent <i>L</i>. <i>donovani</i>. Intracellular cytokine analysis was done as shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004963#pntd.0004963.g006" target="_blank">Fig 6A</a> and divided into seven distinct subpopulations. (A) Cytokine analysis of CD4 T cells from 8-wk post immunized and 4-wk post challenged mice. (B) Cytokine analysis of CD8 T cells of 8-wk post immunized and 4-wk post challenged mice. (C) IL-10 secreting CD4 T cells and (D) the ratio of IFN-γ to IL-10 producing CD4 T cells from spleens at the time of challenge [(naive and immunized (8W)] and after challenge [(naive-challenged and immune-challenged (8WI plus 4WPC)]. The data presented are representative of two experiments with similar results. Mean and SEM of six mice in each group are shown. *<i>p < 0</i>.<i>05</i>, **<i>p < 0</i>.<i>005</i>. Black bar indicates young mice and white bar indicates aged mice.</p