Relationships between drug concentrations and <i>P</i>. <i>carinii</i> viability inhibition of pentamidine, atovaquone and trimethoprim-sulfamethoxazole (TMP/SMX).

<p>Values of <i>P</i>. <i>carinii</i> viability inhibition are calculated from the SYTO-13 live-cell staining assay. To calculate the percentage of viability inhibition in relation with drug concentrations, one drug-free control was included in each assay. All susceptibility assays were set up in triplicate.</p

Proportions of sporocyte and mature cyst stages of <i>Pneumocystis carinii</i>.

<p>Whole organism population was isolated from the lungs of immunosuppressed animals as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020935#pone.0020935-Aviles1" target="_blank">[27]</a>. Sporocyte and mature cyst stages were counted from RAL-555-stained smears according to the number of nuclei in each life cycle stage. Percentages of sporocyte and cystic stages with N nuclei in the fraction of sporocytes and mature cysts were given as mean of three independent experiments (n = 70, n = 55, n = 50). N = number of nuclei per fungal cell. SD = standard deviation.</p>(a)<p>Early sporocytes.</p>(b)<p>Sum of intermediate sporocytes with 2, 3 and 4 nuclei.</p>(c)<p>Sum of late sporocytes and mature cysts with 5, 6, 7 and 8 nuclei.</p

Histogram representing mean areas of nuclei of <i>P. carinii</i> organisms according to their DNA contents.

<p>Following cell sorting according to <i>P. carinii</i> DNA content, images of Giemsa-like stained <i>P. carinii</i> organisms were analysed using the automated ImageJ software. Statistical analysis using F-test and Tukey test was performed. The p-values are indicated for each pairwise comparison. A p-value<0.05 is deemed to be significant. NS = non significant.</p

SYTO-13 labelling of <i>P</i>. <i>carinii</i> trophic and cystic forms.

<p>All <i>P</i>. <i>carinii</i> life cycle stages were stained in red (panels A–D) with a home-made anti-<i>Pneumocystis</i> polyclonal antibody, recognized by an Alexa-647-conjugated secondary antibody. SYTO-13 nuclear staining of viable <i>P</i>. <i>carinii</i> is displayed for corresponding fields (panels E–H). Differential Interference Contrast (DIC) pictures of corresponding fields are also shown (I–L). Panels (B, F, J) and (D, H, L), show an isolated cystic form with one or several labelled nuclei. In the other panels, cystic forms (arrowheads) and trophic forms are well visible. The white arrows show non-viable <i>Pneumocystis</i> organisms stained in red but did not display any nuclear green fluorescence (Panels C, G, K). Bar = 5 μm.</p

Representative cell cycle analysis histograms of sorted <i>P. carinii</i> organisms.

<p>DNA contents of (A) all sorted <i>P. carinii</i> life cycle stages; (B) sorted <i>P. carinii</i> trophic forms; (C) haploid <i>S. cerevisiae</i> reference strain; (D) sorted <i>P. carinii</i> cystic forms; (E) diploid <i>S. cerevisiae</i> reference strain. For all histograms, the number of cells (Count, Y-axis) was plotted against the fluorescence intensity of DNA-bound SYTOX® Green (channel number (area), X-axis). Cell acquisition was done as follows: (A) and (B) 100,000 cells; (D) 20,000 cells; (C) and (E) 10,000 cells.</p

Transmission electronic microscopy of untreated or sorted parasite stages.

<p>(A) A <i>P. carinii</i> sorted cystic form; (B) an untreated intermediate sporocyte : the thick cell wall and two nuclei are well visible; (C) a <i>P. carinii</i> sorted trophic form; (D) an untreated trophic form. Trypsin treatment, required for antibody labeling of the sorted cystic forms, altered their cell wall external dense layer. The electron dense layer of the sorted trophic form cell wall was also altered but not completely removed while the plasma membrane remained intact (see insets C and D). Cell wall lucent layers of cystic forms, as well as cytoplasmic or nuclear structures of both life-cycle stages, were ultrastructurally unaltered. Bar = 1 µm. N = Nucleus, Mi = Mitochondrion, S = Spore.</p

Interpretation scheme of <i>P. carinii</i> life cycle and cell cycle.

<p>Two haploid trophic forms (T.f.) mate (Ma) to produce a zygote with an enlarged single diploid nuclei. DNA synthesis (S) occurs to give rise to diploid early sporocytes (E.s.) which undergo the first meiotic division (MI), subsequently leading to intermediate sporocytes (I.s.) with 2 haploid nuclei each containing one set of chromosomes with two chromatides per chromosome. The second meiotic division leads to the separation of chromatides in four haploid nuclei within each intermediate sporocyte. DNA content (4C) does not change during meiotic divisions. A final mitosis produces late sporocytes (L.s.) and mature cysts (M.c.) with 8 contents of DNA shared between the 8 nuclei. Once fully matured, the cysts release 8 haploid spores (Sp.). Ploidy values (n) indicate the number of chromosome sets per nucleus. Time laps of each phase of the <i>P. carinii</i> life cycle are arbitrary. (A) Nuclear fusion of two trophic forms. Spindle pole bodies are clearly visible. Drawn according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020935#pone.0020935-Yoshida2" target="_blank">[58]</a>. (B) Early sporocyte in which a synaptonemal complex is indicated (arrow). Drawn according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020935#pone.0020935-Itatani1" target="_blank">[40]</a>. (C) Intermediate sporocyte. (D) Late sporocyte. (E) Mature cyst. (F) Released spore.</p

<i>In vitro</i> pharmacodynamic parameters of pentamidine, atovaquone and trimethoprim-sulfamethoxazole (TMP/SMX) calculated using the SYTO-13 assay.

<p><i>In vitro</i> pharmacodynamic parameters of pentamidine, atovaquone and trimethoprim-sulfamethoxazole (TMP/SMX) calculated using the SYTO-13 assay.</p

SYTOX® Green labeling of nuclei of <i>P. carinii</i> trophic and cystic forms.

<p>Home-made polyclonal antibody, recognized by Alexa-647 conjugated secondary antibody, labeled all <i>P. carinii</i> life cycle stages in red (A–E). Corresponding fields showed SYTOX® Green discrete nuclear staining (F–J). Differential Interference Contrast (DIC) pictures of corresponding fields were shown (K–O). Panels A, F, K, showed an isolated cystic form with two labeled nuclei. In the other panels, cystic forms (thick-walled stages, arrowheads) and trophic forms (thin-walled stages) are well visible. Bar = 5 µm.</p

Viability assessment of <i>P</i>. <i>carinii</i> using SYTO-13 by flow cytometry analysis.

<p>The data are displayed as histograms displaying 10,000 <i>P</i>. <i>carinii</i> collected events. The axes represent the relative number of cells (y axis) and the cell-associated fluorescence intensity on a logarithmic scale (x axis). Panel A shows the gated (R1) <i>P</i>. <i>carinii</i> population labelled with a rat specific polyclonal antibody and a goat anti-rat IgG antibody conjugated to Alexa-647 (FL4 channel). The intensity of SYTO-13 live-cell nucleic acid staining is analyzed within the gated <i>P</i>. <i>carinii</i> population (FL1 channel) for <i>P</i>. <i>carinii</i> organisms cultured during 4 days without (panel B), with 0.15 μM (panel C) or 90 μM (panel D) of pentamidine. The gated population R2 represents the viable <i>Pneumocystis</i> cells. The percentages of viability are indicated for each histogram. The presented histograms are representative of one experiment.</p