Appendices 1-2-3

Appendices 1-2-

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

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

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

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

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

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

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

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