Fig 1.
TcIPCS1 model prediction and topology.
(A) Rosetta prediction of TcIPCS Esmeraldo-like allele (TcCLB.506885.124). Color palette indicates per-residue confidence score (pLDDT), which ranges from 0 to 100. (B) Topology diagram (schematic) of TcIPCS. (C) Key residues for the catalytic triad (H202, H245, D249) and substrate selectivity (S244). Predicted location of the TcIPCS active site with residues and loops are indicated. (D) Quantitative RT-PCR analysis of the TcIPCS transcript levels obtained from RNA isolated from amastigotes and tissue culture derived trypomastigote stages as well as epimastigote cultures. Statistical significance was determined by one-way ANOVA (n = 5; ***, P < 0.001).
Fig 2.
CRISPR-Cas9 strategy to generate TcIPCS knockout in T. cruzi epimastigotes.
(A) Schematic representation of the Cas9 ribonucleoprotein complexes with the two sgRNAs and their annealing position in the TcIPCS locus. A DNA fragment containing the TcIPCS homologous sequences flanking a Neomycin resistance cassette to allow homologous recombination repair is also shown. After epimastigote transfection, replacement of the TcIPCS coding region by the Neomycin resistance gene is indicated. (B) PCR analysis of wild type and TcIPCS knockout epimastigotes. The annealing position of primers are shown in the schematic representation of one wild type and one disrupted TcIPCS allele with the predicted sizes for the amplicons indicated below the figure. Agarose gel electrophoresis analyses of PCR products obtained with DNA extracted from WT epimastigotes and from two TcIPCS knockout (KO) cloned cell lines, KO4 and KO8 and the indicated primer sets. (C) Relative expression levels of TcIPCS transcripts quantified by RT-qPCR using RNA extracted from WT and two TcIPCS KO cell lines, KO4 and KO8. (D) Negative ion ES-MS lipidomic analysis of WT and TcIPCS KO4 lipid extracts. Spectra show survey scans (600-1000m/z) of WT (upper figure) and KO4 epimastigotes (bottom figure) showing a heterogeneous mixture of PI, IPC, PS and PE. (E) Positive ion ES-MS lipidomic analysis of WT and TcIPCS KO4 lipid extracts. Spectra show survey scans (600-1000m/z), of WT (upper figure) and KO4 epimastigotes (bottom), showing a mixture of PC and PG species.
Fig 3.
Metacyclogenesis and infection capacity of TcIPCS knockout mutants.
(A) Analyses by light microscopy of WT epimastigotes and one TcIPCS KO cell line (KO4) cultured in LIT medium. Bar = 10 μm. (B) Growth curve of epimastigote cultures from WT and two TcIPCS knockout cell lines, KO4 and KO8. Parasites were diluted in LIT medium to a concentration of 2.5 x106 cells/mL and parasite numbers were determined over 9 days. Statistical significance could not be established for the analysis shown in B as described in Methods. (C) Percentages of metacyclic trypomastigotes on day 9 of cultures of WT and the two cloned TcIPCS KO cell lines (KO4 and KO8). Statistical significance was analyzed by one-way ANOVA (n = 3; ***, P < 0.001). (D-G) In vitro infection of Vero cells with tissue culture-derived trypomastigotes from WT and the TcIPCS KO cell lines KO4 and KO8. The percentages of infected cells at 24 hours post-infection are shown in D; the numbers of intracellular amastigotes per infected cell are shown in E; the numbers of trypomastigotes released in the supernatant of infected cells at different time points post-infection are shown in F. The percentages of amastigotes present in the supernatants of cultures on days 8 and 10 post-infection as well as their morphology are shown in G and H, respectively. Multiplicity of infection (MOI) was 10 parasite per cell. Statistical significance was analyzed by one-way ANOVA for data shown in D and G (**P < 0.01, *** P < 0.001). Statistical significance could not be established for the analyses shown in E and F as described in Methods.
Fig 4.
Sensitivity of epimastigotes to inhibitors of Leishmania IPCS.
Epimastigotes cultures of WT and TcIPCS KO cell lines KO4 and KO8 were incubated in the presence of increasing concentrations of tamoxifen (A), benzazepane compound 7 (B) and benznidazole (C). Viability of the cells were determined by Alamar blue assay. Data points are mean values ± SD of three determinations. Representative experiments performed in triplicates are shown. IC50 values were determined as the mean ± SD of at least 3 independent biological replicates. There were no statistically significant differences between IC50 values, as determined by the Kruskal-Wallis test.
Fig 5.
Generation of TcIPCS addback cell lines, protein localization and enzyme activity.
(A) Schematic representation of the linearized TcIPCS-HA expression vector used for transfection and integration in the tubulin locus to generate the addback line. (B) PCR analysis of WT, TcIPCS KO cell line (KO8) and two cloned addback cell lines (AB1 and AB3) obtained after transfection of the TcIPCS KO8 clone with the TcIPCS-HA expression vector. Annealing positions of the primers and the expected sizes of the amplicons are indicated in the schematic representation of one wild type TcIPCS allele (top figure), one disrupted TcIPCS allele (medium) and in the addback sequence inserted in the tubulin locus (bottom). Agarose gel electrophoresis analyses showed PCR products obtained with DNA extracted from WT epimastigotes, the TcIPCS KO8 cell lines and the two cloned addback cell lines AB1 and AB3. (C) Relative expression levels of TcIPCS transcripts quantified by RT-qPCR using RNA extracted from WT, from the TcIPCS KO cell line KO8 and from the addback cell line AB1. (D) Western blot analysis with anti-HA antibody of HA-tagged TcIPCS and total protein extracts from WT, TcIPCS KO cell lines KO4 and KO8, a transfected population derived from the KO8 cell line (pop. addback) and a transfected population derived from WT epimastigotes (pop super-expressor). The bands corresponding to the HA-tagged TcIPCS protein with a MW of 37 KDa are indicated. (E-H) Fluorescence microscopy analyses of epimastigotes incubated with NBD C6-Ceramide to stain ceramide rich regions (red), such as the Golgi apparatus (E). Parasites showing the immunostaining using anti-HA antibody (green) indicate TclPCS protein localization (F). Merged image showing the co-localization of the protein with ceramide-rich regions, close to the kinetoplast, indicates that TcIPCS localizes to the Golgi apparatus (G). White arrows indicate co-localization of the TclPCS protein with NBD C6-ceramide close, but not at the same location to the parasite kinetoplast, where the Golgi apparatus is localized (H). (I) HP-TLC analysis of IPC present in extracts from T. cruzi epimastigotes, Vero cells (negative control) and L. major (positive control). (J) HP-TLC analysis of IPC present in T. cruzi epimastigotes extracts derived from WT, TcIPCS KO4 and KO8 cell lines as well as in the TcIPCS addback cell line AB1. In I and J, cells were incubated in presence of fluorescent ceramide and immediately used for preparation of lipid extracts. The migration positions of fluorescent inositol phosphorylceramide (IPC), sphingomyelin (SM) as well as the origin (O) and the front (F) are indicated. The presence of an unknown putative lipid was also identified.
Fig 6.
Metacyclogenesis and infection capacity of TcIPCS knockout mutants and addback cell lines.
(A) Growth curve of WT, TcIPCS knockout (KO8) and TcIPCS addback (AB1) parasites. Parasites were diluted to a concentration of 2.0 x106 parasite/mL, and parasite density was determined over 13 days. Statistical significance could not be established for A as described in Methods. (B) Percentages of metacyclic trypomastigotes on day 9 of cultures of WT, one cloned TcIPCS KO cell line (KO8) and one TcIPCS addback cell line (AB1) in LIT medium. Statistical significance was analyzed by one-way ANOVA (n = 3; **, P < 0.01, ***, P < 0.001). (C-E) In vitro infection of Vero cells with tissue culture-derived trypomastigotes from WT, one cloned TcIPCS KO cell line (KO8) and one TcIPCS addback cell line (AB1). The percentages of infected cells (C) and the numbers of intracellular amastigotes per infected cell (D) at 24- and 72-hours post-infection are shown; the numbers of trypomastigotes released in the supernatant of infected cells at different time points post-infection are shown in E. Multiplicity of infection (MOI) was 10 parasite per cell. Statistical significance was analyzed by one-way ANOVA in C and D, but data from E could not be statistically assessed within the current ANOVA, as described in Methods. (F-G) Interferon gamma knockout mice were infected with 10,000 culture-derived trypomastigotes of the WT, TcIPCS-KO (KO8) and TcIPCS addback (AB1). Parasitemia (F) and survival rates (G) were followed for 20- and 30-days post infection (DPI). Data points are mean values ± SD from four mice per group. Data from F and G could not be statistically assessed. (H) Thirty days after challenge infection, IFN-g KO mice were sacrificed, their hearts were harvested, and DNA extracted. The number of parasites was determined by quantitative PCR (qPCR). Statistical significance was analyzed by two-tailed unpaired t-test (n = 5; ***, P <0.001).