Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite

Abstract

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Keywords: RNA sequencing; cytoplast; fibroblasts; leishmania.

FIG 1

Cytoplasts generated from L929 fibroblasts by enucleation. (A) Epifluorescence microscopy of viable cells from enucleation cell culture. Enucleated cells (cytoplasts) and nucleated cells (postenucleation residual fibroblasts) were distinguished using the Hoechst 33342 nuclear fluorescent probe. Scale bar, 100 μm. (B) Confocal microscopy of cytoplasts stained for mitochondria using the MitoTracker fluorescent probe. MitoTracker staining is shown in red, and Hoechst 33342 staining is shown in blue (absent in the cytoplasts). Scale bar, 10 μm. (C) Number of cells and cytoplasts counted per microscopic field throughout 48 h of in vitro cultivation after enucleation. Multiplication of fibroblasts in nonenucleated cultures is indicated by the dotted black line and residual fibroblasts by the blue line. The stability in enucleated cultures is shown. The example shown is representative from three independent experiments. (D) Epifluorescence microscopy of cultures of live fibroblasts (upper panel) and cytoplasts (bottom panel) 96 h postenucleation, following staining with the cell viability dye carboxyfluorescein diacetate succinimidyl ester (green) and annexin V (red) to identify apoptotic cells (CFDA-se⁺Annexin V⁺). The inset shows an apoptotic, enucleated cell. Scale bar, 20 μm.

FIG 2

Transcriptomic profile and functional enrichment analysis comparison of enucleated cells (cytoplasts) and nucleated cells (fibroblasts). (A) Principal component analysis (PCA) of M. musculus transcripts in noninfected fibroblasts (blue) and noninfected cytoplasts (red). Each point represents a biological replicate, i.e., independent experiments. (B) Volcano plots in a pairwise format showing the –log₁₀ (P value) and fold change (log₂) of transcripts detected in a pairwise comparison between fibroblasts and cytoplasts. Transcripts with a negative fold change signal (in blue, adjusted P < 0.1) correspond to transcripts that are more preserved/enriched in cytoplasts or less prevalent in fibroblasts. Transcripts with a positive fold change signal (in orange, adjusted P < 0.1) correspond to transcripts that are more frequently detected in fibroblasts or are more absent/degraded in cytoplasts. Transcripts with lowest adjusted P values are identified by their gene names. (C) Top-ranking biological pathways by GO enrichment. GO functional enrichment analysis performed by GOseq showing the top GO terms enriched in the list of transcripts that are more preserved/enriched in cytoplasts, and the percentage of differentially detected transcripts fitting each category (Hits%). Coordinates are represented by dots whose size and color indicate the number of differentially detected transcripts fitting the GO category, and whose P value is determined by a colorimetric scale of P values (darker for higher and lighter for lower P values).

FIG 3

Cytoplasts support L. amazonensis amastigote multiplication in enlarging parasitophorous vacuoles. (A) Fibroblast (upper) and cytoplast (bottom) cultures after interaction with green fluorescent protein-expressing L. amazonensis or L. major for 6 and 48 h postinteraction. The phase-contrast images are merged with the green fluorescent signal of the parasites. In contrast to L. major, L. amazonensis amastigotes are internalized by cytoplasts, within which they develop large parasitophorous vacuoles (PVs) that are visible by phase-contrast imaging. Scale bar, 10 μm. (B) Quantification of the number of internalized parasites per microscopic field (left graph) or parasites per fibroblast/cytoplast (right graph) after 6 or 48 h of host cell-parasite interaction using cytoplasts or fibroblasts and L. amazonensis or L. major lesion-derived amastigotes. A Student t test was used to compare 6- and 48-h time points under each condition. The asterisk indicates P < 0.01 (n = 3 independent experiments). (C) Live imaging of infected cytoplasts showing the multiplication of one intracellular parasite (amastigote form, round, no apparent flagellum) into four parasites (arrowheads and numbers representing parasites) in an enlarging PV (red asterisk). Image acquisition began 2 h after cytoplast-parasite interaction and is displayed as “day-hours:minutes” (dhh:mm). Scale bar, 20 μm. (D) Quantification of the number of internalized parasites per PV in fibroblasts (blue) or cytoplasts (magenta) showing mean multiplication of parasites from approximately one to two amastigotes per PV. A Student t test was used to compare 6- and 48-h time points or comparing parasite number per PV in fibroblasts and cytoplasts at 48 h postinfection. The asterisk indicates P < 0.01 (n = 3 independent experiments). (E) Number of cytoplasts hosting (red) or not hosting (blue) parasites counted per microscopic field in the same infected cytoplasts preparation at 6 and 48 h after cytoplast-parasite interaction. A Student t test was used to compare 6- and 48-h time points under each condition. ns, nonsignificant (P > 0.05; n = 3 independent experiments).

FIG 4

Transcriptomic profile and functional enrichment comparison of infected cytoplasts and noninfected cytoplasts. (A) PCA of M. musculus transcripts found in noninfected fibroblasts (yellow) and infected fibroblasts (fibroblasts plus L. amazonensis, purple; each point represents a biological replicate, i.e., independent experiments) and volcano plot in a pairwise format showing the –log₁₀ (P value) and fold change (log₂) of transcripts detected in a pairwise comparison between these two groups. No statistically significant difference in differential gene expression was detected. (B) PCA of M. musculus transcripts found in noninfected cytoplasts (blue) and infected cytoplasts (cytoplasts plus L. amazonensis, magenta). Each point represents a biological replicate, i.e., independent experiments. (C) Volcano plot in a pairwise format showing –log₁₀ (P value) and fold change (log₂) of transcripts detected in a pairwise comparison between infected cytoplasts and noninfected cytoplasts. Transcripts with negative fold change signal (in orange, adjusted P < 0.1) correspond to those that are absent/degraded more by intracellular infection or present more in noninfected cytoplasts. Transcripts with a positive fold change signal (in blue, adjusted P < 0.1) correspond to transcripts that are more preserved/enriched by intracellular infection. Transcripts with adjusted P < 0.1 are identified by their gene names. (D and E) Top-ranking biological pathways by GO enrichment. GO functional enrichment analysis performed using GOseq showing the top GO terms enriched in the list of cytoplasts transcripts that are more absent/degraded (D) or more preserved/enriched by intracellular infection (E). Graphs show the ranked top GO terms and the percentage of differentially detected transcripts that fit in each category (Hits%). Coordinates are represented by dots whose size and color indicate the number of differentially detected transcripts fitting the GO category and whose P value is determined by a colorimetric scale (darker for higher and lighter for lower P values).

FIG 5

Transcriptomic profile and functional enrichment analysis of intracellular parasites hosted by cytoplasts and fibroblasts. (A) Volcano plot in a pairwise format showing –log₁₀ (P value) and fold change (log₂) of L. amazonensis transcripts detected in a pairwise comparison between parasites hosted by fibroblasts and cytoplasts. Transcripts with fold change negative signal correspond to parasite transcripts upregulated when parasites are hosted by cytoplasts (top lowest adjusted P value transcripts and other interesting transcripts with an adjusted P value of <0.1 identified in orange). Transcripts with positive fold change signal correspond to parasite transcripts upregulated when parasites are hosted by fibroblasts (top lowest adjusted P value transcripts and other interesting transcripts with adjusted P < 0.1 identified in blue). Parasite transcripts indicated by colors are also identified by gene annotations (orthology, protein family, and domain detection) and prediction for secretion (Sec, green) or RNA-binding properties (RBP, red). (B) Functional enrichment analysis performed by ClueGO and GeneMANIA software using parasite transcripts homologous to M. musculus and stratifying the analysis in parasite functional enrichment in cytoplasts (orange data) or parasite functional enrichment in fibroblasts (blue data). The upper panel shows the predicted connection between detected functional clusters formed with enriched GO terms that are described in the tables in the bottom panel.

FIG 6

In silico hybrid M. musculus-L. amazonensis protein-protein interactomes constructed by host-parasite transcriptomic profiles of infected fibroblasts and cytoplasts. (A) Infected fibroblast hybrid interactome formed between the 1,950 most upregulated/preserved host transcripts detected in noninfected fibroblasts compared to noninfected cytoplasts (subset 1, gray nodes, protein-protein interactions indicated by gray lines) and the 27 most upregulated parasite transcripts expressed when hosted by fibroblasts compared to expression of parasites hosted by cytoplasts, whose products are at the same time predicted for secretion and homologous to M. musculus proteins (subset 2). Among these selected 27 parasite transcripts, 19 interacted directly or indirectly with host transcripts from subset 1 (blue nodes). A direct protein-protein interaction between parasite transcript products is indicated by blue lines. Protein-protein interactions between parasite and host transcript products are also indicated by gray lines. The generated hybrid network was submitted to functional enrichment analysis by STRING enrichment software allowing for localizing enriched functional clusters identified by colors and GO terms. (B) Infected cytoplast hybrid interactome formed between (i) the 1,950 most upregulated/preserved host transcripts detected in noninfected cytoplasts compared to noninfected fibroblasts (subset 3, gray nodes, protein-protein interactions indicated by gray lines); (ii) the 39 most preserved host transcripts in infected cytoplasts compared to noninfected cytoplasts (subset 4, magenta nodes); and (iii) the 11 most upregulated parasite transcripts expressed when hosted by cytoplasts compared to expression of parasites hosted by fibroblasts whose products are at the same time predicted for secretion and homologous to M. musculus proteins (subset 5). Among the 11 selected parasite transcripts, nine interacted directly or indirectly with host transcripts from subsets 3 and 4 (orange nodes). Protein-protein interactions between parasite and host transcript products are indicated by gray lines. A direct protein-protein interaction between parasite transcript products (orange nodes) and between these and parasite-mediated more preserved host transcript products (orange and magenta nodes interaction) is highlighted by the orange line. The generated hybrid network was submitted to functional enrichment analysis by STRING enrichment software allowing localization of enriched functional clusters identified by colors and GO terms.

FIG 6

In silico hybrid M. musculus-L. amazonensis protein-protein interactomes constructed by host-parasite transcriptomic profiles of infected fibroblasts and cytoplasts. (A) Infected fibroblast hybrid interactome formed between the 1,950 most upregulated/preserved host transcripts detected in noninfected fibroblasts compared to noninfected cytoplasts (subset 1, gray nodes, protein-protein interactions indicated by gray lines) and the 27 most upregulated parasite transcripts expressed when hosted by fibroblasts compared to expression of parasites hosted by cytoplasts, whose products are at the same time predicted for secretion and homologous to M. musculus proteins (subset 2). Among these selected 27 parasite transcripts, 19 interacted directly or indirectly with host transcripts from subset 1 (blue nodes). A direct protein-protein interaction between parasite transcript products is indicated by blue lines. Protein-protein interactions between parasite and host transcript products are also indicated by gray lines. The generated hybrid network was submitted to functional enrichment analysis by STRING enrichment software allowing for localizing enriched functional clusters identified by colors and GO terms. (B) Infected cytoplast hybrid interactome formed between (i) the 1,950 most upregulated/preserved host transcripts detected in noninfected cytoplasts compared to noninfected fibroblasts (subset 3, gray nodes, protein-protein interactions indicated by gray lines); (ii) the 39 most preserved host transcripts in infected cytoplasts compared to noninfected cytoplasts (subset 4, magenta nodes); and (iii) the 11 most upregulated parasite transcripts expressed when hosted by cytoplasts compared to expression of parasites hosted by fibroblasts whose products are at the same time predicted for secretion and homologous to M. musculus proteins (subset 5). Among the 11 selected parasite transcripts, nine interacted directly or indirectly with host transcripts from subsets 3 and 4 (orange nodes). Protein-protein interactions between parasite and host transcript products are indicated by gray lines. A direct protein-protein interaction between parasite transcript products (orange nodes) and between these and parasite-mediated more preserved host transcript products (orange and magenta nodes interaction) is highlighted by the orange line. The generated hybrid network was submitted to functional enrichment analysis by STRING enrichment software allowing localization of enriched functional clusters identified by colors and GO terms.

FIG 7

Summary of the dual host-parasite transcriptomic profiling in the presence or absence of host cell nucleus. The left section shows the predicted landscape of host-parasite mutual stimulation generated by host de novo mRNA synthesis promoted by the nucleus in response to the parasite and the expression of parasite transcripts that are differentially regulated by the nuclear host response. The right section shows the predicted landscape of host-parasite cross talk when parasites established in cells without nucleus that display a self-limiting amount of resources for the parasite, promoting a parasite-mediated preservation of specific transcripts involved in maintaining host cell functions that are beneficial to the parasite. Functional clusters identified by in silico analysis of hybrid interactomes appear as colored regions.