Toxoplasma gondii Me49 and NED strains arrest host cell cycle progression and alter chromosome segregation in a strain-independent manner

Abstract

Toxoplasma gondii is an obligate intracellular parasite that modulates a broad range of host cell functions to guarantee its intracellular development and replication. T. gondii includes three classical clonal lineages exhibiting different degrees of virulence. Regarding the genetic diversity of T. gondii circulating in Europe, type II strains and, to a lesser extent, type III strains are the dominant populations, both in humans and animals. Infections with the type I strain led to widespread parasite dissemination and death in mice, while type III is considered avirulent. Previously, we demonstrated that primary endothelial cells infected with the T. gondii RH strain (haplotype I) were arrested in the G2/M-phase transition, triggering cytokinesis failure and chromosome missegregation. Since T. gondii haplotypes differ in their virulence, we here studied whether T. gondii-driven host cell cycle perturbation is strain-dependent. Primary endothelial cells were infected with T. gondii Me49 (type II strain) or NED (type III strain), and their growth kinetics were compared up to cell lysis (6-30 h p. i.). In this study, only slight differences in the onset of full proliferation were observed, and developmental data in principle matched those of the RH strain. FACS-based DNA quantification to estimate cell proportions experiencing different cell cycle phases (G0/1-, S-, and G2/M-phase) revealed that Me49 and NED strains both arrested the host cell cycle in the S-phase. Cyclins A2 and B1 as key molecules of S- and M-phase were not changed by Me49 infection, while NED infection induced cyclin B1 upregulation. To analyze parasite-driven alterations during mitosis, we demonstrated that both Me49 and NED infections led to impaired host cellular chromosome segregation and irregular centriole overduplication. Moreover, in line with the RH strain, both strains boosted the proportion of binucleated cells within infected endothelial cell layers, thereby indicating enhanced cytokinesis failure. Taken together, we demonstrate that all parasite-driven host cell cycle arrest, chromosome missegregation, and binucleated phenotypes are T. gondii-specific but strain independent.

Keywords: Me49 strain; NED strain; Toxoplasma gondii; cell cycle arrest; cell cycle dysregulation; haplotypes.

Figure 1

Toxoplasma gondii Me49 and NED tachyzoite replication kinetics in primary bovine umbilical vein endothelial cells (BUVEC). BUVEC isolates (n = 3) were simultaneously infected at subconfluence with either Me49 or NED tachyzoites (MOI 1:2) and monitored for parasite proliferation by analyzing the numbers of tachyzoites/parasitophorous vacuole (PV, white circle) from 6 to 30 h p. i. (A) Representative illustration of different stages of proliferation. Host cell nuclei were stained by DAPI (blue) and tachyzoites by T. gondii-specific antibodies (red). (B) Estimation of the proliferation status of Me49 and NED strains at 6, 12, 18, 24 and 32 h p. i. The scale bar represents 5 μm.

Figure 2

Toxoplasma gondii Me49 and NED infections induce a binucleated phenotype and centriole overduplication in infected host cells. Subconfluent BUVEC isolates (n = 3) were infected with either Me49 or NED tachyzoites at an MOI 1:2. After 24 h p. i., samples were stained for DNA/chromosomes (DAPI, blue) and centrioles (γ-tubulin, green). (A) Percentages of host cells with a binucleated phenotype. (B) Mitotic rate (cells undergoing mitosis/total number of cells) of T. gondii-infected BUVEC and control cells. (C) Exemplary illustration of the main aberrant mitotic structures observed for both Me49 and NED strain infections. Additionally, the intensity of centriole-related signals was assessed and plotted as a graph showing intensity value vs. distance (white line). Yellow arrows: centriole overduplication; asterisks: chromosome bridge. The scale bar represents 5 μm.

Figure 3

Toxoplasma gondii Me49 and NED infections arrest the host cell cycle in S-phase. Subconfluent BUVEC isolates (n = 6) were infected with either Me49 or NED tachyzoites at an MOI 1:2 and evaluated for DNA content at 24 h p. i. (A) Exemplary flowchart of FACS analysis showing the total number of host cells in G0-1 (one genomic DNA copy), G2- (two genomic copies), and S-phase (DNA synthesis, the cell population in between both phases). The number of cells positive for DNA staining (PI) was graphed as a histogram to obtain the total number of cells in each peak. (B) Percentages of Me49- and NED-infected BUVEC and control cells in each cell cycle phase were estimated via FACS-based DNA quantification. Bars represent the median ± SD.

Figure 4

Cyclin A2 and cyclin B1 expression in Toxoplasma gondii Me49 and NED strain-infected BUVEC. Confluent BUVEC isolates (n = 6) were infected with either Me49 (A) or NED (B) tachyzoites. At 24 h p. i., protein extracts were analyzed by Western blotting to estimate cyclin A2 (indicative for S-phase) and cyclin B1 (mitosis marker) expression. Densities of protein signals were quantified and graphed as a relative ratio to vinculin expression (loading control). Bars represent the median ± SD of six biological replicates.