TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii

Abstract

Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

Keywords: Mec-17; acetyltransferase; cytoskeleton; endodyogeny; lysine acetylation; microtubules.

FIG 1

Ablation of K40 acetylation is not tolerated unless it is replaced with the K40Q acetyl-lysine mimic. (A) Diagram of TgTUBA1 genomic locus aligned with the construct used to replace the endogenous locus by double homologous recombination. The construct contains the nonstabilizing V252L oryzalin resistance mutation and the K40K, K40R, or K40Q mutation. (B) IFAs of K40 mutants stained for acetyl-K40–α-tubulin (red) or β-tubulin (specific to Toxoplasma, green). The K40Q mutation results in complete loss of K40 acetylation in the parasite but not its host cell. Images were merged with the DNA stain DAPI (blue). Scale bars, 3 μm. (C) Western blot (WB) assay of parental RH and mutant parasites showing loss of K40 acetylation in K40Q mutants. The blot was probed with anti-acetyl-K40–α-tubulin and anti-SAG1 antibodies as a loading control. (D) Doubling assays performed to assess the growth of parental RH and mutant parasites. Replication rates were determined by counting the parasites within 100 random vacuoles at 24 h postinfection. Three independent trials were conducted, and the average percentage of vacuoles with the indicated number of parasites ± the standard error of the mean is shown (no significant difference between mean percentages of vacuoles at each stage between strains as determined by two-way analysis of variance).

FIG 2

K40 acetylation is dispensable in the presence of the T239I oryzalin resistance mutation. (A) Diagram of the TgTUBA1 genomic locus aligned with the allelic replacement construct containing the oryzalin resistance mutation T239I and the K40K, K40R, or K40Q mutation. (B) IFAs of mutant parasite lines stained for acetyl-K40–α-tubulin (red) or β-tubulin (green). Images were merged with the DNA stain DAPI (blue). Scale bars, 3 μm. (C) Western blot (WB) assay of parental RH and mutants confirms the loss of K40 acetylation in K40Q and K40R parasites. The blots were probed with anti-acetyl-K40–α-tubulin and anti-SAG1 antibodies as a loading control. (D) Doubling assays performed as described in the legend to Fig. 1D to assess the growth of parental RH and mutant parasites.

FIG 3

Comparison of ATAT/Mec-17 homologues. (A) Depiction of ATAT protein sequences from T. gondii (TgATAT, TGME49_31600), Plasmodium falciparum (PfATAT, PF3D7_0924900), Homo sapiens (HsMec17, XP_005249477.1), Tetrahymena thermophila (TtMec17, TTHERM_00355780), and C. elegans (CeATAT2, CELE_W06B11.1), with the number of amino acids (aa) in parentheses. Gray boxes represent the lysine acetyltransferase domain. (B) Amino acid sequence alignment of the KAT domain of the indicated ATAT homologues with identical residues highlighted in black and similar residues highlighted in gray. The asterisk denotes an aspartic acid residue previously shown to be important for ATAT activity (43).

FIG 4

Expression of TgATAT and acetylated α-tubulin during the tachyzoite cell cycle. (A) Western blot (WB) assay of lysates from the parental strain (RHΔhxΔku80) and parasites containing endogenously HA-tagged TgATAT (TgATAT^HA). The blot was probed with antibodies recognizing the HA epitope or acetyl-K40–α-tubulin. β-Tubulin was also probed as a loading control. (B) IFAs of TgATAT^HA parasites stained for HA (red, TgATAT^HA) or β-tubulin (green) at the indicated stages of the parasite cell cycle. Images were merged with the DNA stain DAPI (blue). (C) IFAs of RH parasites stained for acetyl-K40–α-tubulin (red) or β-tubulin (green) at the indicated stages of the parasite cell cycle. Note that K40 acetylation is present on both spindle microtubules during mitosis and in the daughter subpellicular microtubules throughout replication. Scale bar, 3 μm.

FIG 5

Selective targeting of GFP-Cas9 to the TgATAT locus eliminates K40 acetylation. (A) Diagram showing the site on TgATAT targeted by GFP-Cas9. The 20-bp TgATAT sgRNA sequence is shown; it is immediately upstream of the protospacer adjacent motif sequence (PAM, red). The dsDNA oligomer used for recombination is shown, and in brackets are the numbers of bases of homology flanking the PAM site. Underlined is the exogenous sequence introduced, including the four stop codons in bold. IFAs of dividing parasites expressing GFP-Cas9 confirm that disruption of TgATAT and loss of K40 acetylation occur only when Cas9 is targeted to the TgATAT locus (sgTgATAT). (B) IFA of TgATAT^HA parasites 40 h posttransfection with dsDNA oligomers and GFP-Cas9 targeted to either the UPRT (sgUPRT) or the TgATAT (sgTgATAT) locus. Scale bar, 3 μm. (C) Bar graph showing the percentage of GFP-Cas9-positive vacuoles that are acetyl-K40 negative in parasites in which GFP-Cas9 was targeted to UPRT (sgUPRT) versus TgATAT (sgTgATAT) 20 or 40 h after transfection. Error bars show the standard errors of the means (n = 3).

FIG 6

Defects in nuclear division and segregation in parasites lacking α-tubulin K40 acetylation. (A) TgATAT^HA parasites were transfected with GFP-Cas9-sgTgATAT and imaged at 40 h posttransfection. Shown are transfectants lacking GFP-Cas9 expression, which display normal replication and have microtubules containing K40 acetylation, as visualized with anti-β-tubulin (green) and acetyl-K40–α-tubulin (red) antibodies, with DAPI costain in blue. (B) Parasites expressing GFP-Cas9 lose K40 acetylation and contain abnormal nuclear morphology compared to that of parasites possessing K40 acetylation. Nuclei were visualized by DAPI staining (blue). Insets of acetyl-K40-positive (i) and -negative (ii) parasites are shown and expanded in the lower panels. The DAPI-stained structures was measured with ImageJ (n = 100 nuclei). Double asterisks indicate a significant difference in mean area, as determined by unpaired t test with Welch’s correction for unequal variance (P = 0.0085). (C) Vacuoles containing parasites lacking acetylated microtubules (red) and showing aberrant phenotypes detected by staining all of the microtubules (green) and DNA (blue) are shown. Inset i shows that parasites lacking K40 acetylation have defects in microtubule structures and fail to partition nuclear material into daughter parasites. Anucleate parasites are marked by arrowheads, while the improperly segregated nuclear mass is indicated by the asterisk. Inset ii shows parasites containing multiple β-tubulin structures resembling daughter cell conoids (arrows). Scale bar, 3 μm.

FIG 7

Centrosome duplication and apicoplast division in parasites lacking K40 acetylation. (A) Duplication of the centrosomes occurs in the presence (top row, arrowhead) or absence of K40 α-tubulin acetylation (bottom row, arrowheads), as visualized by IFA staining for centrin 1 (red), acetyl-K40–α-tubulin (green), and DNA (DAPI, blue). K40-acetylated α-tubulin and GFP-Cas9 (localized to the nucleus) were detected in the same channel (green). Note the loss of acetylated α-tubulin and GFP-Cas9-expressing parasites. Dotted lines encircle individual parasites with arrowheads indicating multiple centrosomes. (B) IFAs of parasites stained for apicoplast membrane protein Atrx1 (red), acetyl-K40–α-tubulin (green), DNA (DAPI, blue), and GFP-Cas9 (green, nuclear). Apicoplasts that underwent normal division are visible in acetyl-K40-positive parasites (top row, arrowhead). Apicoplasts that failed to divide in parasites lacking K40 acetylation (bottom row) are indicated by arrows. Scale bars, 3 μm.

FIG 8

Daughter cytoskeleton completion is impaired upon loss of K40 acetylation. (A) IFA visualizing ISP1 (red), acetyl-K40–α-tubulin (green), DNA (DAPI, blue), and GFP-Cas9 (green, nuclear) in normal parasites (top) versus those lacking acetylated α-tubulin (bottom). Dotted lines encircle an individual parasites containing excess daughter buds (bottom row, arrowhead), as visualized by numerous ISP1-positive apical cap structures. (B) IFA stained for IMC3 (red), acetyl-K40–α-tubulin (green), DNA (DAPI, blue), and GFP-Cas9 (green, nuclear). Arrowheads indicate multiple IMC3-positive structures in parasites lacking K40 α-tubulin acetylation, indicative of partially formed daughter cells that failed to complete division. Scale bars, 3 μm.