Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions

Abstract

Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth.

Keywords: Amino acid hydroxylase; Apicomplexan; Metabolism.

Figure 1. Exogenous tyrosine is essential for Toxoplasma plaque formation independently of AAH2

A. Absence of tyrosine results in a defect in plaque formation that is similar for wild type and Δaah2 parasites. Tachyzoites were cultured for 14 days on HFFs in DMEM titrated with varying concentrations of tyrosine. Plaques were fixed with methanol at 14 days post-infection, stained with crystal violet, and visualized by photomicroscopy. B. Schematic of the vector used to replace the AAH2 coding region with the hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) selectable marker via double homologous recombination. Arrow indicates promoter. UTR indicates untranslated region. C. Confirmation of AAH2 deletion. PCR was performed on genomic DNA from PruΔku80 wildtype and Δaah2 clones using primers specific for AAH2. Primers specific to AAH1 were used to confirm that AAH1 was not deleted. Size markers (M) are in base pairs.

Figure 2. Absence of tyrosine does not affect host cell viability

Confluent HFFs were cultured in tyrosine-rich or tyrosine-depleted DMEM for 14 days and stained with calcein-AM and ethidium homodimer-1 (Eth HD-1) to measure cell viability (green) and death (red), respectively. HFFs cultured in complete DMEM or treated with 70% methanol were used as controls. Scale bar indicates 5 μm.

Figure 3. Exogenous tyrosine is required for replication but not invasion of both wild type and Δaah2 tachyzoites

A. The absence of tyrosine results in fewer parasites per vacuole using both wild type and Δaah2 tachyzoites. The indicated tachyzoites were cultured on HFFs in DMEM containing or lacking tyrosine. Infected coverslips were fixed with formaldehyde at 36 hours post-infection. The number of tachyzoites per vacuole was quantified based on mCherry expression and counting parasites per vacuole in random fields at 100x magnification. Only vacuoles with two or more parasites were scored. B. The absence of tyrosine results in fewer doublings using both wild type and Δaah2 tachyzoites. The data on the average numbers of parasites per vacuole shown in 3A were converted to parasite doublings using a log base 2 scale. C. The absence of tyrosine results in fewer vacuoles per field using both wild type and Δaah2 tachyzoites. The number of vacuoles containing two or more parasites was scored on random fields as described above. D. The absence of tyrosine does not affect invasion efficiency of wild type and Δaah2 parasites. Tachyzoites were allowed to invade HFFs for one hour in DMEM containing or lacking tyrosine. The samples were then fixed with formaldehyde and extracellular parasites were stained with anti-SAG1 antibody and Alexa-488-conjugated secondary antibody without permeabilization. Total parasites were quantified by cytosolic mCherry expression. Extracellular parasites were quantified based on SAG1 surface staining. The results are plotted as the percentage of all parasites seen that are intracellular (IC) versus extracellular (EC). Data shown are the average values in one experiment that is representative of three or more repeated experiments. Error bars show standard deviation. Values with statistically significant differences are labeled by brackets and asterisks as follows: *, p < 0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The averages shown were compared by two-way ANOVA and Tukey’s multiple comparisons test.

Figure 4. Exogenous tyrosine is required for bradyzoite growth but not differentiation

A. The absence of tyrosine results in fewer parasites per cyst for both wild type and Δaah2 bradyzoites. Tachyzoites were allowed to infect HFFs on coverslips in DMEM for 3.5 hours, after which the monolayers were washed three times with PBS and cultured in tyrosine-titrated, alkaline switch media. Infected monolayers were fixed with methanol at three days post-infection and stained with fluorescein-conjugated dolichos biflorus agglutinin (DBA), a cyst wall marker, and anti-SAG1 antibodies. DBA-positive vacuoles with two or more parasites were scored. Data shown are from one experiment that is representative of three or more repeated experiments. Error bars indicate standard deviation. B. The absence of tyrosine results in fewer doublings for both wild type and Δaah2 bradyzoites. Data for the average numbers of parasites per vacuole shown in (4A) were converted to parasite doublings using a log base 2 scale. Error bars indicate standard deviation. C. The absence of tyrosine results in fewer vacuoles per field using both wild type and Δaah2 strains. Wild type and Δaah2 tachyzoites were induced to switch and stained as described in (4A). The number of vacuoles containing two or more parasites was scored at 100× magnification on random fields. Data shown are averages from one experiment that is representative of three or more repeated experiments. Error bars indicate standard deviation. D. The absence of tyrosine does not significantly affect switch efficiency of wild type and Δaah2 strains. Tachyzoites were induced to switch in tyrosine-titrated, alkaline media, fixed with methanol at three days post-infection, and stained as described in (4A). Switch efficiency was measured by counting the number of vacuoles stained with DBA over the number of vacuoles staining with SAG1. Data shown are the combined averages of three independent experiments on triplicate coverslips using the same conditions. Error bars indicate standard error of the mean. Values with statistically significant differences are labeled by brackets and asterisks as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The averages shown were compared by two-way ANOVA and Bonferroni’s or Tukey’s multiple comparisons test.