Phenotypic analysis of knock out parasites ( Δ andh1/Andh1-myc)

The growth rate of ∆ndh2-i/ndh2-i-myc and ndh2-i/ndh2-i-myc parasites was compared in the absence and presence of Atc (Fig. 3.26). The ndh2-i/ndh2-i-myc parasites showed no difference in the replication rate compared to the parental RH-TATi strain under both conditions, with and without Atc. The depletion of NDH2-I in

∆ ndh2-i/ndh2-i-myc parasites did not result in a reduced intracellular replication rate, suggesting that NDH2-I is not essential for parasite growth. Continuous passage of

∆ndh2-i/ndh2-i-myc parasites in the presence of Atc also did not decrease growth rate, excluding the possibility that an insufficient dilution of formerly expressed enzyme prevents an effect. Moreover, depletion of NDH2-I in ∆ ndh2-i/ndh2-i-myc parasites did not have an effect on the ability of these parasites to invade HFF host cells as monitored by microscopy, number of vacuoles per field were 14.75 for ∆ndh2-i/ndh2-i-myc+Atc, 12.75 for ndh2-i/ndh2-i-myc with or without Atc.

0 5 10 15 20 25 30 35 40

1 2 4 8 16

number of parasites/vacoule

Numberof vacoules; n=100 Δndh2-I/Ndh2-I-myc+ Atc

Ndh2-I/Ndh2-I-myc Ndh2-I/Ndh2-I-myc+ Atc

Figure 3.26: NDH2-I depletion does not result in a decreased growth rate. The growth rate of Δandh1/Andh1-myc parasites in the presence of Atc was compared with those from parental andh1/Andh1-myc parasites in the absence or presence of Atc. Parasites were cultured at least one round before in the absence or presence of Atc. The number of parasites per parasitophorous vacuole was determined 24 h post infection from 100 vacuoles by immunofluorescence microscopy. The distribution of vacuole sizes is shown in the figure.

The average number of parasites/vacuole was 5.50 for Δandh1/Andh1-myc/+Atc, 5.62 for andh1/Andh1-myc/+Atc, 5.59 for andh1/Andh1-myc/-Atc parasites.

RESULTS 88.

3.3.2.2 Expression of NDH2-II in knockout parasites (∆ndh2-i/ndh2-i-myc) Since there is another mitochondrial isoform of alternative NADH dehydrogenase (TgNDH2-II) in T. gondii, which is believed to have same function as the depleted isoform, we studied its expression in the tachyzoites of ∆ndh2-i/ndh2-i-myc+Atc.

Quantitative real time RT-PCR has shown a slight upregulation of the gene expression of TgNDH2-II in the knock out parasites (∆ndh2-i/ndh2-i-myc+Atc) in comparison to the wild type parasites (ndh2-i/ndh2-i-myc+Atc) when normalized to the expression of both house keeping genes β-tubulin and GAPDH (Fig. 3.27). This slight upregulation in the level of mRNA transcripts of ndh2-II might contribute to compensate the loss of NDH2-I activity leading to an absence of effect on the growth rate of knock out parasites (∆ndh2-i/ndh2-i-myc) in comparison to wild type (ndh2-i/ndh2-i-myc) parasites.

1 1

1.93

2.7

4.98

2.3

0 1 2 3 4 5 6

NDH2-II (TUB) NDH2-II (GAPDH)

Expression level (x-fold)

ndh2-i/ndh2-i-cmyc

?ndh2-i/ndh2-i-cmyc (clone 6)

?ndh2-i/ndh2-i-cmyc (clone 5)

Figure 3.27: Quantitative real time RT-PCR analysis of ndh2-II mRNA expression.

Tachyzoites of clones 5 and 6 of the mutant (∆ndh2-i/ndh2-i-myc) and parental (ndh2-i/ndh2-i-myc) strains were allowed to infect HFF monolayers and followed by cultivation for 24 h in the presence of Atc. RNA was isolated 24 h post infection. Lightcycler PCR was performed to amplify cDNAs of tgndh2-II, β-tubulin, and GAPDH. Values represent the percentage of bag1 mRNA expression in the knock out (∆ndh2-i/ndh2-i-myc) compared to the parental (ndh2-i/ndh2-i-myc) strains after normalization for β-tubulin and GAPDH mRNA expression.

RESULTS 89.

3.3.2.3 In vitro differentiation of knock out parasites (∆ndh2-i/ndh2-i-myc) To study the effect of NDH2-I disruption on the ability of these parasites to differentiate in vitro from tachyzoite to bradyzoites, we compared the expression profile of the bradyzoite marker bag1 (Bohne, 1995) normalized to the housekeeping gene tubulin. Quantitative real time RT-PCR analysis by a Lightcycler has shown no significant difference in the expression of bag1 gene in both knock out (∆ndh2-i/ndh2-i-myc) and parental (ndh2-(∆ndh2-i/ndh2-i-myc) parasites (Fig. 3.28)

100

109

10 30 50 70 90 110 130

ndh2-I/Ndh2-I-myc + Atc ∆ndh2-I/Ndh2-I-myc + Atc

Expression of bag1 (%)

Figure 3.28: Quantitative real time RT-PCR analysis of bag1 mRNA expression.

Tachyzoites of both mutant (∆ndh2-i/ndh2-i-myc) and parental (ndh2-i/ndh2-i-myc) strains were allowed to infect HFF monolayers, followed by cultivation in pH shift media (pH 8.3) with Atc. RNA was isolated 4 days post infection. Light cycler PCR was performed to amplify cDNAs of bag1 and β-tubulin. Values represent the percentage of bag1 mRNA expression in the knock out (∆ndh2-i/ndh2-i-myc) compared to the parental (ndh2-i/ndh2-i-myc) strains after normalization for β-tubulin mRNA expression.

RESULTS 90.

3.3.2.4 NDH2-I depletion leads to HDQ hypersensitivity

The susceptibility of Δndh2-i/ndh2i-myc and ndh2-i/ndh2i-myc parasites to HDQ was compared in the absence and presence of Atc (Fig. 3.29). Δandh1/Andh1-myc parasites displayed in the presence of Atc (ectopic Andh1 copy “OFF”) a significant higher sensitivity to HDQ. The IC50 in NDH2-I depleted parasites is in the pM range.

In contrast, if Δandh1/Andh1-myc parasites were cultured without Atc (ectopic Andh1 copy “ON”), the phenotype was restored and parasites revealed the same sensitivity to HDQ as the parental RH-TATi strain with an IC50 of ~ 2-3 nM. The additional expression of the ectopic Andh1 in andh1/Andh1-myc parasites had no influence on the IC50 of HDQ.

0 1 2 3 4 5 6 7 8 9 10

10 1 0.1 0.01 0.001 control

HDQ [µM]

numberof parasites/vacoule Δndh2-I/Ndh2-I-myc-Atc Δndh2-I/Ndh2-I-myc+Atc ndh2-I/Ndh2-I-myc +Atc

Figure 3.29: NDH2-I depletion results in HDQ hypersensitivity. The HDQ sensitivity of Δandh1/Andh1-myc parasites in the presence or absence of Atc was compared with parental andh1/Andh1-myc parasites +/- Atc. The average number of parasites per parasitophorous vacuole was determined 24 h post infection by immunofluorescence microscopy. NDH2-I depletion in Δndh2-I/ndh2-II-my/+Atc parasites results in a strongly increaed HDQ susceptibility with a complete inhibition of parasite replication at 1 nM.

DISCUSSION 91.