Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;8(11):e1003049.
doi: 10.1371/journal.ppat.1003049. Epub 2012 Nov 29.

A Forward Genetic Screen Reveals That Calcium-Dependent Protein Kinase 3 Regulates Egress in Toxoplasma

Affiliations
Free PMC article

A Forward Genetic Screen Reveals That Calcium-Dependent Protein Kinase 3 Regulates Egress in Toxoplasma

Erin Garrison et al. PLoS Pathog. .
Free PMC article

Abstract

Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutation in TgCDPK3 of strain MBE1.1.
A. DNA sequence chromatogram of bases 4,684,206-18 of chromosome IX for both the parental (RHΔhpt) and the iiEgress mutant (MBE1.1) strains are shown. Gray box highlights the SNP found in the mutant strain. B. A comparison of the equivalent of protein kinase superfamily subdomain VIII from TgCDPK3 (accession number ABA60892), TgCDPK1 (accession number XP_002371666) and PfCDPK1 (accession number XP_001349680) is shown. The Threonine (T) changed to an Isoleucine (I) in TgCDPK3 from the mutant strain MBE1.1 is indicated. The numbers of the amino acid shown are listed in parenthesis next to the name of each protein.
Figure 2
Figure 2. Phenotypic complementation of MBE1.1.
A. Intracellular parasites of the parental (RHΔhpt), mutant (MBE1.1), complemented (MBE1.1+sagCDPK3::HA) strains, as well as MBE1.1 parasites complemented with TgCDPK3 carrying the T239I mutation (MBE1.1+sagCDPK(T239I)::HA) were exposed to 1 µM A23187 for the time points indicated in the graph. Percentage egress represents the number of lysed vacuoles divided by the total number of vacuoles (lysed+intact). B. Exogenous copy of CDPK3::HA in the strains complemented with the wild type (MBE1.1+sagCDPK3::HA) or mutant gene (MBE1.1+sagCDPK3(T239I)::HA) was detected by western blot analysis using HA antibodies. Antibodies against the surface protein Sag1 were used to show equal loading. C. Extracellular parasites of the indicated strains were exposed to 1 µM A23187 for 45 minutes before being added to cells. Percentage survival was calculated by dividing the number of plaques formed by parasites treated with A23187 divided by the number of plaques formed by untreated parasites. For each treatment, all plaques in a well of a 12-well plate were counted. In B and C, each data point represents the average of four independent experiments and the error bars represent the standard deviation.
Figure 3
Figure 3. Localization of TgCDPK3.
A. Intracellular and extracellular parasites of the MBE1.1 strain complemented with a wild-type copy of TgCDPK3 were stained with antibodies against the hemagglutinin (HA) tag present in the exogenous copy of TgCDPK3. B. Parasites with an HA tag in the endogenous copy of TgCDPK3 (RHΔku80Δhpt CDPK3::HA) were stained with HA antibodies while intracellular and extracellular. In addition, intracellular parasites of the same strain were exposed to 1 µM A23187 for 2 minutes to induce iiEgress (two far right panels). In the iiEgress panels arrows point at parasites in the process of exiting or invading cells, arrow head points at a parasite that has remained inside of a host cell, while all other parasites have already completed egress. C. RHΔku80Δhpt CDPK3::HA parasites were co-stained with HA antibodies and antibodies against the inner-membrane protein 3 (a gift of MJ Gubbels). Scale bar 10 µm.
Figure 4
Figure 4. Effect of mutating myristoylation and palmitolation sites in TgCDPK3 localization and function.
A construct with TgCDPK3 carrying mutations in amino acids 2 and 3 (TgCDPK3::HA(G2A,C3A)) expressed by either the Sag1 promoter (A) or the TgCDPK3 promoter (B) was introduced into the MBE1.1 strain. Left panels show localization of TgCDPK3::HA(G2A,C3A) using HA antibodies. Right panels show percentage of parasites of the strain indicated that undergo egress after 2 minutes exposure to 1 µM A23187. Each data point represents the average of four independent experiments and the error bars represent the standard deviation. Scale bar is 10 µm.
Figure 5
Figure 5. Mutations in TgCDPK3 in mutant strains MBD1.1, 52F11 and 91E4.
A. The positions of the amino acids mutated in each of the four iiEgress mutants are shown within the TgCDPK3 structure. Schematic was rendered in Protein Workshop using the published structure of TgCDPK3 bound to the ATP analog 5-[(Z)-(5-chloro-1, 2-dihydro-2-oxo-3H-indol-3- ylidene)methyl]-N-[2-(diethylamino)ethyl]-2, 4-dimethyl-1H-pyrrole-3-carboxamide (accession 3HZT). B. The protein sequences show the amino acids in TgCDPK3 surrounding the glycine (G) to aspartic acid (D) change in 52F11 (top), those around the leucine (L) to proline (P) change in MBD1.1 (bottom left) and the HRD catalytic domain of TgCDPK3 with the Asparagine mutated in 91E4 (bottom right), all compared to the equivalent regions in homologs TgCDPK1 and PfCDPK1. C. Intracellular parasites of the parental (RHΔhpt for MBD1.1, PruΔhpt for 52F11 and 91E4), mutants (MBE1.1, 52F11 and 91E4) and the strains complemented with the wild-type copy of TgCDPK3 were exposed to 1 µM A23187 for 2 minutes. Percentage egress represents the number of lysed vacuoles divided by the total number of vacuoles (lysed+intact). Each data point represents the average of four independent experiments and the error bars represent the standard deviation.
Figure 6
Figure 6. In vitro kinase activity of wild-type and mutant TgCDPK3.
Recombinant wild-type (WT) or mutant (G88D, L184P, N204K and T239I) TgCDPK3 were tested for activity against syntide-2 in the presence of CaCl2. Whereas wild-type TgCDPK3 shows robust activity against the substrate, mutagenesis of G88D, L184P, N204K or T239I inactivates kinase activity. Error bars represent standard deviation from three independent experiments.
Figure 7
Figure 7. Effect of purfalcamine on iiEgress and TgCDPK3 activity.
A. Intracellular parasites were pre-treated with the indicated amount of purfalcamine for 15 minutes before being exposed to 1 µM A23187 for 3 minutes to induce egress. B. Parasites were pre-incubated with 25 µM A23187 for 15 minutes before treatment with 1 µM A23187 for the time indicated in the X-axis. Percentage egress represents the number of lysed vacuoles divided by the total number of vacuoles (lysed+intact). Each data point represents the average of four independent experiments and the error bars represent the standard deviation. C. The effect of purfalcamine on TgCDPK3 activity was tested in in vitro kinase assays. Each data point indicates the mean of 4 experiments; error bars represent the standard deviation.
Figure 8
Figure 8. Establishment and analysis of a Tgcdpk3 knockout strain.
A. Schematic diagram of the TgCDK3 genomic locus and the region disrupted in the knockout strain. The top black line represents the genomic DNA region with each tick being 1,000 bases. The gray box represents the region of the TgCDPK3 locus that is replaced by the hpt selectable marker (dhfrHXGPRTdhfr), which is transcribed in the opposite direction from TgCDPK3 (as indicated by arrow pointing left). The relative positions of exons (white boxes) and introns (dashed lines) are shown below. Black arrowhead indicates position of start codon while the white arrowhead points at the stop codon. Lines labeled a, b, c and d represent the regions amplified by PCR to test for the disruption of TgCDPK3. B. PCR products of the parental strain, RHΔhptΔku80, and the TgCDPK3 KO strain, RHΔhptΔku80Δcdpk3+HPT are shown. Labels above lanes indicate the DNA fragment amplified. Fragments a, b and c cannot be amplified if the TgCDPK3 locus has been disrupted by the hpt marker. Fragment d is expanded from both the parental and knockout strain and it serves as a control. C. Intracellular parasites of either the parental or knockout strain were treated with 1 µM A23187 for the time indicated. Percentage egress represents the number of lysed vacuoles divided by the total number of vacuoles (lysed+intact). Each data point represents the average of four independent experiments and the error bars represent the standard deviation. D. Extracellular parasites of either the parental or the knockout strain were exposed to 1 µM A23187 for 45 minutes before being added to cells. Percentage survival was calculated by dividing the number of plaques formed by parasites treated with A23187 divided by the number of plaques formed by untreated parasites. Each data point represents the average of three independent experiments and the error bars represent the standard deviation. E. Parasites were allowed to settle on glass in intracellular buffer, which was changed to extracellular buffer to initiate motility. After 15 minutes parasites were fixed and the percentage of parasites that had moved was determined by counting parasites that were associated with trails of surface proteins left behind during gliding. Data is from 3 independent studies and the error bars are the standard deviation.
Figure 9
Figure 9. Propagation related phenotypes of TgCDPK3 knockout strain.
A. Parental or knockout parasites were allowed to invade confluent HFFs for the amount of time indicated. Cultures were then fix and the percentage of parasites that were inside of the cells were determine for each data point. The data represents the average of 3 independent assays and the error bars represent the standard deviation. B. Parasites were allowed to divide for 24 hours and a total of at least 100 vacuoles were inspected to determine the number of vacuoles with 2, 4, 8 or 16 parasites within. Data are the average of 3 independent experiments and the error bars are the standard deviations.

Similar articles

See all similar articles

Cited by 60 articles

See all "Cited by" articles

References

    1. Israelski DM, Remington JS (1993) Toxoplasmosis in patients with cancer. Clin Infect Dis 17 Suppl 2: S423–435. - PubMed
    1. Luft BJ, Remington JS (1992) Toxoplasmic encephalitis in AIDS. Clin Infect Dis 15: 211–222. - PubMed
    1. Slavin MA, Meyers JD, Remington JS, Hackman RC (1994) Toxoplasma gondii infection in marrow transplant recipients: a 20 year experience. Bone Marrow Transplant 13: 549–557. - PubMed
    1. Wong SY, Remington JS (1994) Toxoplasmosis in pregnancy. Clin Infect Dis 18: 853–861. - PubMed
    1. Black MW, Boothroyd JC (2000) Lytic cycle of Toxoplasma gondii. Microbiol Mol Biol Rev 64: 607–623. - PMC - PubMed

Publication types

Feedback