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Toxoplasma Actin Is Required for Efficient Host Cell Invasion.

Drewry LL, Sibley LD - MBio (2015)

Bottom Line: We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed.Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.Our results have important implications for the interpretation of the apicomplexan invasion model and also highlight significant considerations when analyzing the phenotypes of inducible knockout parasites generated using Cre-Lox technology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.

No MeSH data available.


Related in: MedlinePlus

Testing sensitivity of invasion to actin inhibitor cytochalasin D (CytD). (A and B) Parasites were classified as intracellular or extracellular and as ACT1f intact or Δact1 using the differential staining protocol described in the legend to Fig. 1. The invasion rates of ACT1f intact and Δact1 parasites and CytD-resistant act1A136 parasites were compared in CytD-sensitive HFF cells (A) and CytD-resistant Cyt-1 cells (B). Data shown are mean values ± standard errors of the means (SEM) from 3 independent experiments with the ACT1f parasites and 2 independent experiments with the act1A136 parasites, each with 3 to 5 technical replicates. For each group, the number of intracellular parasites was normalized to the mean invasion rate of that group with no CytD. ****, P ≤ 0.0001; two-way ANOVA with Dunnett’s multiple-comparison test. All experiments were performed with ACT1f-2 4 days after rapamycin induction of gene excision.
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fig4: Testing sensitivity of invasion to actin inhibitor cytochalasin D (CytD). (A and B) Parasites were classified as intracellular or extracellular and as ACT1f intact or Δact1 using the differential staining protocol described in the legend to Fig. 1. The invasion rates of ACT1f intact and Δact1 parasites and CytD-resistant act1A136 parasites were compared in CytD-sensitive HFF cells (A) and CytD-resistant Cyt-1 cells (B). Data shown are mean values ± standard errors of the means (SEM) from 3 independent experiments with the ACT1f parasites and 2 independent experiments with the act1A136 parasites, each with 3 to 5 technical replicates. For each group, the number of intracellular parasites was normalized to the mean invasion rate of that group with no CytD. ****, P ≤ 0.0001; two-way ANOVA with Dunnett’s multiple-comparison test. All experiments were performed with ACT1f-2 4 days after rapamycin induction of gene excision.

Mentions: Parasite invasion is known to be sensitive to the actin polymerization inhibitor cytochalasin D (CytD) (2). If Δact1 parasites rely on residual ACT1 for invasion, we reasoned that these Δact1 parasite invasions would retain CytD sensitivity. To test this, we used our modified invasion assay to track parasite invasion into both human foreskin fibroblast (HFF) cells and a CytD-resistant epithelial cell line, Cyt-1 (24). Four days after the induction of gene excision, we observed dose-dependent CytD inhibition of invasion into HFF cells by both ACT1f intact and Δact1 parasites, confirming that invasion by Δact1 parasites is indeed actin dependent (Fig. 4A). CytD is a reversible inhibitor of actin polymerization. Because Δact1 parasites contain less ACT than ACT1f intact parasites, we predicted that Δact1 parasites would be inhibited at lower CytD concentrations than ACT1f intact parasites. Consistent with this prediction, when invading HFF cells, Δact1 parasites were inhibited by 200 nM CytD in 3 independent experiments, while ACT1f intact parasites never showed significant sensitivity below 500 nM CytD (Fig. 4A).


Toxoplasma Actin Is Required for Efficient Host Cell Invasion.

Drewry LL, Sibley LD - MBio (2015)

Testing sensitivity of invasion to actin inhibitor cytochalasin D (CytD). (A and B) Parasites were classified as intracellular or extracellular and as ACT1f intact or Δact1 using the differential staining protocol described in the legend to Fig. 1. The invasion rates of ACT1f intact and Δact1 parasites and CytD-resistant act1A136 parasites were compared in CytD-sensitive HFF cells (A) and CytD-resistant Cyt-1 cells (B). Data shown are mean values ± standard errors of the means (SEM) from 3 independent experiments with the ACT1f parasites and 2 independent experiments with the act1A136 parasites, each with 3 to 5 technical replicates. For each group, the number of intracellular parasites was normalized to the mean invasion rate of that group with no CytD. ****, P ≤ 0.0001; two-way ANOVA with Dunnett’s multiple-comparison test. All experiments were performed with ACT1f-2 4 days after rapamycin induction of gene excision.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471557&req=5

fig4: Testing sensitivity of invasion to actin inhibitor cytochalasin D (CytD). (A and B) Parasites were classified as intracellular or extracellular and as ACT1f intact or Δact1 using the differential staining protocol described in the legend to Fig. 1. The invasion rates of ACT1f intact and Δact1 parasites and CytD-resistant act1A136 parasites were compared in CytD-sensitive HFF cells (A) and CytD-resistant Cyt-1 cells (B). Data shown are mean values ± standard errors of the means (SEM) from 3 independent experiments with the ACT1f parasites and 2 independent experiments with the act1A136 parasites, each with 3 to 5 technical replicates. For each group, the number of intracellular parasites was normalized to the mean invasion rate of that group with no CytD. ****, P ≤ 0.0001; two-way ANOVA with Dunnett’s multiple-comparison test. All experiments were performed with ACT1f-2 4 days after rapamycin induction of gene excision.
Mentions: Parasite invasion is known to be sensitive to the actin polymerization inhibitor cytochalasin D (CytD) (2). If Δact1 parasites rely on residual ACT1 for invasion, we reasoned that these Δact1 parasite invasions would retain CytD sensitivity. To test this, we used our modified invasion assay to track parasite invasion into both human foreskin fibroblast (HFF) cells and a CytD-resistant epithelial cell line, Cyt-1 (24). Four days after the induction of gene excision, we observed dose-dependent CytD inhibition of invasion into HFF cells by both ACT1f intact and Δact1 parasites, confirming that invasion by Δact1 parasites is indeed actin dependent (Fig. 4A). CytD is a reversible inhibitor of actin polymerization. Because Δact1 parasites contain less ACT than ACT1f intact parasites, we predicted that Δact1 parasites would be inhibited at lower CytD concentrations than ACT1f intact parasites. Consistent with this prediction, when invading HFF cells, Δact1 parasites were inhibited by 200 nM CytD in 3 independent experiments, while ACT1f intact parasites never showed significant sensitivity below 500 nM CytD (Fig. 4A).

Bottom Line: We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed.Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.Our results have important implications for the interpretation of the apicomplexan invasion model and also highlight significant considerations when analyzing the phenotypes of inducible knockout parasites generated using Cre-Lox technology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.

No MeSH data available.


Related in: MedlinePlus