calcimycin and oryzalin

We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcimycin; Cell Division; Cells, Cultured; Cytochalasin D; Cytoskeleton; Dinitrobenzenes; Fibroblasts; Growth Inhibitors; Humans; Ionophores; Microscopy, Electron; Microtubules; Nocodazole; Organelles; Paclitaxel; Sulfanilamides; Thiazoles; Thiazolidines; Toxoplasma

Submicromolar concentrations of several dinitroaniline herbicides have been found to specifically inhibit intracellular replication of the protozoan parasite Toxoplasma gondii. IC50 concentrations for T. gondii survival were approximately 100 nM for ethalfluralin and oryzalin and approximately 300 nM for trifluralin. Primary human fibroblasts employed as host cells for parasite culture were unaffected at > 100-fold higher concentrations. Extracellular parasites were unaffected by these drugs, but within 8 hr after treatment of infected cell cultures, intracellular tachyzoites formed large amorphous bodies containing distorted nuclei. Parasite cytokinesis was completely blocked by drug treatment; nucleic acid synthesis, however, continued at near-normal levels for several days in the continuous presence of drug. All dinitroanilines appear to block nuclear division by inhibition of intranuclear spindle formation, but other cytoskeletal components were differentially affected by the various drugs tested. Subpellicular microtubules were absent in oryzalin-treated parasites, and large fragments of the inner membrane complex were observed throughout the parasite cytoplasm. In contrast, subpellicular microtubules and the inner membrane complex remained intact in ethalfluralin-treated parasites, but the endoplasmic reticulum and nuclear envelope were highly distended. Cytoskeletal elements associated with the conoid were not affected by any of the dinitroanilines tested, and treatment with the Ca2+ ionophore A23187 failed to trigger release of drug-treated parasites from infected cells. Mutant parasites resistant to oryzalin, ethalfluralin, or trifluralin were selected by chemical mutagenesis and examined for cross-resistance. An ethalfluralin-resistant mutant displayed cross-resistance to both oryzalin and trifluralin, while a trifluralin-resistant mutant was sensitive to oryzalin and only partially resistant ethalfluralin; an oryzalin-resistant mutant exhibited higher resistance to ethalfluralin and trifluralin than to oryzalin itself. Similarities between Apicomplexan and plant tubulin are discussed.

Topics: Animals; Anti-Infective Agents; Calcimycin; Cells, Cultured; Dinitrobenzenes; Fibroblasts; Herbicides; Humans; Ionophores; Mitosis; Mutation; Pyrimethamine; Structure-Activity Relationship; Sulfanilamides; Toxoplasma; Trifluralin