Comparison of mutants of Toxoplasma gondii selected for resistance to azithromycin, spiramycin, or clindamycin

Abstract

Azithromycin and spiramycin markedly inhibited the growth of Toxoplasma gondii in cultured human fibroblasts. However, 3 days of treatment were required to reveal their full antitoxoplasma activity. This delayed onset of inhibition was similar to that previously reported for clindamycin. Mutants of T. gondii resistant to azithromycin (AziR_l) and spiramycin (SprR-1) were isolated and compared with a previously described mutant resistant to clindamycin (ClnR-2). Mutant ClnR-2 was cross-resistant to all three antibiotics, while AziR_1 was cross-resistant only to spiramycin and SprR-1 was cross-resistant only to azithromycin. In short-term studies of protein synthesis by freshly prepared extracellular parasites, clindamycin and azithromycin were effective only at concentrations much greater than their 50% inhibitory concentrations in infected cultures and the resistant mutants did not differ from the wild type in antibiotic sensitivity. Thus, protein synthesis on cytoplasmic ribosomes of the parasite did not seem to be the target of these antibiotics. To determine whether mitochondrial protein synthesis in T. gondii was inhibited by clindamycin or azithromycin, wild-type parasites were grown in cultured cells in the presence of antibiotic concentrations well above the 50%o inhibitory concentrations. Mitochondrial function, measured by oxygen uptake per purified extracellular parasite, did not decrease substantially, after the parasites had multiplied 11-fold in the presence of antibiotic.Thus, mitochondrial protein synthesis did not seem to be the target of clindamycin or azithromycin. An alternative target is protein synthesis in the putative apicomplexan organelle that has a 35-kb genome.Toxoplasmic encephalitis is common among AIDS patients. The standard treatment of this opportunistic infection with pyrimethamine and sulfadiazine is generally effective. However, since many AIDS patients cannot tolerate prolonged administration of these drugs (11), alternative treatments are needed. Among the antibiotics known to be active against Toxoplasma gondii are the lincosamide clindamycin and the macrolides spiramycin and azithromycin. Clindamycin reduces mortality in infected mice (1,12,18) and, in combination with pyrimethamine, is as effective as pyrimethamine-sulfadiazine in the treatment of toxoplasmic encephalitis in AIDS patients (7,14). Spiramycin is active against murine toxoplasmosis (2) and is thought to reduce the incidence and severity of congenital disease when used to treat women who acquired toxoplasmosis during pregnancy (17). Azithromycin reduces mortality in experimental murine toxoplasmosis (2). The mechanism of action of these antibiotics against T. gondii is unknown. An understanding of how they exert their antiparasitic activity might allow the design of more potent derivatives. One way to approach the mechanism of action is to characterize antibiotic-resistant mutants. We have isolated a mutant of T. gondii that is markedly resistant to clindamycin (21) and describe here mutants selected for resis...

Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii. Implications for the target of macrolide antibiotics.

“…These levels are nearly two orders of magnitude greater than required for killing intracellular parasites (3,4), and an order of magnitude higher than achieved in the parasite cytosol with usual doses (5 Optimization ofprotein synthesis in extracellular parasites. To determine whether the drug sensitivity of cytoplasmic ribosomes might be altered under the ionic environment prevailing within infected host cells, we modified parasite incubation conditions to more closely mimic the intracellular milieu.…”

“…The observation that > 100-fold higher drug concentrations are needed to even partially inhibit total protein synthesis in extracellular parasites (3) has been taken to imply that parasites growing inside the parasitophorous vacuole must concentrate these compounds extensively within the parasite cytoplasm (4). We have recently demonstrated that azithromycin taken up by T. gondii-infected fibroblasts is indeed localized, but that the drug is concentrated within lysosomes of the host cell and in acidified organelles of the parasite (5).…”

“…Extremely low concentrations of clindamycin or azithromycin (10 and 30 ng/ml, respectively) are sufficient to inhibit parasite growth in extended tissue culture assays (2,3), and it has been suggested that the activity of azithromycin against Toxoplasma results from the inhibition of total protein synthesis (4). The observation that > 100-fold higher drug concentrations are needed to even partially inhibit total protein synthesis in extracellular parasites (3) has been taken to imply that parasites growing inside the parasitophorous vacuole must concentrate these compounds extensively within the parasite cytoplasm (4).…”

The Apicoplast: A Review of the Derived Plastid of Apicomplexan Parasites

“…One interesting feature of some of these inhibitors is a "delayed death" phenotype seen in Toxoplasma gondii (see Table 1) (Pfefferkorn and Borotz, 1994;Fichera et al, 1995;Fichera and Roos, 1997). This effect is identical to the growth pattern of apicoplast segregation mutants lacking an apicoplast (He et al, 2001).…”

In Vivo Efficacy of Drugs against <i>Toxoplasma gondii</i> Combined with Immunomodulators

“…Particularly in adults, less toxic and more effective novel drugs are required to treat T. gondii-associated chorioretinitis and congenital toxoplasmosis (3,4,8,9). Recently, in vitro resistance against pyrimethamine (PYR), clindamycin, spiramycin, and azithromycin has been demonstrated in T. gondii mutants, and this has increased the attention towards new antimicrobials (10,11). A treatment that is particularly effective against the bradyzoite forms of T. gondii in the tissues might eliminate the life-threatening potential of the infection.…”