Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay

Chamberland, Suzanne; Kirst, Herbert A.; Current, William L.

doi:10.1128/aac.35.5.903

Cited by 50 publications

(36 citation statements)

References 18 publications

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“…If the antibiotic treatment immediately blocked mitochondrial protein synthesis and allowed no further increase in mitochondrial activity, the 9-to 13-fold increase in parasite number should reduce the per-parasite oxygen uptake to 11 to 8% of the level seen of the control parasites. As shown in 1,000-fold greater IC50s previously reported for azithromycin (8 ,g/ml) and spiramycin (20 ,g/ml) in 2-day experiments (6). The observation that mutants selected for resistance to clindamycin, azithromycin, or spiramycin show a pattern of partial or complete cross-resistance (Table 1) suggests that these antibiotics share a common target in T. gondii.…”

Section: Resultssupporting

confidence: 55%

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

Pfefferkorn

Borotz

1994

Antimicrob Agents Chemother

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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...

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Section: Resultssupporting

confidence: 55%

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

Pfefferkorn

Borotz

1994

Antimicrob Agents Chemother

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“…Host cells were cultivated in tissue culture flasks using minimum essential medium supplemented with 10 % horse serum. Cells were infected with tachyzoites at a final hostto-parasite ratio of 1 : 5 ; parasites were harvested 2-3 days after infection and purified as described previously [22]. The protein concentration was determined by the biuret assay [23] in the presence of 0.2 % deoxycholate.…”

Section: Culture Methodsmentioning

confidence: 99%

Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

Moreno

Zhong

et al. 1998

Biochemical Journal

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Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

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“…In mice treated with azithromycin plus sulfadiazine and azithromycin plus pyrimethamine, parasite burdens in blood and organs, relapses after cessation of therapy, and mortality were all markedly reduced relative to mice treated with any of the agents alone. These results show that azithromycin, which is remarkably active on pulmonary Toxoplasma infection, significantly potentiates the curative effect of sulfadiazine or pyrimethamine.Macrolides are inhibitory for Toxoplasma gondii tachyzoites in vitro, but a significant effect is only obtained at high concentrations (3,4,6 …”

mentioning

confidence: 99%

“…Macrolides are inhibitory for Toxoplasma gondii tachyzoites in vitro, but a significant effect is only obtained at high concentrations (3,4,6 …”

mentioning

confidence: 99%

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Synergistic activity of azithromycin and pyrimethamine or sulfadiazine in acute experimental toxoplasmosis

Derouin

Almadany

Chau

et al. 1992

Antimicrob Agents Chemother

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The efficacy of azithromycin administered alone or combined with pyrimethamine or sulfadiazine was examined in a murine model of acute toxoplasmosis. Outbred Swiss mice acutely infected with tachyzoites of the virulent RH strain were treated for 10 days from day +1 postinfection. The efficacy of each regimen was assessed in terms of survival rates and sequential titration of parasites in blood, brain, and lungs by using a tissue culture method. Administration of azithromycin at 300, 150, or 75 mg/kg of body weight per day resulted in prolonged survival relative to that of untreated controls; sequential examination of parasite burden showed early eradiaction of Toxoplasma gondii from the lungs, whereas dissemination to the brain was not prevented. A remarkable synergistic effect was observed when azithromycin (150 mg/kgtday) was administered in combination with pyrimethamine or sulfadiazine at noncurative dosages, i.e., 12.5 and 200 mg/kg/day, respectively. In mice treated with azithromycin plus sulfadiazine and azithromycin plus pyrimethamine, parasite burdens in blood and organs, relapses after cessation of therapy, and mortality were all markedly reduced relative to mice treated with any of the agents alone. These results show that azithromycin, which is remarkably active on pulmonary Toxoplasma infection, significantly potentiates the curative effect of sulfadiazine or pyrimethamine.Macrolides are inhibitory for Toxoplasma gondii tachyzoites in vitro, but a significant effect is only obtained at high concentrations (3,4,6

show abstract

Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay

Cited by 50 publications

References 18 publications

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

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

Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

Synergistic activity of azithromycin and pyrimethamine or sulfadiazine in acute experimental toxoplasmosis

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