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Localization of azithromycin in Toxoplasma gondii-infected cells
J C Schwab1, Y Cao, M R Slowik
1Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022.
Abstract:
Agents effective against intracellular pathogens must enter infected cells, crossing vacuolar membranes surrounding the organisms and then penetrating into the microbe and localizing to the microbial target site. We have characterized these parameters for azithromycin entry into Toxoplasma gondii-infected Chinese hamster ovary cells and murine macrophage-like J774 cells. Azithromycin uptake into infected host cells was concentrative and was dependent upon proton gradients. Subcellular fractionation of azithromycin-loaded infected CHO cells demonstrated > 95% intracellular drug in host cell lysosomes and cytosol, with < 5% associated with the parasite. Uptake of azithromycin into the T. gondii vacuole increased if parasites were coated with antibody prior to internalization by murine J774 cells, conditions which result in the formation of acidified phagolysosomes. No redistribution or retention of azithromycin in the parasite was observed when drug efflux from antibiotic-loaded infected CHO cells was monitored. Azithromycin entry into extracellular T. gondii was concentrative, was temperature and pH dependent, and was not different when azithromycin-sensitive and -resistant parasites were compared. These results demonstrate that azithromycin concentrates primarily in acidified compartments in parasites and host cells. The high concentration of azithromycin within these compartments may not be biologically relevant to inhibition of intracellular parasite growth by this agent.
Insights
Azithromycin concentrates in host cell lysosomes and parasite vacuoles, but not within the parasite itself. This accumulation in acidified compartments may not be key for inhibiting intracellular parasite growth.
Area of Science:
- Microbiology
- Pharmacology
- Cell Biology
Background:
- Effective antimicrobial agents targeting intracellular pathogens require cellular entry and localization within the pathogen.
- Understanding azithromycin's intracellular pharmacokinetics is crucial for its efficacy against pathogens like Toxoplasma gondii.
Purpose of the Study:
- To characterize the cellular entry and localization parameters of azithromycin in Toxoplasma gondii-infected host cells.
- To determine if azithromycin accumulates within the parasite or host cell compartments.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells and murine macrophage-like J774 cells infected with T. gondii.
- Performed subcellular fractionation to quantify intracellular azithromycin distribution.
- Investigated azithromycin uptake and efflux under various conditions, including antibody opsonization and extracellular parasite exposure.
Main Results:
- Azithromycin uptake into infected host cells was concentrative and proton gradient-dependent.
- Over 95% of intracellular azithromycin localized to host cell lysosomes and cytosol, with less than 5% associated with the parasite.
- Uptake into the T. gondii vacuole increased in acidified phagolysosomes, but no drug retention within the parasite was observed.
- Azithromycin entry into extracellular T. gondii was concentrative and pH-dependent, irrespective of parasite sensitivity.
Conclusions:
- Azithromycin primarily concentrates in acidified compartments within host cells and parasites.
- The observed high concentration of azithromycin in these compartments may not be directly relevant for inhibiting intracellular parasite growth.