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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.

We investigated the effects of the extracellular matrix proteins laminin and fibronectin on the attachment of tachyzoites of Toxoplasma gondii to the murine macrophage cell line J774. Laminin but not fibronectin increased parasite attachment to J774 cells in a dose-dependent fashion. Cyclic YIGSR, a laminin-derived peptide which inhibits laminin binding to the 32/67-kDa laminin-binding protein on host cells, blocked the laminin-mediated enhancement of parasite attachment. An antiserum to the 32/67-kDa laminin-binding protein also inhibited binding of parasites to J774 cells. These results, in conjunction with our previous observations (G. C. Furtado, F. L. Collins, and K. A. Joiner, submitted for publication), demonstrate that tachyzoites bearing surface laminin bind to multiple laminin receptors in attaching to different target cells.