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Tryptophan depletion resulting from indoleamine 2,3-dioxygenase (IDO) activity within the kynurenine pathway is one of the most prominent gamma interferon (IFN-␥)-inducible antimicrobial effector mechanisms in human cells. On the other hand, nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) serves a more immunoregulatory role in human cells and thereby interacts with tryptophan depletion in a number of ways. We investigated the effects of NO on IDO gene transcription, protein synthesis, and enzyme activity as well as on IDO-mediated bacteriostasis in the human epithelial cell line RT4. IFN-␥-stimulated RT4 cells were able to inhibit the growth of Staphylococcus aureus in an IDO-mediated fashion, and this bacteriostatic effect was abolished by endogenously produced NO. These findings were supported by experiments which showed that IDO activity in extracts of IFN-␥-stimulated cells is inhibited by the chemical NO donors diethylenetriamine diazeniumdiolate, S-nitroso-L-cysteine, and S-nitroso-N-acetyl-D,L-penicillamine. Furthermore, we found that both endogenous and exogenous NO strongly reduced the level of IDO protein content in RT4 cells. This effect was not due to a decrease in IDO gene transcription or mRNA stability. By using inhibitors of proteasomal proteolytic activity, we showed that NO production led to an accelerated degradation of IDO protein in the proteasome. This is the first report, to our knowledge, that demonstrates that the IDO is degraded by the proteasome and that NO has an effect on IDO protein stability.The indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step in the kynurenine pathway of tryptophan degradation, the conversion of tryptophan to N-formyl-kynurenine. IDO is induced by gamma interferon (IFN-␥) in the course of an inflammatory response in many human cell types, including macrophages, astrocytes, fibroblasts, and epithelial cells. A strong IDO activity leads to a nearly complete depletion of the essential amino acid tryptophan at the site of infection, which results in growth arrest of several tryptophandependent microorganisms. Among these IDO-sensitive microorganisms are eukaryotic pathogens such as Toxoplasma gondii (10, 36) and bacteria such as group B streptococci (28) and enterococci (29). Furthermore, the immunoregulatory role of tryptophan depletion has recently received much attention. Mellor and colleagues found that T cells are unable to proliferate in a tryptophan-depleted environment and that in vivo IDO activity in the mouse placenta protects allogeneic concepti from being rejected by a T-cell-driven mechanism (33). It has been suggested that first-time activation of T cells in the absence of tryptophan may even result in the development of tolerance to the antigen presented (31).The role of nitric oxide (NO) production by the inducible isoform of NO synthase (iNOS) in human cells is controversial. While having a clearly illustrated antimicrobial potential against a variety of pathogens in rodent cells (reviewed in refer...

Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.Members of the phylum Apicomplexa are obligate intracellular parasites that replicate in a variety of cell types. Some, including the human pathogens Plasmodium and Babesia, replicate primarily in the bloodstream, whereas others such as Cryptosporidium, a cause of chronic gastritis among the immune-compromised, and Eimeria, an agricultural parasite, replicate in the intestinal epithelium. Only parasites in the isosporoid coccidian clade, which includes Toxoplasma gondii, the causative agent of toxoplasmosis, replicate in deep tissues (2). Thus, it can be expected that each clade has its own complement of proteins facilitating survival in a specific habitat, along with conserved proteins that play fundamental roles in events common to all apicomplexans.Many invasion studies have been performed in T. gondii, as it is more amenable to in vitro manipulation than other members of the Apicomplexa. Upon contact with a host cell, T. gondii tachyzoites discharge the contents of apically localized microneme (MIC) organelles (13). MIC adhesive proteins contribute to binding host cell receptors, and blocking micronemal secretion dramatically reduces invasion (9). These proteins, which often cluster into multiunit complexes, are transiently deployed to the apical surface during apical attachment and invasion. Transmembrane (TM) MIC proteins, such as MIC2, MIC6, and MIC8, are thought to act as bridging molecules that connect host receptors with the parasite's motility apparatus, the glideosome (37). By translocating MIC-receptor complexes backwards toward the posterior end, the parasite "pulls" itself into the target cell, invaginating the host plasma membrane to form the nascent parasitophorous vacuole (PV). As they treadmill posteriorly,...

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