| Siderophores are chelators with an extraordinarily high affinity for ferric iron. Produced by bacteria and some species of fungi during periods of iron limitation, siderophores often play a crucial role in the survival and, in pathogens, virulence of the producing species. Although more than 500 different siderophores have been isolated to date, only a few have been studied in detail, and many questions regarding their in vivo targets, properties, and effects on host systems remain unanswered.;In Chapter 2, a new method of analyzing siderophore-protein interactions is presented. This method, which utilizes HPLC-MS/MS, is shown to be a sensitive, general, and accurate means for detecting and monitoring iron removal from mammalian proteins by siderophores. Additionally, the results presented in this chapter support a reanalysis of previous kinetic data for the siderophore acinetoferrin, and suggest that iron contamination may have biased the earlier interpretation of acinetoferrin-transferrin kinetic behavior.;In Chapter 3, the properties of mycobactin J, a member of a little-studied class of hydrophobic siderophores called the mycobactins, are explored relative to the synthetic iron chelator TrenCAM. The results indicate that mycobactin J is an unexpectedly strong iron chelator that is capable of removing iron from TrenCAM in nonaqueous solvents. As a result, an updated model for the function of the mycobactin siderophores in vivo is presented based on the newly described kinetic and thermodynamic properties of mycobactin J.;Chapter 4 describes the effects of mycobactin J on a model host system, murine macrophage cells. Results show that mycobactin J is cytotoxic at concentrations far less than that of the iron chelators desferrioxamine B and TrenCAM; further analysis indicates that this cytotoxicity is consistent with an iron starvation response, which supports the finding in Chapter 3 that mycobactin J has a very high affinity for iron. This confirms that mycobactin J is a strong iron chelator under physiologically relevant conditions, and supports the proposal in Chapter 3 of an expanded role for mycobactin siderophores in vivo .;As a whole, these results demonstrate the importance of studying siderophore properties in vitro and in vivo, and of finding new methods for doing so. Because of their insolubility in water, the study of many hydrophobic siderophores, and their role under physiological conditions (including within hydrophobic membrane spaces) have been neglected. Future work should encompass two-phase systems that more accurately represent the native working environments of siderophores and allow for a more complete description of their actual role in vivo. |
