Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects one-third of humanity and is one of the world's most important infectious agents. M. tuberculosis is a facultative intracellular pathogen. In the host, it multiplies intracellularly in mononuclear phagocytes, and it also appears to multiply extracellularly at least in lung cavities. Its capacity to infect the host is closely linked to its ability to acquire iron. Serum containing poorly saturated transferrin, such as human serum, is tuberculostatic, and its tuberculostatic effect is neutralized by the addition of iron (1, 2).Free iron is very limited in the host, particularly in extracellular sites, owing to the high affinity with which it is held by host iron-binding proteins, chiefly transferrin and lactoferrin. To obtain iron at sites where it is limited, many pathogens have developed high-affinity iron-binding molecules of their own called siderophores, which can remove iron from host ironbinding molecules. Mycobacteria have been shown by Macham, Ratledge, Barclay, and colleagues (3-5) to produce small water-soluble siderophores called exochelins. This group of investigators has proposed that exochelins bind iron in the extracellular aqueous environment and transport the metal to another high-affinity iron-binding molecule located in the cell wall ofM. tuberculosis-mycobactin (3). Mycobactin is a highly lipophilic molecule thought to facilitate the transport of iron across the cell wall to the interior of the bacterium (6).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Both exochelins and mycobactins are induced by low concentrations of iron in broth medium (4, 7). There are two general types of exochelins, classified according to their extractability in organic solvents (8). The chloroform-insoluble exochelins, produced by saprophytic mycobacteria, are not extractable into any organic solvent. The chloroform-soluble exochelins, produced by slow-growing pathogenic mycobacteria, including M. tuberculosis, are extractable into chloroform (5).Mycobactins have been extensively studied and their structures delineated (7). In contrast, individual exochelins have not been purified previously and neither their structure nor composition has been described. In this paper, we describe the purification of exochelins of M. tuberculosis from both a virulent (Erdman) and an avirulent (H37Ra) strain and their characterization by MS. MATERIALS AND METHODSMedium and Reagents. Modified iron-deficient Sauton's broth medium (9) was prepared with 1-10 ,uM Fe3+ and without Tween. Mycobactin J was purchased from Allied Monitor (Fayette, MO).Bacteria. M. tuberculosis Erdman (ATCC catalog no. 35801) and H37Ra (A...

SummaryTo multiply and cause disease in the host, Mycobacterium tuberculosis must acquire iron from the extracellular environment at sites of replication. To do so, the bacterium releases high-affinity iron-binding siderophores called exochelins. In previous studies, we have described the purification and characterization of the exochelin family of molecules. These molecules share a common core structure with another type of high-affinity iron-binding molecule located in the cell wall of M. tuberculosis: the mycobactins. The water-soluble exochelins differ from each other and from the water-insoluble mycobactins in polarity, which is dependent primarily upon the length and modifications of an alkyl side chain. In this study, we have investigated the capacity of purified exochelins to remove iron from host high-affinity iron-binding molecules, and to transfer iron to mycobactins. Purified desferri-exochelins rapidly removed iron from human transferrin, whether it was 95 or 40% iron saturated, its approximate percent saturation in human serum, and from human lactoferrin. Desferri-exochelins also removed iron, but at a slower rate, from the iron storage protein ferritin. Purified ferri-exochelins, but not iron transferrin, transferred iron to desferri-mycobactins in the cell wall of live bacteria. To explore the possibility that the transfer of iron from exochelins to mycobactins was influenced by their polarity, we investigated the influence of polarity on the iron affinity of exochelins. Exochelins of different polarity exchanged iron equally with each other. This study supports the concept that exochelins acquire iron for M. tuberculosis by removing this element from host iron-binding proteins and transferring it to desferri-mycobactins in the cell wall of the bacterium. The finding that ferri-exochelins but not iron transferrin transfer iron to mycobactins in the cell wall underscores the importance of exochelins in iron acquisition. This study also shows that the variable alkyl side chain on the core structure of exochehns and mycobactins, the principal determinant of their polarity, has little or no influence on their iron affinity.

Reperfusion injury, which occurs upon the reintroduction of blood f low to an ischemic organ, is responsible for considerable damage in heart attacks and strokes. However, no treatment for reperfusion injury is currently available. A major cause of reperfusion injury is the ironmediated generation of hydroxyl radical (⅐OH). In this study we have explored the capacity of novel iron chelators called ''exochelins'' to prevent reperfusion injury. Exochelins, siderophores of Mycobacterium tuberculosis, are unique iron chelators because they are lipid soluble, and hence able to enter cells rapidly. In the iron-free state, exochelins prevented ⅐OH formation. Desferri-exochelins prevented oxidative injury to cultured cardiac myocytes, and did so more rapidly and effectively than the nonlipid soluble iron chelator deferoxamine. The capacity of various desferri-exochelins to protect myocytes from oxidative injury varied directly with their solubility in lipid. Infused into isolated rabbit hearts during reperfusion after a period of ischemia, desferri-exochelins dramatically improved systolic and diastolic left ventricular function, preserved coronary f low, reduced release of the cardiac enzyme lactic dehydrogenase, and reduced myocardial concentrations of ⅐OH metabolites. Thus, highly diffusible desferri-exochelins block injury caused by ⅐OH production and have potential for the treatment of reperfusion injury.Reperfusion injury occurs in tissues that have been temporarily deprived of blood flow, as occurs in heart attack, stroke, and cardiopulmonary bypass and organs removed for transplantation. Paradoxically, although re-establishment of blood flow to ischemic tissue is necessary for its survival, reperfusion initiates a second phase of injury. In animal models of ischemia and reperfusion, up to 60% of the total damage to the heart (1) and 73% of the damage to the brain (2) is caused by processes initiated during reperfusion.

Mycobacterium avium secretes iron-binding siderophores called exochelins. The exochelins from M. avium have previously been reported to have unsaturated side chains that terminate in carboxylic acid. In contrast, our data show the side chains to be both saturated and unsaturated and to terminate with either a carboxylate or methyl ester.Mycobacterium avium is a slowly growing mycobacterium and an important human pathogen. Because M. avium is one of the most common opportunistic pathogens associated with AIDS, the prevalence of M. avium infection has skyrocketed in recent years (5).Like many pathogens, M. avium produces high-affinity ironbinding molecules known as siderophores to help it acquire iron in the host (9). Free iron is extremely limited in the host because of the high affinity with which it complexes to host iron-binding proteins. Other investigators (2,7,8,10) have shown that mycobacteria produce small water-soluble siderophores called exochelins. They have proposed that exochelins bind iron in the extracellular environment and deliver the iron to mycobactin, another high-affinity iron-binding molecule, which is located in the cell wall (or envelope) of the mycobacterium (8). Gobin and Horwitz have recently demonstrated that the exochelins of Mycobacterium tuberculosis remove iron from human transferrin and lactoferrin and donate iron to mycobactins in living intact organisms (3).Exochelins can be classified into two general categories depending on their extractability into organic solvents (11). Saprophytic mycobacteria produce exochelins which cannot be extracted into any organic solvent. In contrast, slowly growing, pathogenic mycobacteria, like M. tuberculosis and M. avium, produce exochelins that are extractable into chloroform (2). Mycobactins have been extensively studied (14), but because of purification problems, physiological and structural studies of exochelins have only recently been performed. The structures of chloroform-insoluble exochelins from the nonpathogens Mycobacterium smegmatis (13) and Mycobacterium neoaurum (12) have recently been described. These structures differ greatly from those of the chloroform-soluble exochelins isolated from the pathogens M. tuberculosis (4) and M. avium (6). More precisely, the exochelins of the nonpathogens are peptides, whereas the exochelins from the pathogenic mycobacteria resemble mycobactins and contain both amino acid and non-amino acid moieties. Although both our group and Lane et al. (6) found that the core structure of the exochelins of the mycobacteria resembles that of the mycobactins, the results of our two groups differ with respect to the R 1 alkyl side chain, which critically influences the water solubility of exochelins and allows them to function in the extracellular environment. Whereas we previously had reported that the R 1 alkyl side chain of the exochelins of M. tuberculosis exists in both saturated and unsaturated forms that terminate predominantly with a methyl ester but additionally with a free carboxylic acid, Lane et al. (...