Iron acquisition and metabolism by mycobacteria.

Iron is a prerequisite for in vitro growth of mycobacteria. Iron is an obligate cofactor for at least 40 different enzymes encoded in the Mycobacterium tuberculosis genome (8). It is required for the cytochromes involved in electron transport and other hemoproteins involved in oxygen metabolism, such as catalase-peroxidase KatG. Most of the intracellular iron in mycobacteria, however, is in the form of nonheme iron (48). One important form of nonheme iron is the iron-sulfur clusters that are cofactors of proteins involved in amino acid and pyrimidine biogenesis, as well as in enzymes involved in the tricarboxylic acid cycle and electron transport. Iron is also required for DNA synthesis in ribonucleotide reductase, superoxide dismutase, and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Rv2178c; DAHP synthase [Every gene in the genome of M. tuberculosis H37Rv has been assigned a gene identifier beginning with Rv which can be used to retrieve information on line; see reference 30a]). It has been estimated that 7 to 64 mg of Fe/g of mycobacterial cell mass is required to support growth (39, 48). Iron limitation in vitro to levels below these can result in growth restriction in many species of mycobacteria, including pathogens such as M. tuberculosis. Since iron is the fourth most abundant element, difficulty in its acquisition may seem counterintuitive. However, the extremely low solubility of ferric ion in aqueous solution makes obtaining it difficult and so microorganisms have evolved many strategies for acquiring sufficient soluble iron for aerobic growth. Iron sequestration represents a formidable challenge to in vivo growth of pathogenic mycobacteria. An important component of the mammalian host defense against bacterial pathogens involves restricting access of such organisms to iron (35). M. tuberculosis is a highly specialized pathogen of humans and must contend with iron sequestration in order to survive in the human lung. Pulmonary tuberculosis patients are often anemic, suggesting sequestration of available iron by the host (5). The serum of many mammals, including humans, is tuberculostatic because of its ability to sequester iron from the bacilli (32, 34). The addition of iron to such sera relieves the bacteriostatic effect completely. In a tragic attempt to rectify what was perceived as a debilitating iron deficiency in infected patients in Somalia, iron supplementation was found to actually promote the development of active tuberculosis (54). Other clinical studies in Africa have also established a strong correlation between dietary iron overload and an enhanced risk of death from tuberculosis (20, 52). Purified transferrin inhibits the growth of M. tuberculosis in vitro (33), and haptoglobin may play a role in restricting access to heme iron (29, 57). Thus, iron availability or sequestration and the bacterium’s efforts to circumvent this restriction are critical determinants of the outcome of infection with M. tuberculosis. Infections with mycobacteria of the M. avium complex (MAC) occur predominantly in human immunodeficiency virus-infected patients and are a major source of mortality for such patients. Iron storage in macrophages is known to be increased in patients with AIDS (although overall iron levels in serum are lower than those of patients without AIDS), and this observation has been proposed to underlie the predisposition of such patients to MAC infections (11, 12, 22). Animal model experiments support this hypothesis in that mice fed iron-rich diets more rapidly develop disease from MAC (11). In cultured human macrophages, the rate of MAC replication has been shown to be directly correlated with the concentration and iron loading of transferrin (12). Mycobacterial iron acquisition is mediated by siderophores. The problem of iron acquisition has been solved by many microorganisms, including mycobacteria, by producing small, soluble iron chelators known as siderophores. These substances are generally secreted by the organism to compete with environmental iron binding molecules for the small amount of available iron. Siderophore synthesis is typically controlled by extracellular iron abundance, and these molecules display a high affinity for ferric iron. Four types of structurally distinct molecules are thought to be involved in iron acquisition in mycobacteria, salicylic acid and citric acid being the simplest, followed by two classes of more complex siderophores. The first, the mycobactins (MBs), are distinguished primarily by the presence of a phenyloxazolidine ring, while the second class, the exochelins, are peptidic siderophores whose iron-chelating ability is associated with ornithine-derived hydroxamates. MBs and exochelins are complex molecules with very high affinities for iron and are integral to the bacterial strategy for obtaining iron from the environment. Early work focused on the dramatic accumulation of salicylic acid secreted into the growth medium coincident with iron starvation of either slow-growing or fast-growing mycobacterial species (60). Salicylate was postulated to be an important extracellular siderophore, but the stability of the salicylate-iron complex appears to be too low to support this hypothesis. However, the amount of salicylate secreted into growth media upon iron starvation reaches as much as 2.28 mg/100 ml of medium and this concentration suggests a secondary physiological role, perhaps in facilitating iron solubilization (60). Citric acid has also been proposed to be involved in iron acquisition in M. smegmatis on the basis of cell association of an iron-citrate complex (51). However, the very high concen* Corresponding author. Mailing address: Tuberculosis Research Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Twinbrook II, Rm. 239, 12441 Parklawn Dr., MSC 8180, Rockville, MD 20852-1742. Phone: (301) 435-7509. Fax: (301) 402-0993. E-mail: [email protected].