TEXT

Top Abstract Text References

Exochelins are water-soluble iron siderophores secreted by mycobacteria to scavenge iron from their extracellular milieu (9, 10). Mycobacteria produce two general types of exochelins (12). The pathogenic mycobacteria produce chloroform-soluble exochelins which have a structure similar to that of mycobactin, another high-affinity iron-binding molecule of mycobacteria that is water insoluble and is located in the cell wall (1). The saprophytic mycobacteria produce chloroform-insoluble exochelins which have a peptide-like structure (14, 15).

In previous studies, we have isolated and characterized by mass spectrometry (MS) the exochelins of the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium avium (7, 20). These mycobactin-like exochelins contain 3 amino acid residues2 N-hydroxylysines and 1 serine or threonine. In M. avium, the 3rd amino acid is always threonine (20). The difference between the exochelins and mycobactins of the same species resides exclusively in the R₁ side chain (7). In mycobactins, the R₁ side chain is a long-chain fatty acid. In exochelins, the side chain is a shorter aliphatic chain, either saturated or unsaturated, that terminates in either a methyl ester or a carboxylic acid moiety. These differences render the exochelins more polar than mycobactins and hence water soluble.

Exochelins of the pathogenic mycobacteria are thought to function by scavenging iron from host iron-binding molecules and transferring the iron to mycobactins associated with the cell wall. Exochelins of M. tuberculosis have been shown capable of removing iron from human transferrin and lactoferrin and donating it to mycobactins in the cell wall (6).

The exochelins of Mycobacterium bovis and its attenuated descendant bacillus Calmette-Guérin (BCG) have not been previously characterized. Therefore, in this study, we sought to isolate them and characterize them by MS.

M. bovis is a slow-growing pathogenic mycobacterium that belongs to the M. tuberculosis complex (M. africanum, M. bovis, M. microti, M. tuberculosis, and M. ulcerans). M. bovis is highly related to M. tuberculosis on the basis of DNA homology (19). M. bovis derives its importance from several factors. First, as the primary causative agent of bovine tuberculosis, it is of major commercial importance to the cattle and dairy industry. Second, it can cause human tuberculosis, mostly via ingestion of unpasteurized cow's milk (5). With the advent of pasteurization and government programs to eradicate bovine tuberculosis, such cases are now rare in immunocompetent individuals. However, human immunodeficiency virus-infected persons appear to be at increased risk for such disease (2, 13, 16, 21). Third, M. bovis BCG remains the only available vaccine against tuberculosis. Although its efficacy has been questioned, the live vaccine has been administered to nearly 3 billion persons since 1945 (4). BCG is also administered intravesicularly in the treatment of bladder cancer. In rare cases, BCG can cause serious and sometimes fatal disseminated disease in immunocompromised persons (3, 8).

Isolation and purification of M. bovis exochelins. We selected for study the M. bovis type strain (ATCC 19210) and five BCG substrains of different geographic origins that are currently being used by pharmaceutical companies in vaccines or for research purposes: Copenhagen (ATCC 27290), Glaxo (ATCC 35741), Japanese (ATCC 35737), Pasteur (ATCC 35734), and Tice (ATCC 35743). To obtain exochelins of M. bovis and M. bovis BCG, we cultured the bacteria in modified iron-deficient Sauton's broth medium (1 µM iron; no Tween) in 1.9-liter tissue culture flasks, 300 ml per flask, without shaking at 37°C in 5% CO₂-95% air for 3 to 8 weeks (7). All bacteria were grown in iron-deficient medium to enhance exochelin production, from an A₅₄₀ of 0.05 to an A₅₄₀ of >1. We purified the exochelins at a point at which production was maximal6 weeks for M. tuberculosis and 8 weeks for M. bovisas previously described (7). Briefly, the supernatant fluid was saturated with iron, and ferri-exochelins were extracted into chloroform. The chloroform extract was dried, and the exochelins were purified by reverse-phase high-pressure liquid chromatography (HPLC) on a C₁₈ column (Vydac, Western Analytical, Temecula, Calif.) with a 50 to 100% gradient of buffer B (0.1% trifluoroacetic acid-50% acetonitrile) at a flow rate of 1 ml/min. Individual exochelins were further purified on an alkyl phenyl column (Waters, Bedford, Mass.) by using the same buffers and flow rate.

MS analysis of exochelins. Peaks isolated from the HPLC and identified as potential exochelins were first subjected to mass analysis by matrix-assisted laser desorption ionization MS. For these experiments, a Voyager linear time-of-flight (TOF) mass spectrometer (PerSeptive Biosystems, Framington, Mass.) equipped with a 337-nm N₂ laser was used. M. bovis exochelins were analyzed under delayed-extraction conditions as described by Vestal et al. (17). The improved optics of delayed extraction allowed for increased resolving power (1,500 M/M) and isotopic resolution of the exochelin molecular ions. Data were recorded with a 500-MHz digitizer. Samples were concentrated under a vacuum, and small aliquots (1 ml) were mixed 1:1 with the matrix (a saturated solution of -cyano-4 hydroxycinnamic acid in 70% acetonitrile-0.1% trifluoroacetic acid). The instrument was externally calibrated by using a mixture of standard peptides consisting of angiotensin II and bombesin.

Characterization of the exochelins of M. bovis and M. bovis BCG. As with M. tuberculosis Erdman and H37Ra, the chloroform extract of the culture filtrate of the M. bovis type strain contained a large family of exochelins. More than 20 peaks exhibiting a high A₄₅₀ eluted from the C₁₈ reverse-phase HPLC column (Fig. 1). Based on the similarity of their elution pattern to that of exochelins from M. tuberculosis (Fig. 1) (7), we tentatively identified 18 of these peaks as ferri-exochelins and subsequently confirmed that they were exochelins by MS analysis (only the major peaks were analyzed). The patterns of elution were very similar at 3, 6, and 8 weeks of culture (data not shown). The yield was highest at 8 weeks (750 µg/liter).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Elution profile of an 8-week culture filtrate from the M. bovis type strain (top) and of a 6-week culture filtrate from M. tuberculosis Erdman (bottom) on a C18 reverse-phase HPLC column. The chloroform extract of 1 liter of the M. bovis culture filtrate or 750 ml of the M. tuberculosis culture filtrate was loaded onto the column. Iron-binding molecules were monitored at 450 nm. The dashed line in each graph represents the concentration of buffer B. Each labeled peak was analyzed by MS and shown to contain an exochelin. Exochelin designations are given above each peak (see the legend to Fig. 3).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Elution profiles of 8-week culture filtrates from M. bovis BCG Copenhagen, Glaxo, Japanese, Pasteur, and Tice on a C18 reverse-phase HPLC column. The chloroform extract of 250 ml of each culture filtrate was loaded onto the column. Iron-binding molecules were monitored at 450 nm. Peaks 1, 2, and 3 correspond to exochelins 772TM, 800SC, and 814SC, respectively.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   General structure of exochelins of the M. bovis type strain. The exochelins of M. bovis differ from each other at R1 and R3. R1 is either saturated or singly unsaturated and terminates with either a methyl ester (COOCH3) or a carboxylic acid (COOH) moiety. R3 is either H or CH3. The exochelins of M. bovis and M. tuberculosis display the same variations at R1 and R3 and also are identical at R4 and R5. The exochelins of M. avium display the same variations at R1 as M. bovis and M. tuberculosis, but R3 is always CH3. In addition, M. avium differs from the other two species at R4, which is CHCH2CH3, and R5, which is CHCH3. The exochelins are named according to (i) their mass in daltons in the iron-loaded form, (ii) whether R3 is H (serine) (S) or CH3 (threonine) (T), and (iii) whether R1 terminates in a methyl ester (M) or carboxylate (C) moiety.

Conclusions. This study demonstrates that the pathogenic mycobacteria M. tuberculosis and M. bovis produce the same set of iron-binding exochelins. Moreover, BCG, an attenuated strain of M. bovis, produces the same set of exochelins as the pathogenic mycobacteria M. tuberculosis and M. bovis. There are only two differences between the exochelins of M. tuberculosis and M. bovis among the strains studied. One difference is in the overall amount of exochelins produced: greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG. The second difference is in the relative amounts of specific exochelin species produced, as each mycobacterial species and strain has its own unique pattern of exochelin production.