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Separation of 55Fe(lll)-exochelin-labelled envelope proteins from M. smegmatis by CHAPS-PAGE. A cell envelope fraction of M. smegmatis, grown iron-deficiently, was prepared by sonication and was separated from intact cells and the cytoplasmic fraction by differential centrifugation. The envelope fraction was incubated for 16 h at 4 "C with 8 mM CHAPS in buffer to extract protein. Soluble and insoluble material were then separated by centrifugation (105000g, 30 min, 4 "C). The soluble extract was analysed by CHAPS-PAGE chromatography on a gel (14x 16 cm) with composition 10% T (total monomer concentration) at 35 mA constant current for 2-3 h. The gel was fixed and visualized by silver staining. Lane A, 100 pg extracted envelope protein from iron-starved cells; lane B, 100 pg extracted envelope protein from iron-replete cells. Lane C: a ferri-exochelin-receptor protein complex was formed by incubation of a sam le of native envelope, nmol-'). The complex was then extracted using 8 mM CHAPS for 16 h at 4 "C. The complex was separated by CHAPS-PAGE on a 10% resolving gel at 35 mA for 2-3 h. The gel was then dried and visualized by autoradiography. containing 100 pg protein, with P Fe(ll1)-exochelin (8332 Bq
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Evidence of a direct association between ferri-exochelin, the major extracellular siderophore of Mycobacterium smegmatis, and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of 55Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a compl...
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Citations
... They demonstrated uptake of ferri-exochelin by M. smegmatis as an active transport process (Messenger and Ratledge 1982) and reported the identification of a 29-kDa iron-regulated protein as a potential receptor for ferri-exochelin (Hall et al. 1987). The 29-kDa protein was reported to co-elute with a 25-kDa band (Dover and Ratledge 1996) but further details on the identity of the two proteins are not known. It is suggested that the 29-kDa protein is the HupB homologue as it reacted with antibodies against HupB of M. tuberculosis (unpublished findings in our laboratory). ...
... Preparation of liposomes Cell wall fractions from high and low iron M. smegmatis were prepared as mentioned above. They were solubilized and incorporated into liposomes as described (Dover and Ratledge 1996). The non-ionic detergent CHAPS {3- dimethylammonio]-1-propanesul-fonate} was used to solubilize the cell wall proteins. ...
... In the pull down assay of the cell wall proteins of ironlimited M. smegmatis, the 21-kDa IrpA and the 29-kDa HupB were detected in the immunoblots developed with the respective antibodies and the corresponding gel (Fig. 4). This experiment was done based on the report of the association and co-elution of the 29-kDa protein with another protein (Dover and Ratledge 1996). The specificity of the anti-HupB antibodies for the 29-kDa protein is shown inFig. ...
Aims:
To characterize the 21 kDa iron-regulated cell wall protein in Mycobacterium smegmatis co-expressed with the siderophores mycobactin, exochelin and carboxymycobactin upon iron limitation.
Methods and results:
M. smegmatis, grown in the presence of 0.02 μg Fe ml-1 (low iron) produced high levels of all the three siderophores, which were repressed in bacteria supplemented with 8 μg Fe ml-1 (high iron). Exochelin, the major extracellular siderophore was the first to rise and was expressed at high levels during log phase of growth. Carboxymycobactin, a minor component in log phase iron-starved M. smegmatis continued to rise when cultured for longer periods, reaching levels greater than exochelin. Iron-starved bacteria expressed a 21 kDa iron-regulated protein (IrpA) that was identified as Clp protease subunit (MSMEG_3671) and characterized as a receptor for ferri-exochelin.
Conclusions:
Ferri-exochelin is the preferred siderophore in M. smegmatis and this ferri-exochelin: IrpA machinery is absent in M. tuberculosis.
Significance and impact of the study:
Exochelin machinery is functional in M. smegmatis and the carboxymycobactin-mycobactin machinery is the sole iron uptake system in M. tuberculosis. The absence of the ferri-exochelin: IrpA system in the pathogen signifies the importance of the carboxymycobactin-mycobactin system machinery in M. tuberculosis.
... In M. smegmatis, the 29 kDa IREP was shown as a potential receptor for the exochelin MS by specifically blocking its uptake using protein specific antibodies against the 29 kDa protein. This was further substantiated by the specificity of this protein as a ferri-exochelin receptor (Dover & Ratledge, 1996). In M. neoaurum, the 21 kDa IREP, ...
... Liposomes were prepared with cell wall proteins isolated from iron-limited and iron replete M. smegmatis and uptake of 55 Fe-exochelin was done essentially as per reported protocol (Dover & Ratledge, 1996) In order to study if the iron from the ferri-exochelin was transferred to mycobactin, test liposomes were prepared by including desferri-mycobactin. Iron was removed from the mycobactin by gently shaking the chloroform suspension with 1 mL of methanol and 1 mL of 6 M HCI. The wine red colour of the ferri-mycobactin was replaced with the colourless desferri-mycobactin in the chloroform layer. ...
... They were taken along with 100 nM desferri-Mb, dissolved in chloroform and dried as a thin film in an inert atmosphere using nitrogen gas were dried onto the walls of a test-tube. The following liposomes were prepared based as described (Dover & Ratledge, 1996) 1. Empty liposomes -these served as controls and were prepared by adding 1 mL of phosphate buffer to the dried lipid film and shaking vigorously for 30 s to get the milky suspension. ...
... A 29-kDa ferriexochelin receptor in M. smegmatis. Among several IREPs expressed by M. smegmatis, a 29-kDa protein was characterized as a receptor for ferriexochelin (51,52), both by demonstrating direct interaction of the purified protein with ferriexochelin MS and by inhibiting ferriexochelin-mediated iron uptake by preincubating live organisms with specific antibodies against the 29-kDa IREP. Several mycobacteria express this protein (53), and it was also identified in an in vivo-derived Mycobacterium avium strain isolated from the liver of experimentally infected C57 black mice (53). ...
Mycobacterium tuberculosis requires iron for its normal growth but faces limitation of the metal ion due to its low solubility at biological pH and the withholding of iron by the mammalian host. The pathogen expresses Fe 3+ -specific siderophores mycobactin and carboxymycobactin to chelate the metal ion from insoluble iron and the host proteins transferrin, lactoferrin and ferritin. Siderophore-mediated iron uptake is essential for the survival of M. tuberculosis as knock out mutants, defective in siderophore synthesis or uptake failed to survive in low iron medium and inside macrophages. But as excess iron is toxic due to its catalytic role in the generation of free radicals, regulation of iron uptake is necessary to maintain optimal levels of intracellular iron. The focus of this review is to present a comprehensive overview of iron homeostasis in M. tuberculosis that is discussed in the context of mycobactin biosynthesis, transport of iron across the mycobacterial cell envelope and storage of excess iron. The clinical significance of the serum iron status and the expression of the iron-regulated protein HupB in tuberculosis (TB) patients is presented, highlighting the potential of HupB as a marker, notably in extrapulmonary TB cases.
... At least five IREPs were detected in membrane extracts from M. smegmatis grown in iron-deficient conditions including the extensively studied 29 kDa IREP (Hall et al ., 1987). This protein can associate directly in vitro with ferriexochelin, and the addition of a polyclonal antiserum generated against it to M. smegmatis cells significantly inhibits ferri-exochelin-mediated iron uptake (Dover and Ratledge, 1996). Based on these observations, the 29 kDa protein has been postulated to be a ferri-exochelin receptor in M. smegmatis. ...
The role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron-rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron-mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron-dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.
... The mechanisms involved in the uptake and transport of Fe-containing siderophores across the mycobacterial membrane are not understood and are likely complex. They are thought to involve specific receptors and transport molecules (7,13,15,26). The efficient internalization of Fe 3ϩ is thought possibly to be linked to its reduction to the ferrous form either enzymatically via a reductase or nonenzymatically (13). ...
Mycobacterium tuberculosis and M. aviumcomplex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture
media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both
M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part
to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga3+) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by
substituting for Fe3+and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways
of mycobacteria. We find that Ga(NO3)3 and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal forM. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal
M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO3)3disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular
mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate
between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.
To obtain host iron, Staphylococcus aureus secretes siderophores staphyloferrin A (SA) or staphyloferrin B (SB), and accesses heme iron through use of iron-regulated surface determinant proteins. While iron transport in S. aureus is well documented, there is scant information about proteins required to access iron from complexes in the cytoplasm. In vitro studies identified a pyridine nucleotide-disulfide oxidoreductase, named IruO, as an electron donor for the heme monooxygenases IsdG and IsdI, promoting heme degradation. Here, we show that an iruO mutant was not debilitated for growth on heme, suggesting involvement of another reductase. NtrA is an iron-regulated nitroreductase and, as with the iruO mutant, a ntrA mutant grew on heme comparable to wildtype. In contrast, a iruO ntrA double mutant was severely debilitated for growth on heme, a phenotype that was complemented by expression of either iruO or ntrA in trans, demonstrating their overlapping role in heme-iron utilization. Contrasting the involvement of multiple reductases for heme iron utilization, ntrA was shown essential for iron utilization using SA, although not SB or other siderophores tested, and an iruO mutant was incapable deferoxamine-mediated growth. Accordingly, virulence of wildtype S. aureus, but not an iruO mutant, was enhanced in mice receiving deferoxamine.
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Background:
HupB is a 28 kDa cell-wall-associated protein co-expressed with the siderophores mycobactin and carboxymycobactin in iron-limited Mycobacterium tuberculosis. HupB is expressed in vivo and anti-HupB antibodies are present in the serum of TB patients.
Methods:
The aims of this study were to evaluate the serodiagnostic potential of HupB and to correlate levels of anti-HupB antibodies with the serum iron status in TB patients, household contacts and normal healthy controls.
Results:
TB patients from Hyderabad (India) showed high levels of anti-HupB antibodies compared with household contacts and normal healthy controls. Interestingly, the levels were maximal in extrapulmonary TB patients, with a two-fold higher titre than pulmonary TB patients. Serum iron levels, total iron-binding capacity (TIBC) and percent saturation of serum transferrin were low in subjects with active TB, whilst serum ferritin was notably high in pulmonary TB patients compared with normal controls.
Conclusions:
There is a strong negative correlation between serum iron levels and TIBC with the titre of anti-HupB antibodies in subjects with active TB. This study reflects the usefulness of screening for anti-HupB antibodies for diagnosis of pulmonary and extrapulmonary TB in this endemic region.
The 160, 190 and 270 kDa outer sheath proteases of Treponema denticola ATCC 35404 were found to be multiple forms of the major 91 kDa phenylalanine protease (PAP) by immunoblotting using anti-91kDa specific antibodies. Multiple forms of the phenylalanine protease were also found in 2 other T. denticola strains studied, ATCC 33520 and the clinical isolate GM-1. Protein, proteolytic and Western blot analyses using antibodies against the PAP and the major outer sheath protein (MSP) indicated that the 190 and 270 kDa proteases were protein complexes formed by the MSP and the PAP. These complexes dissociated by storage in 0.3% or higher SDS concentrations. The purified PAP was found to completely degrade keratin, but was unable to degrade native actin either in its monomeric or polymerized form. The association of the MSP adhesin with a protease capable of degrading host native proteins may benefit the obtention of protein-based nutrients necessary to support the growth of these treponemes. These complexes may also play a role in the structural organization of T. denticola outer sheath.
