Gallium Disrupts Iron Metabolism of Mycobacteria Residing within Human Macrophages

Abstract

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophoremediated 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 (Ga 3؉ ) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe 3؉ 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(NO 3 ) 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 for M. 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(NO 3 ) 3 disruption 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.

Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcγRII and the mannose receptor

“…Mononuclear cells were separated on a Ficoll (Amersham Biosciences) cushion and the PBMCs were cultured for 5 days in Teflon wells (Savillex) containing RH (RPMI medium, 20 mM HEPES, 20 mM -glutamine) plus 20 % autologous serum. The resulting MDMs were adhered to glass or plastic (see electron-microscopy section ; Waco Pure Chemical Industries) coverslips, or they were adhered directly to wells of a 24-well (Becton Dickinson Labware) or a 6-well (Corning) tissue-culture plate for 2 h. Non-adherent cells were removed from the plates by washing, and the MDMs were cultured for an additional 7 days in RH plus 20 % autologous serum, prior to the addition of Mtb (Olakanmi et al, 2000). Each experimental condition was performed with 2 donors.…”

Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

“…Infected monolayers were washed and either repleted with RH containing 1% human autologous serum and further incubated for 24, 48, or 72 h or lysed (0 h time point) as described (27). After the 0 time point, the supernatant (containing detached infected MDMs) and monolayers were lysed separately and then pooled.…”

“…Intracellular Growth of Strains Erdman and HN885 in Macrophages-For measurements of intracellular growth in macrophages, 12-day-old MDM monolayers were washed, and M. tuberculosis bacilli were added to MDMs (multiplicity of infection 1:1; duplicate wells) in either RH containing 2.5% serum or RHH and incubated for 2 h at 37°C in 5% CO 2 (27). Infected monolayers were washed and either repleted with RH containing 1% human autologous serum and further incubated for 24, 48, or 72 h or lysed (0 h time point) as described (27).…”

Preparation, structural characterisation and antibacterial properties of Ga-doped sol–gel phosphate-based glass

“…[2] It was also shown that gallium is effective in two murine lung infection models. Other studies have demonstrated that gallium is effective against the organisms causing tuberculosis [3] and malaria [4] in human beings, and in the treatment of Rhodococcus equi caused pneumonia in foals. [5] Recently, we have shown that phosphate-based glasses (PBGs) in the system CaO-Na 2 O-P 2 O 5 system provide a means to deliver Ga 3+ ions in a controlled way.…”