Resistance mechanisms of Mycobacterium tuberculosis against phagosomal copper overload

Abstract

Mycobacterium tuberculosis is an important bacterial pathogen with an extremely slow growth rate, an unusual outer membrane of very low permeability and a cunning ability to survive inside the human host despite a potent immune response. A key trait of M. tuberculosis is to acquire essential nutrients while still preserving its natural resistance to toxic compounds. In this regard, copper homeostasis mechanisms are particularly interesting, because copper is an important element for bacterial growth, but copper overload is toxic. In M. tuberculosis at least two enzymes require copper as a cofactor: the Cu/Zn-superoxide dismutase SodC and the cytochrome c oxidase which is essential for growth in vitro. Mutants of M. tuberculosis lacking the copper metallothionein MymT, the efflux pump CtpV and the membrane protein MctB are more susceptible to copper indicating that these proteins are part of a multipronged system to balance intracellular copper levels. Recent evidence showed that part of copper toxicity is a reversible damage of Fe-S clusters of dehydratases and the displacement of other divalent cations such as zinc and manganese as cofactors in proteins. There is accumulating evidence that macrophages use copper to poison bacteria trapped inside phagosomes. Here, we review the rapidly increasing knowledge about copper homeostasis in M. tuberculosis and contrast those with similar mechanisms in Escherichia coli. These findings reveal an intricate interplay between the host which aims to overload the phagosome with copper and M. tuberculosis which utilizes several mechanisms to reduce the toxic effects of excess copper.

Fig. 1. Copper homeostasis in macrophages

A. In resting macrophages CTR1 imports Cu(I) which is bound by the chaperone ATOX1. ATP7A is localized to the Golgi Network to deliver Cu to copper-requiring proteins. B. Under copper-stress ATP7A is trafficked to the membrane to export excess copper, but its transcription and translation are not increased. C. Initially upon phagocytosis into macrophages, M. tuberculosis suppresses phagolysosome formation. D. Once infected macrophages are activated with IFN-γ, phagolysosome fusion proceeds, the pH of the M. tuberculosis containing vacuole is reduced to pH4.5, CTR1 and ATP7A expression is increased and ATP7A is trafficked to the phagolysosome.

Fig. 2. Copper homeostasis in M. tuberculosis and E. coli

The thicknesses of the membranes were drawn according to measurements derived from cryo-electron microscopy. The size of the periplasmic space in mycobacteria is larger compared to E. coli, but is not shown proportionally in this figure. A. In M. tuberculosis outer membrane proteins involved in copper uptake or efflux are unknown. CtpV is an inner membrane (IM) transporter which likely functions as a copper efflux pump and whose expression is regulated by CsoR (PDB: 2HH7). Rv0846c is a putative multi-copper oxidase. The exact localization of the membrane protein Rv1698 (MctB) which reduces intracellular copper levels is unknown. MymT encodes a cytoplasmic copper metallothionein whose expression is regulated by RicR. Other genes in the RicR regulon (rv2963, socAB and lpqS) have undetermined functions. RicR and CsoR are both repressors which are induced by copper binding. B. In E. coli the tripartite efflux pump CusABC transports copper from the cytoplasm to the extracellular space (CusAB, PDB: 3NE5; CusC, PDB: 3PIK). CusF (PDB: 2VB2) may act as a periplasmic copper chaperone. CueO (PDB 1KV7) is a multi-copper oxidase and CopA is an IM copper transporting P-type ATPase. The two-component regulator CusSR activates transcription of cusCFBA, but does not regulate its own transcription. CueR (PDB 1Q05) binds copper to activate transcription of copA and cueO. Molecular structures were prepared using the UCSF Chimera program.