Copper transport and trafficking at the host-bacterial pathogen interface

Abstract

CONSPECTUS: The human innate immune system has evolved the means to reduce the bioavailability of first-row late d-block transition metal ions to invading microbial pathogens in a process termed "nutritional immunity". Transition metals from Mn(II) to Zn(II) function as metalloenzyme cofactors in all living cells, and the successful pathogen is capable of mounting an adaptive response to mitigate the effects of host control of transition metal bioavailability. Emerging evidence suggests that Mn, Fe, and Zn are withheld from the pathogen in classically defined nutritional immunity, while Cu is used to kill invading microorganisms. This Account summarizes new molecular-level insights into copper trafficking across cell membranes from studies of a number of important bacterial pathogens and model organisms, including Escherichia coli, Salmonella species, Mycobacterium tuberculosis, and Streptococcus pneumoniae, to illustrate general principles of cellular copper resistance. Recent highlights of copper chemistry at the host-microbial pathogen interface include the first high resolution structures and functional characterization of a Cu(I)-effluxing P1B-ATPase, a new class of bacterial copper chaperone, a fungal Cu-only superoxide dismutase SOD5, and the discovery of a small molecule Cu-bound SOD mimetic. Successful harnessing by the pathogen of host-derived bactericidal Cu to reduce the bacterial load of reactive oxygen species (ROS) is an emerging theme; in addition, recent studies continue to emphasize the importance of short lifetime protein-protein interactions that orchestrate the channeling of Cu(I) from donor to target without dissociation into bulk solution; this, in turn, mitigates the off-pathway effects of Cu(I) toxicity in both the periplasm in Gram negative organisms and in the bacterial cytoplasm. It is unclear as yet, outside of the photosynthetic bacteria, whether Cu(I) is trafficked to other cellular destinations, for example, to cuproenzymes or other intracellular storage sites, or the general degree to which copper chaperones vs copper efflux transporters are essential for bacterial pathogenesis in the vertebrate host. Future studies will be directed toward the identification and structural characterization of other cellular targets of Cu(I) trafficking and resistance, the physical and mechanistic characterization of Cu(I)-transfer intermediates, and elucidation of the mutual dependence of Cu(I) trafficking and cellular redox status on thiol chemistry in the cytoplasm. Crippling bacterial control of Cu(I) sensing, trafficking, and efflux may represent a viable strategy for the development of new antibiotics.

Figure 1

(a) Fenton reaction;, (b) chemical structure of reduced glutathione (GSH), and (c) chemical structure of yersiniabactin (Ybt) from uropathogenic E. coli (UPEC) and proposed reaction mechanism for the Cu-dependent superoxide dismutase (SOD) activity of Ybt.

Figure 2

Pathways of copper transport, trafficking, sensing, and resistance in several well-studied bacterial pathogens, including the Gram positive pathogen S. pneumoniae, M. tuberculosis, and two similar Gram negative pathogens, E. coli and Salmonella spp., at the host–pathogen interface. Inset (upper left), cartoon representation of a host macrophage engulfing a bacterial pathogen, ultimately sequestered in an intracellular phagolysosomal compartment. The red box highlights the plasma, phagolysosomal, and outer/inner membranes of the bacterium (from left to right), expanded in the main body of the figure. E. coli CueO, plasmid-encoded PcoA, and mycobacterial MmcO are multicopper oxidases (MCOs). Both MmcO and an outer membrane channel MctB are required for mycobacterial copper resistance. CtpV is a copper exporting ATPase that is required for full virulence of M. turberculosis in murine models of infection, while Msp is a porin on the outer membrane of M. tuberculosis. Overexpression of Msp genes induces copper stress in M. turberculosis, consistent with a role in Cu uptake. Representative metalloregulatory proteins are also shown (right).^,,,

Figure 3

Molecular structures of (a) Cu(I)- and (b) Cu(II)-bound Candida albicans SOD5.

Figure 4

Molecular structures of two Cu(I)-specific metalloregulatory proteins: (a) Cu(I)-bound E. coli CueR; (b) Cu(I)-bound CsoR from Geobacillus thermodenitrificans. Subunits are differentially shaded with the Cu(I) binding sites circled and expanded (right).

Figure 5

Molecular structures of selected periplasmic copper homeostasis proteins: (a) Cu(I)-bound E. coli CusF; (b) Cu(I)/Cu(II) bound CopC.

Figure 6

Molecular structures of two cytoplasmic Cu(I) chaperones and the apo-structure of the Cu(I)-effluxing P_1B-type ATPase CopA from L. pneumophila. (a) B. subtilis CopZ, representative of the Atox1-like ferredoxin-like fold metallochaperones. The S–Cu–S bond angle of 120° suggests a third ligand from solvent to complete a trigonal coordination structure. (b) The Cu(I) chaperone CupA from S. pneumoniae that harbors a binuclear Cu(I) center. (c) L. pneumophila CopA with the proposed copper entry, transmembrane (TM-MBS), and exit sites indicated.^, The MA and MB helices are shaded cyan, with the actuator (A, green), nucleotide-binding (N, red) and phosphorylated (P, blue) domains also highlighted.

Figure 7

Cartoon of a mechanistic model of Cu(I) efflux across the bacterial inner membrane that summarizes recent structural and biochemical studies on CopA from E. coli, A. fulgidus and L. pneumophila. CopA is postulated to transport two Cu(I) ions per ATPase cycle. E. coli CusF has been shown to be metalated upon Cu(I) release from CopA through a protein–protein interaction.