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Molecular Microbiology (1998) 28(3), 403–412 MicroReview Metal ion homeostasis and intracellular parasitism Daniel D. Agranoff and Sanjeev Krishna* Division of Infectious Diseases, Department of Cellular and Molecular Sciences, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. Summary Bacteria possess multiple mechanisms for the transport of metal ions. While many of these systems may have evolved in the first instance to resist the detrimental effects of toxic environmental heavy metals, they have since become adapted to a variety of important homeostatic functions. The ‘P’-type ATPases play a key role in metal ion transport in bacteria. A Cu þATPase from the intracellular bacterium Listeria monocytogenes is implicated in pathogenesis, and similar pumps in Mycobacterium tuberculosis and M . leprae may play a comparable role. Intracellular bacteria require transition metal cations for the synthesis of superoxide dismutases and catalases, which constitute an important line of defence against macrophage-killing mechanisms. The macrophage protein Nramp1 , which confers resistance to a variety of intracellular pathogens, has also been shown recently to be a divalent amphoteric cation transporter. Mycobacterial homologues have recently been identified by genomic analysis. These findings suggest a model in which competition for divalent cations plays a pivotal role in the interaction between host and parasite. Introduction The dynamic relationship between pathogenic bacteria and their hosts is an area of profound medical and scientific interest. The long-recognized phenomenon of ‘nutritional immunity’, in which sequestration of iron and possibly other metals occurs as a non-specific host response to infection (Beisel, 1977), hints in general terms at the possibility of keen competition between host and parasite for essential metal ions. However, much of the extensive literature on metal ion homeostasis in bacteria has addressed Received 22 November, 1997; revised 20 January, 1998; accepted 23 January, 1998. *For correspondence. E-mail s.krishna@sghms. ac.uk; Tel. (0181) 725 5836; Fax (0181) 725 3487. Q 1998 Blackwell Science Ltd itself to mechanisms of environmental heavy metal resistance (Silver and Phung, 1996), and it is not clear how, if at all, this relates to pathogenicity. The antiquity of major metal transporter families is evident from their high degree of sequence, and presumably functional, conservation between bacteria and complex eukaryotes. Bacterial resistances to a wide variety of toxic heavy metal ions, including Agþ, AsO2– , AsO43– , Cd 2þ, Co 2þ, CrO42– , Cu 2þ, Hg2þ, Ni 2þ, Pb 2þ, Sb 3þ, TeO32– , Tl þ and Zn 2þ, are well recognized, and their genetic determinants are frequently plasmid borne (Silver and Phung, 1996). Four broad strategies for minimizing toxicity from metal ions are discernible in bacteria. These involve: (i) the ATP-driven efflux pumps comprising the ‘P’-type ATPases and ATP-binding cassette (ABC) superfamilies (examples are the copB Cu þ efflux and the cadA Cd 2þ /Zn 2þ efflux ‘P’-type ATPases of Enterococcus hirae and Staphylococcus aureus , respectively, and the ABCbased arsenic efflux pumps in a number of Gram-positive and Gram-negative bacteria); (ii) chemiosmotic systems (metal ion/proton antiporters), in which the energy for the secondarily active transport of the metal ion is provided by proton gradients, e.g. the Cd 2þ, Zn 2þ, Co2þ (Czc) threecomponent systems found in the soil bacteria Alcaligenes spp.; (iii) redox systems, of which the merA mercuric reductase of Pseudomonas aeruginosa is the prototype; and (iv) metallothioneins – small metal-binding proteins previously believed to be confined to animal cells but recently identified in the cyanobacterium Synechococcus (Silver and Phung, 1996). Over the course of evolution, these systems have been selected to perform physiological functions extending beyond those in bacteria. Thus, for example, the proteins associated with human copper metabolism, whose genes are mutated in the copper transport disorders Menkes and Wilson’s diseases, are copper-transporting ‘P’-type ATPases that bear a closer resemblance to bacterial heavy metal ‘P’-type ATPases than they do to other mammalian ATPases (Bull and Cox, 1994). In recent years, it has become apparent that the regulation of metal ions plays a key role in a much broader sphere of bacterial homeostasis. These areas include the generation of electrochemical ionic gradients across membranes, which, in turn, provide the driving force for the secondarily active transport of metabolic precursors; the regulation of 404 D. D. Agranoff and S. Krishna cell volume, which depends on the control of intracellular K þ in response to changes in the osmolality of the external milieu; and the regulation of trace metal ions such as copper, manganese, zinc and iron, which are essential components of metalloenzymes necessary for the survival of both prokaryotic and eukaryotic cells. Copper, for example, is an essential cofactor in a variety of redox enzyme systems, including lysyl oxidase, cytochrome c oxidase and several bacterial superoxide dismutases (SODs), as are iron, zinc and manganese (Sehn and Meier, 1994). Iron is located at the active centre of catalase, an enzyme important for the disposal of toxic peroxide anions. These redox systems protect the organism against reactive oxygen intermediates, which may be generated either endogenously during aerobic respiration or exogenously by neutrophils or macrophages, the preferred host cell targets of a number of medically important human intracellular pathogens, such as Mycobacterium tuberculosis and M . leprae , Legionella pneumophila, Listeria monocytogenes , Mycoplasma spp. and Chlamydia spp. Exposure to such metal ions, however, is a double-edged sword, for it is precisely those redox properties harnessed in the context of SODs that, in other circumstances, render them highly toxic through interference with the functioning of intracellular macromolecules and the generation of toxic free radicals. Thus, mechanisms for metal ion homeostasis or, more specifically, metal ion transport may constitute major adaptations to intracellular survival and replication among pathogenic bacteria. These concepts are reinforced by new findings (Gunshin et al., 1997) which indicate that Nramp1 (natural resistanceassociated macrophage protein), which is associated with resistance to a variety of unrelated bacterial and protozoan intracellular pathogens (Vidal et al ., 1995), is probably itself a heavy amphoteric metal ion transporter. The ‘P’-type ATPases, a ubiquitous and highly conserved superfamily of cation transporters, are also prime candidates for a central role in intracellular survival in addition to their housekeeping functions. Indeed, recent evidence for the role of a putative copper-importing ‘P’-type ATPase as a virulence factor in L . monocytogenes (Francis and Thomas, 1997a,b) points to their potential importance for intracellular pathogens. Knockout mutants exhibited no phenotypic impairment when grown in competition with wild types in liquid media or in J774 or HeLa cell monolayers but were cleared much more rapidly from the tissues of infected mice. Thus, it now becomes possible to bridge the conceptual gap between the hitherto ill-defined importance of metal ions in host resistance and bacterial pathogenicity. ‘P’-type ATPases The ‘P’-type ATPases are fundamental components of the cell’s homeostatic apparatus. They constitute a large, ubiquitous and diverse superfamily of integral membrane proteins, containing 8–10 hydrophobic membrane-spanning helices (Moller et al., 1996). Their core structure is conserved throughout the three major domains of living organisms: the Archaea, Prokaryota and Eukaryota. Most are involved in the transport of cations across cellular and subcellular membrane systems. Identified substrate specificities encompass Naþ, K þ, Hþ, Ca 2þ, Mg 2þ, Cu þ, Cd 2þ and Zn 2þ as well as aminophospholipids (Tang et al ., 1996). Their common biochemical characteristics include the utilization of energy derived from hydrolysis of the terminal pyrophosphate bond of ATP for the vectorial transport of cations across the cell membrane and a dual-energy state catalytic cycle involving a phosphorylated acyl-intermediate (hence ‘P’ type). The basic mechanism of ion translocation has been worked out for the Naþ /K þ and sarcoplasmic reticulum Ca 2þ ATPases and probably applies in general terms to all ‘P’-type ATPases. ATP-dependent phosphorylation at a well-defined site on the cytoplasmic side of the protein is coupled with binding and occlusion of one or more intracellular cations. This generates a highenergy intermediate and induces conformational changes causing translocation of the cation across the membrane. Binding of an extracellular counterion to the now lowenergy-state protein is associated with dephosphorylation and transport of this ion into the cell. The orientation of the ATPase in the membrane is, therefore, defined by the fact that ATP is only likely to be available intracellularly. Consequently, as ATP-dependent phosphorylation appears to be a common feature of ‘P’-type ATPases, those pumps whose function appears to be the uptake of a physiologically important ion (see below) must presumably export another intracellular cation. In many cases, this secondary ion has yet to be identified. Once thought to be poorly represented in bacteria, recent genome-sequencing initiatives have to date identified more than 50 bacterial ATPase sequences, including representatives of all the major subgroups. Although highly divergent in many regions, they display several extraordinarily wellconserved amino acid motifs at the phosphorylation and phosphatase sites and at the ATP-binding and cation transduction domains. These sequences can be regarded as signature motifs essential for classification (Fig. 1). ‘P’-type ATPases are subclassified on the basis of cation specificity and membrane topology as proposed by Lutsenko and Kaplan (1995). The subgroups encompass the putative heavy metal transporters such as E . hirae copA (designated type I ATPases), a second subgroup, designated type II ATPases [the Naþ /K þ and Hþ /K þ ATPases of animals, the sarco(endo)plasmic reticulum calcium ATPases (SERCA), plasma membrane calcium ATPases (PMCA), the proton ATPases of fungi and lower plants (HA) and aminophospholipid translocases], and the Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 Bacterial ion transport 405 Fig. 1. Summary of alternative nomenclatures for the ‘P’-type ATPases with schematic representations of the three subclasses of bacterial ATPases showing their proposed membrane topologies, configuration of putative membrane-spanning helices and conserved motifs. Note that the type I ATPases possess an additional pair of transmembrane helices in their N-terminal region, while the type II proteins have a much extended C-terminus, accommodating 4–6 transmembrane helices. The several designations for each subclass have been proposed by various authors on the basis of different criteria, i.e. membrane topology, function, motifs etc., but are essentially overlapping. The motifs conserved among all subclasses are the ‘TGE’ or phosphatase site, the proline residue in the M4 transmembrane helix flanked by subclass-specific residues (Cpx, PEGL, PTTI) believed to form part of the ion channel, the phosphorylation site motif DKTGTXT, and the hinge region motif, GDGXND, forming part of the ATP-binding domain. GMXCXXC is the heavy metal binding motif occurring in many but not all type I ATPases. M1–M6, transmembrane helices. SERCA, sarco(endo)plasmic reticulum calcium ATPase; PMCA, plasma membrane calcium ATPase; HA, proton ATPase of fungi and lower plants. high-affinity K þ uptake ATPase exemplified by the kdpB protein found only in bacteria (type III ATPases). Among bacterial sequences, cation specificities for Cd 2þ, Zn 2þ, Cu þ/2þ, Agþ and Mg 2þ have been demonstrated, while presumptive Hþ and Ca 2þ transporters have been recognized on the basis of sequence homology. Many remain functionally uncharacterized. The majority of bacterial sequences are monosubunit members of the type I or heavy metal subclass and are probably involved in tolerance to heavy metal ions or in the scavenging of essential trace metals. Whether this apparent predominance of type I pumps is a true reflection of the distribution of ‘P’type ATPases in bacteria or whether it is an artifact of selective approaches to cloning will be clarified by the systematic sequencing of bacterial genomes currently in progress. Functional studies on bacterial ‘P’-type ATPases In this section, we review those ‘P’-type ATPases that have been investigated experimentally. Table 1 summarizes the properties of all ‘P’-type ATPases identified in the 10 prokaryotic genomes sequenced to date, as well as those of other functionally characterized ATPases discussed below. The E. coli KdpB ATPase The inducible K þ uptake system of E . coli has been well Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 characterized (Altendorf et al ., 1992) and consists of a complex of three proteins, kdpABC , which are encoded as a single operon. kdpA contains a putative periplasmic K þ binding site, while kdpC may be analogous to the beta subunit of the mammalian heterodimeric Naþ /K þ ATPase. The catalytic subunit, kdpB , is a ‘P’-type ATPase. K þ transport by kdpB is electrogenic (Fendler et al ., 1996). Homologues in numerous other Gram-negative bacteria, one Gram-positive bacterium, Bacillus acidocaldarius (Treuner-Lange et al ., 1997), and in M . tuberculosis (accession no. Z92539) have been identified. kdpB differs sufficiently from the type I and type II ‘P’-type ATPases by virtue of its membrane topology (see Fig. 1) to merit its inclusion in a subclass of its own (the type III ‘P’-type ATPases). The kdpABC system is a high-affinity system (reflected by a K m for K þ of 2 mM) for the uptake of K þ at low external K þ concentrations, in contrast to the constitutively active TrK and Kup systems with only a modest affinity for K þ (K m 1.5 mM). In this respect, it resembles several of the other bacterial ‘P’-type ATPase-based ion transport mechanisms in providing a recruitable and tightly regulated system for reinforcing existing constitutive transporters under extreme or rapidly changing environmental conditions. It is likely that the kdp system plays a role in the regulation of cell volume, as the transcriptional regulation of the kdp operon is stimulated by low, and inhibited by high, turgor pressures (Epstein, 1992). This regulatory system appears to be mediated by two proteins, kdpD and KdpE , which are encoded as an adjacent operon copA hpcopA copA cadA pacL mgtA cop1 cop2 cadA1 cadA2 pacL1 pacL2 pacL3 pma1 kdpB Haemophilus influenzae Helicobacter pylori Mycoplasma genitalium Mycoplasma pneumoniae Synechocystis sp. ykvW yvgW yvgX yloB Bacillus subtilis ATZN HRA-1 HRA-2 ATCU mgtA kdpB ORF Methanococcus jannaschii Escherichia coli pacS copB ATPase name Archaeoglobus fulgidus A: Completed genomes Organism D90904 D90915 D64005 D64005 D64005 D90911 D90905 D90910 D90910 MG071 P47317 HP1072 HP1503 L46864 HI0290 U00039 U16658 U16659 U58330 U14003 K02670 BG13325 BG14105 BG14106 BG13384 U67563 AF0473 AF0152 Acc. no. Table 1. Properties of bacterial ‘P’-type ATPases. 745 780 642 721 945 953 972 905 690 872 874 746 789 687 723 733 732 722 835 899 683 637 702 803 890 805 805 691 No. amino acids 79.9 82.6 68.7 76.7 102.5 103.6 106.1 97.6 73 94.9 96.3 81.8 86.5 74.9 78.1 76.8 78.4 78.5 87.8 99.5 72.1 68.6 75.2 85.8 97.1 89.6 86.4 75.3 MW (kDa) I I I I II II II II III II II I I I I I I I I II III I I I II II I I Class GMTCQSC ?Cu þ/ 2þ ?Cu ?Cu 2þ ?Cd 2þ ?Cd 2þ ?Ca 2þ ?Ca 2þ ?Ca 2þ ?H þ ?K þ 2þ ?Mg GMRCAAC GMKCAGC – GMDCTSC – – – – – – – ?Ca 2þ 2þ GMTCTAC CSHC NLDCPDC Cu efflux? ?Cuþ /2þ ?Cd 2þ efflux þ /2þ GMDCAAC (MXHX/C/GX) Histidine-rich N-terminus GLSCGHC, GMSCASC – – ?Zn 2þ Unknown heavy metal Unknown heavy metal ?Cu 2þ ?Mg 2þ K þ uptake – GLDCSNC (GMTCAAC) × 2 – – ?H þ Unknown heavy metal Unknown heavy metal Unknown heavy metal ?Ca 2þ GMTCAMC Histidine-rich N-terminus Heavy metal binding motif Unknown heavy metal Cu 2þ Transport activity CPC CPC SPC CPC PEGL PEGL PEGL PEGL PTTI PEGL PEGL CPC CPC CPC CPC CPC CPH CPH CPC PEML PTTI SPC CPC CPC PEGL PAVL CPC CPH M4 motif SEHPLA TRHPLA SEHPIG STHPIA – – – – – – – SEHVIA LAHSAI STHPIA ANHPIA ATHPLA SSHPIA STHPLA SSHPLA – – SSHPLA SQHPIA SEHPLG – – SEHPIA SEHPIA HP motif et et et et et et et et et et al . al . al . al . al . al . (1997) (1997) (1997) (1997) (1997) (1997) al. (1997) al . (1997) al . (1997) al . (1997) Kaneko Kaneko Kaneko Kaneko Kaneko Kaneko Kaneko Kaneko Kaneko et et et et et et et et et al . al . al . al . al . al . al . al . al . (1996) (1996) (1996) (1996) (1996) (1996) (1996) (1996) (1996) Himmelreich et al . (1996) Fraser et al . (1995) Tomb et al. (1997) Tomb et al. (1997) Tomb et al. (1997) Fleischmann et al. (1995) Blattner Blattner Blattner Blattner Blattner Blattner Kunst Kunst Kunst Kunst Bult et al . (1996) Klenk et al . (1997) Klenk et al. 1997) References 406 D. D. Agranoff and S. Krishna Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 ctpA ctpB cadA copA copB cadA ctpA fixI mgtA mgtB cadA(p) cadA(c) pacS pacL ctaA ctpA ctpB yhho iod7 y71 2097 * 830 y39 y21 kdpB 781 751 723 727 745 651 654 757 903 909 727 805 748 927 791 856 797 709 Z74025 Z95210 Z92539 Z46257 Z46257 M90750 L13292 L13292 U15554 U15554 M36068 U07843 M57715 J04551 P4738 D16437 D16436 U04356 762 752 772 789 719 657 Z74410 Y07638 Z74025 Z79700 Z92771 AL021184 82.3 78.2 78.2 78.4 81.5 69.2 69.5 79.6 99.8 100.5 78.8 86.9 79.7 99.7 83.7 89.9 85 74.6 79.02 77.5 79.5 80.1 76.5 67.9 I I I I I I I I II II I I I II I I I I I I I I II II III heavy heavy heavy heavy heavy heavy heavy metal metal metal metal metal metal metal Unknown heavy metal Unknown heavy metal Cd 2þ efflux? Cu þ /Ag þ uptake Cu þ /Ag þ efflux Cd 2þ efflux Cu 2þ Unknown heavy metal Mg 2þ Mg 2þ Cd 2þ /Zn 2þ efflux Cd 2þ efflux Cu þ / 2þ efflux? ?Mg 2þ Cu þ / 2þ uptake? Unknown Unknown Unknown Unknown Unknown Unknown Unknown ?Ca 2þ ?H þ ?K þ GMSCSCC GMLCAAC GFTCANC GMTCANC (MXHXXMSGMXHSH) × 3 GFTCANC – NAYCGTC? – – – (GFSCANC) × 2 GMGCAAC – GMKCAGK – – – GMSCSAC GMSCAAC – – – CPC CPC CPC CPC CPH CPC CPC CPC PEML PEML CPC CPC CPC PEGL CPC PEGL PEGL PTTI CPC CPC APC CPC CPC SPC SEHAVA SEHSVA SQHPLA SEHPLG ANHPLA SQHPLA SEHPLA SRHPIA – – SQHPLA SQHPLA SEHPLA – SRHPLA SEHSVA SEHAMA SEHPLA SEHPIG SRHPLA SEHPLG SEHPLG – – – Fsihi and Cole (1995) Fsihi and Cole (1995) Ivey et al . (1992) Odermatt et al. (1993) Odermatt et al . (1993) Lebrun et al . (1994) Francis and Thomas (1997) Khan et al . (1989) Snavely et al . (1991) Snavely et al . (1991) Nucifora et al . (1989) Witte et al . (1986) Kanamaru et al. (1993) Kanamaru et al . (1993) Phung et al . (1994) – – – – – – – – – – Section A lists, by organism, all ‘P’-type ATPases identified in the 10 prokaryotic genomes completely sequenced at the time of writing, permitting comparison of ATPase number and diversity between genomes. Sequences were identified initially through the relevant genome sequencing project’s own open reading frame annotations and checked for completeness through our own similarity searches using the TBLASTN algorithm, which searches translations of all six reading frames against a query protein sequence. Section B summarizes additional sequences discussed in the text. Acc. no: EMBL/GenBank Accession number. No. amino acids, MW, calculated number of amino acids and molecular weight. Class refers to the classificatory scheme based on that of Lutsenko and Kaplan (1995) discussed in the text. The transport activity quoted for most of the proteins derived from the completed genome data is speculative and based solely on sequence similarity to proteins in which the cation specificity has been experimentally verified. The ‘heavy metal binding motif’, GMXCXXC (where X ¼ any amino acid residue), is characteristic of the N-terminal region of most type I ‘P’-type ATPases, although in several type I proteins this is replaced by a histidine-rich repeating motif, while in others no obvious heavy-metal binding region has so far been recognized. The M4 motif refers to the invariant proline residue (and subclass-defined adjacent residues) found in the transmembrane helix (M4) preceding the phosphorylation site motif, DKTGTXT. The ‘HP’ motif is another characteristic type I ATPase motif, located 34–43 residues downstream of the phosphorylated aspartyl residue. P, plasmid encoded; C, chromosomally encoded. *This sequence had only just appeared in the public domain at the time of writing and had not yet appeared on the EMBL/GenBank databases. Since then a further sequence (not shown) has been identified. B: Additional sequences Mycobacterium leprae Mycobacterium leprae Bacillus firmus Enterococcus hirae Enterococcus hirae Listeria monocytogenes Listeria monocytogenes Rhizobium melliloti Salmonella typhimurium Salmonella typhimurium Staphylococcus aureus Staphylococcus aureus Synechococcus sp. 7942 Synechococcus sp. 7943 Synechococcus sp. 7942 Mycobacterium tuberculosis Bacterial ion transport 407 408 D. D. Agranoff and S. Krishna and whose sequences place them in the sensor–effector family of bacterial regulators. Copper-transporting ‘P’-type ATPases There is genetic and biochemical evidence for coppertransporting ‘P’-type ATPases in Enterococcus hirae , Helicobacter pylori , Listeria monocytogenes and Synechococcus spp. In E . hirae , copA and copB form part of a single inducible operon that also incorporates two transcriptional regulators, copY and copZ , located upstream of the ATPase sequences (Odermatt et al ., 1993). copA exhibits 35% sequence identity to copB and, remarkably, 43% identity with the human Menkes disease gene, underscoring the probable role of both encoded proteins in copper transport. The N-termini copA and copB contain the heavy metal binding motifs GMXCXXC and MXHXXMSGXHS respectively. copA mediates Cu þ uptake, as demonstrated by increased Cu þ dependence in copA knockout mutants, whereas copB mediates Cu þ extrusion as demonstrated by enhanced sensitivity to the toxic effects of Cu þ in copB and copAB knockouts, increased Cu þ uptake by copB and copAB knockouts compared with wild-type cells and copA knockouts and the need for a functioning copB gene for the extrusion of 110Agþ (which substitutes for Cu þ ) from previously loaded cells (Odermatt et al ., 1993). Cu þ accumulation by everted membrane vesicles engineered to overexpress copB has been shown to be dependent on ATP hydrolysis and to be inhibitable by vanadate ions, confirming that copB is functionally a ‘P’type ATPase (Solioz and Odermatt, 1995). Knockout studies also show that copY is a transcriptional repressor and copZ a transcriptional activator of the copAB operon. Interestingly, both copY and copZ contain heavy metal binding motifs, reflecting their role in mediating the effect of ambient copper ions on gene expression. Helicobacter pylori and Listeria monocytogenes Cu-ATPases The complete genomic sequence of H . pylori reveals two putative Cu-ATPases, hpcopA and copA (see Table 1). Only hpcopA has been functionally characterized (Ge and Taylor, 1996) and forms part of a single operon with a short, downstream-encoded polypeptide, hpCopP , containing a heavy metal binding motif. Knockout mutants of hpcopA display reduced environmental copper tolerance, supporting a role for copA in copper extrusion. The ctpA gene of the intracellular pathogen L . monocytogenes also appears to be involved in copper homeostasis (Francis and Thomas, 1997a). Unusually, its product is shorter than other bacterial type I ‘P’-type ATPases, with a truncated N-terminus lacking a heavy metal-binding motif. This is reminiscent of the initial report concerning the H . pylori hpcopA gene, which was also thought to lack a portion of the N-terminal region (Ge et al ., 1995). However, no upstream sequence containing the heavy metal motif has so far been identified. Significantly, knockout mutants were cleared more rapidly from the livers of infected mice than wild-type organisms and were rapidly out-competed by wild types in mixed infections (Francis and Thomas, 1997b). This is the first direct evidence for the role of a ‘P’-type ATPase in virulence. Cadmium-transporting ‘P’-type ATPases The Cd 2þ efflux ATPases represent one of several bacterial mechanisms for Cd 2þ resistance. They occur widely in Gram-positive bacteria including Staphylococcus aureus , L . monocytogenes , H . pylori and Bacillus subtilis (the last two recognizable on the basis of homology only). S . aureus possesses a plasmid-borne cadA gene expressed as part of a single operon with a short upstream-encoded open reading frame, cadC , which may represent a metal ion-responsive transcriptional repressor, and a chromosomally encoded ‘P’-type ATPase, which lacks a cadC homologue (Silver et al ., 1993). cadA transports and confers resistance to both Cd 2þ and Zn 2þ, while the chromosomal gene product transports only Cd 2þ. There is direct biochemical evidence for ATP-driven cadmium transport by cadA in everted membrane vesicles of B . subtilis expressing cadA (Tsai et al ., 1992). The cadC,A operon is capable of conferring high levels of Cd 2þ resistance on B . subtilis (a cadmium-sensitive organism more readily manipulated than S . aureus ). cadC,A expression is inducible by Cd 2þ, Zn 2þ, Pb 2þ and Bi 3þ. Cd 2þ is its most powerful inducer. The amino acid sequences of the chromosomally encoded B . firmus cadA and the plasmid-encoded L . monocytogenes cadA are highly similar to those of S . aureus , and both are associated with cadC homologues. In L . monocytogenes , experimental evidence supports the role of cadA in Cd 2þ resistance as well as its induction by Cd 2þ, although it differs from its S . aureus homologue in not transporting Zn 2þ (Lebrun et al ., 1994). Magnesium- and calcium-transporting ‘P’-type ATPases Mg 2þ is the most abundant divalent cation in biological systems. It exhibits the largest hydrated ionic radius of all biological cations, while being the most charge dense in its unhydrated state. In this respect, in differs from the otherwise chemically closely related Ca 2þ. Salmonella typhimurium possesses two Mg 2þ-transporting ‘P’-type ATPases, mgtA and mgtB , both of which mediate Mg 2þ uptake, confer tolerance to Mg 2þ-limiting Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 Bacterial ion transport 409 environments and are tightly regulated by the external Mg 2þ concentration (Roof, 1994). Both bear a far closer resemblance to the eukaryotic SERCA and PMCA ATPases than to the bacterial heavy metal pumps and can clearly be classified as type II ‘P’-type ATPases (Fig. 1). They exhibit homologies to the eukaryotic SERCA ATPases in regions surrounding at least five of the six amino acid residues suggested by Clarke et al . (1989) as being critical for calcium binding and ion transduction in the rabbit SERCA pump. Differences with respect to certain key residues may reflect the different physicochemical properties of Mg 2þ and Ca 2þ. Although both appear to perform the same function, they differ significantly in terms of amino acid sequence (only 50% identity), kinetic parameters and regulation (both are inducible but to greatly differing extents). mgtA displays 91% identity with a putative Mg 2þ transporter in E . coli (mgtA ) (Burland et al ., 1995), suggesting that the two S . typhimurium genes are not the result of a recent gene duplication. Moreover, the mgtB locus occurs within a bicistronic operon, mgtCB , incorporating a smaller gene, mgtC , which encodes a protein essential for the full expression of the Mg 2þ-scavenging phenotype (Snavely et al ., 1991). A further feature of these transporters is the fact that, unlike all other biochemically characterized ‘P’type ATPases, the direction of cation transport is down rather than against the electrochemical gradient. It is unclear why an energy-dependent system should be required for this process. An important clue as to their function is provided by the observation that mgtB transcription is greatly increased during phagocytosis of a pathogenic S . typhimurium strain into the vacuole of an epithelial cell line (Portillo et al ., 1992). More recently, it has been shown that the mgtCB operon forms part of a pathogenicity island in S . typhimurium and that mgtC expression is essential both for intramacrophage survival and for virulence in a mouse model (Blanc-Potard and Groisman, 1997). The intramacrophage survival defect exhibited by mgtC knockout mutants can be rescued by exposure to Mg 2þ, indicating a central role for this ion in the interaction between macrophage and parasite in this instance. Six putative bacterial Ca 2þ ATPases have been identified, although this is an inference so far based on sequence homologies alone (see Table 1). Mycobacterial ‘P’-type ATPases The pathogenicity of the important human bacterial pathogens Mycobacterium tuberculosis and Mycobacterium leprae is at least in part related to their ability to grow and multiply within host macrophages. The intraphagosomal microcellular environment differs markedly from the extracellular milieu in terms of osmolality, metal ion Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 concentrations, pH and nutrient availability, demanding the modulation of the parasite’s homeostatic responses. Thirteen mycobacterial ‘P’-type ATPases have appeared on the databases: 11 in M . tuberculosis and two in M . leprae. Assignation of function is so far based on sequence homology alone, although we are attempting the functional characterization of some of these proteins. The apparently restricted number of ‘P’-type ATPases in M . leprae may be a reflection of the proteomic streamlining characteristic of obligate parasites, in which functional redundancy is kept to a minimum. This characteristic is seen throughout the M . leprae genome in which many members of paralogous gene families have become pseudogenes. Both of the M . leprae ATPases (ctpA and ctpB ) and 7 of the 11 M . tuberculosis proteins (ctpA, ctpB , ioD7 , yhho , y71 , 2097 and 830) are putative ‘heavy metal’ pumps. Sequence alignments place ctpA , ctpB and ioD7 in the Cu 2þ-transporting group, while yhho has some affinities with the Cd 2þ transporters. However, these inferences are highly tentative in the absence of functional data. M . tuberculosis also possesses a putative Ca2þ pump ( y39 ), a putative proton pump ( y21 ) and a kdpB ATPase (see Table 1). Nramp and Mramp – a new class of metal ion transporter The energy expended by one or more ‘P’-type ATPases in maintaining transmembrane electrochemical gradients is also likely to contribute to the functioning of secondarily active transporters of metal ions. The Nramp (natural resistance-associated macrophage protein) family are novel members of the latter class of transporter and constitute a group of homologous hydrophobic membrane proteins associated with resistance to infection in mice by such phylogenetically and immunologically distinct intracellular pathogens as Mycobacterium bovis (BCG), Salmonella typhimurium and Leishmania donovani . The mechanism is independent of immunological resistance. First identified in inbred populations of mice (Vidal et al ., 1995), homologues are now known from a wide variety of organisms, including humans, insects, plants, yeast (Cellier et al ., 1995; 1996) and, most intriguingly, the intracellular pathogens M . leprae and M . tuberculosis (accession nos. U15184 and Z95212 respectively). The latter, which we have designated ‘Mramp ’, was recently identified through the M . tuberculosis genome-sequencing initiative undertaken by the Sanger Centre, UK. Interestingly, no homologues have been identified in the completely sequenced genomes of free-living bacteria with the exception of E. coli . In mammals, two isotypes have been identified – Nramp1 and Nramp2 – the former restricted to cells of the macrophage/monocyte lineage, the latter expressed ubiquitously (Cellier et al ., 1996). 410 D. D. Agranoff and S. Krishna Fig. 2. Model (modified from Supek et al ., 1997; Gunshin et al ., 1997) illustrating the proposed competition between macrophage and intraphagosomal mycobacteria for divalent cations. It is envisaged that Nramp1 , located in the membrane of the early phagosome, functions as a metal ion/proton symporter, which deprives the mycobacteria of divalent cations by export from phagosomes. The mycobacteria may counter this process through the actions of Mramp and the type 1 ‘P’-type ATPases, located in the bacterial plasma membrane, that pump metal ions from the phagosome into the bacterium. The associated accumulation of protons by the mycobacteria could be countered by a putative bacterial proton efflux pump. Divalent cations are required by mycobacteria for the proper functioning of superoxide dismutase (SOD) and catalase (CAT), which serve as a defence against reactive oxygen intermediates such as the superoxide anion (O2¹ ) and hydroxyl radical (.OH) generated by the macrophage. X 2þ, divalent cation, probably Fe 2þ /Cu 2þ /Mn 2þ / Zn 2þ. Myc, Mycobacterium. Accumulating evidence points to the function of the Nramp family as divalent cation transporters of broad specificity (Gunshin et al ., 1997). Thus, the 10–12 hydrophobic membrane-spanning domains common to all members are highly conserved, and the amphipathic character of several of these domains is very suggestive of a transmembrane hydrophilic pathway. Secondly, a consensus transport motif common to a variety of bacterial periplasmic transporters (the prokaryotic binding protein-dependent transport signature) is located in the cytoplasmic loop after transmembrane segment 8 (Fig. 2). Thirdly, the yeast Nramp homologue, SMF1 , has been shown to be associated with high-affinity Mn 2þ uptake (Supek et al ., 1997). Most recently, a ubiquitously expressed rat homologue of Nramp2 (designated DCT1 ) has been shown through formal electrophysiological studies in Xenopus oocytes expressing DCT1 mRNA to function as a divalent cation/ proton symporter of broad specificity (Gunshin et al ., 1997). Fe 2þ uptake may be its main physiological role, although significant uptake of the other predominantly transition element cations, Zn 2þ, Mn 2þ, Cu 2þ, Co 2þ, Ni 2þ, Cd 2þ and Pb 2þ, was also demonstrated. Maximal uptake was observed at an extra-oocytic pH of 5.5. Competition for heavy metal cations may be related to intracellular survival The discovery that Nramp proteins are divalent cation transporters and the observation that many macrophage-inhabiting intracellular pathogens possess active cation-transporting mechanisms of their own permits a new conceptual synthesis. While the Nramp cation transport specificity is fairly broad, it is nevertheless restricted predominantly to the divalent cations of transition elements. Many of these have several stable oxidation states enabling them to take part in redox reactions. As discussed earlier, several of these metals are precisely those required for the functioning of the redox enzymes, superoxide dismutase and catalase. Parasite-encoded versions of these enzymes represent a major line of defence against the reactive oxygen intermediates generated by the macrophage during the ‘respiratory burst’, one of the major bactericidal mechanisms used by the host. Mycobacteria reside within the phagolysosome compartment of the macrophage, the arena in which many of the host’s killing mechanisms operate. This is a dynamic microenvironment, which alters as the phagosome progresses along a well-defined maturation pathway. M . tuberculosis arrests this maturation at the early phagosome stage, preventing the acidification of the phagosome below a pH of about 6.3. Other parasites, such as Leishmania spp., reside in a pH below 5.0 (Russell, 1995). Immunolocalization studies have demonstrated that Nramp1 is recruited to the early phagosome membrane (Gruenheid et al ., 1997). This strongly suggests that Nramp1 is involved in the modulation of the divalent cation Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 403–412 Bacterial ion transport 411 composition of the early phagosome, rendering it hostile to the parasite. It is therefore tempting to speculate that the mechanism of action of Nramp1 is to deplete the phagosome of the essential heavy metal cations required by the parasite for its own defences (Gunshin et al ., 1997). The parasite, in response, may compete against this deprivation using its own armoury of metal ion transporters, which, in the case of M . tuberculosis , may include Mramp and the several heavy metal ‘P’-type ATPases. The delineation of the relative contributions of Mramp and ‘P’-type ATPases to cation homeostasis in M . tuberculosis awaits experimental verification. The proposed macrophage–parasite interaction is illustrated in Fig. 2. Important questions requiring experimental elucidation include the functional characterization of Mramp (and its pH dependence), its localization (probably the mycobacterial plasma membrane), the relative orientations of Nramp , Mramp and the mycobacterial type I ‘P’-type ATPases in their respective membranes and the manner of their expression during infection. If Mramp and Nramp1 behave as proton/divalent cation symporters like DCT1 , then the relatively acidic intraphagosomal environment (even in M . tuberculosis -infected cells) would favour ion fluxes consistent with our model. Concluding remarks The recent identification of Nramp as a divalent cation transporter coupled with the discovery of complementary heavy metal transporters in important intracellular pathogens suggests that metal ion dynamics may prove to play a central role in the preimmunological interaction between macrophage and parasite. The experimental dissection of the details of these processes is an important objective that promises to provide fascinating insights into a fundamental area of biology and that may eventually lead to the identification of new therapeutic strategies. Acknowledgements D.A. is an MRC (UK) Clinical Training Fellow and S.K. is a Wellcome Trust Senior Research Fellow in Clinical Science. References Altendorf, K., Siebers, A., and Epstein, W. 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