Manganese acquisition and homeostasis at the host-pathogen interface

Abstract

Pathogenic bacteria acquire transition metals for cell viability and persistence of infection in competition with host nutritional defenses. The human host employs a variety of mechanisms to stress the invading pathogen with both cytotoxic metal ions and oxidative and nitrosative insults while withholding essential transition metals from the bacterium. For example, the S100 family protein calprotectin (CP) found in neutrophils is a calcium-activated chelator of extracellular Mn and Zn and is found in tissue abscesses at sites of infection by Staphylococcus aureus. In an adaptive response, bacteria have evolved systems to acquire the metals in the face of this competition while effluxing excess or toxic metals to maintain a bioavailability of transition metals that is consistent with a particular inorganic "fingerprint" under the prevailing conditions. This review highlights recent biological, chemical and structural studies focused on manganese (Mn) acquisition and homeostasis and connects this process to oxidative stress resistance and iron (Fe) availability that operates at the human host-pathogen interface.

Keywords: ATP-binding cassette; homeostasis; iron; manganese; metal transport; nutritional immunity.

Figure 1

Inorganic chemistry of the cell. (A) Biologically important first-row transition metals extracted from the periodic table from Mn to Zn and with outer (3d) shell electronic configurations (d⁵–d¹⁰) as indicated. Zn(II) and Cu(I) both have filled d-shells. Coordination number (CN) preferences move from Mn to Cu(I) from high [CN = 6 for Mn(II)] to low [CN = 2–4 for Cu(I)], which tracks with increasing thiophilicity and polarizability of the metal. The approximate trend in metal(II) complex stability for a mixed N/O ligand donor set is indicated for each metal (Fraústo da Silva and Williams, 2001); this trend is inversely related to competitiveness in a cellular environment which itself is inversely related to “bioavailability” of each metal in the cell. Bioavailability is very roughly based on reported metal sensor affinities for their cognate metal and is not a direct measure (Reyes-Caballero et al., 2011). (B) Total cell-associated log (Mn:Fe ratio) plotted for individual bacteria as measured by ICP-MS. The term Mn-centric is operationally defined here as those organisms for which Mn:Fe ≥0.2, or the lower limit obtained for Deinococcus radiodurans under extremely Mn-depleted growth conditions (Daly et al., 2004). Fe-centric refers to those organisms for which Mn:Fe ≤0.2. Most of these analyses were obtained for exponentially growing cells on a rich growth medium (given in parentheses) with no added Mn(II), unless otherwise indicated. These measurements were taken for Lactobacillus plantarum (All Purpose Tween, APT) (Posey and Gherardini, 2000), Borrelia burgdorferi [Barbour-Stoenner-Kelly media (BSK-II) or Modified serum-free media with Exyte (SF-E)] (Posey and Gherardini, 2000), Deinococcus radiodurans (Defined Minimal media + 2.5 μM Mn) (Daly et al., 2004), Streptococcus pneumoniae (Brain Heart Infusion, BHI) (Jacobsen et al., 2011), Neisseria meningitidis (Gonococcal Broth, GCB) (Veyrier et al., 2011), Deinococcus geothermalis (Tryptone Glucose Yeast extract media, TGY) (Daly et al., 2004), Deinococcus radiodurans (TGY) (Daly et al., 2004), Enterococcus faecium (TGY) (Daly et al., 2004), Escherichia coli (Luria Broth, LB) (Outten and O'Halloran, 2001), Saccharomyces cerevisiae (Yeast extract Peptone Dextrose media, YPD) (Rosenfeld and Culotta, 2012), Neisseria gonorrhoeae (GCB) (Veyrier et al., 2011), Caulobacter crescentus (Peptone Yeast Extract media, PYE) (Hughes et al., 2013), Escherichia coli (TGY) (Daly et al., 2004), Shewanella oneidensis (TGY; Mn/Fe 0.0005 ± 0.00004) (Daly et al., 2004), and Pseudomonas putida (TGY; <0.001) (Daly et al., 2004).

Figure 2

Overview of the responses available to a hypothetical bacterial pathogen exposed to host derived exogenous or endogenous ROS (O₂⁻, redox-cycling organic molecules and H₂O₂) and how upregulation of the OxyR (Gram-negative)/PerR (Gram-positive) and SoxRS regulons (Gu and Imlay, 2011) leads to protection against ROS-mediated damage (for a review see Imlay, 2013). Here, we focus on the antioxidant effects that derive from upregulated Mn(II) import. (Note: not all processes are known to occur in all cells). The primary molecular targets of ROS in Fe-centric bacteria are Fe (rust-filled circles)-release from mononuclear Fe enzymes and from 4Fe-4S clusters (pink boxes, lower left), which must be avoided to due to the autocatalytic formation of the freely diffusible hydroxyl radical (OH•). (pink, upper left). Four cellular responses involving changes in Mn or Fe speciation as a result of ROS are schematized and highlighted (bold-face). (1) Fe sequestration: Fe(II) is scavenged by Dps (green triangle) to form Fe-Dps which oxidizes Fe to insoluble Fe oxide using O₂ or H₂O₂ as an oxidant and limiting catalytic formation of OH• (bottom, middle). As a result of PerR/OxyR regulatory responses to ROS, increased expression of the Mn (upper right; ABC transporter schematic shown) importer lead to increased intracellular Mn(II) (green circles) that (2) increases the concentration of LMW-Mn complexes that function to dismutate O₂⁻ and possible to detoxify H₂O₂ (upper middle) (see Figure 4), (3) permits cofactor substitution of mononuclear Fe-containing enzymes (yellow ellipses; right) and perhaps of Dps (lower right) with Mn, and (4) enhances the metallation and increased activity of Mn SOD (middle, right). Hosts attempt to limit these responses through direct competition of the Mn(II) transporter with host-encoded calprotectin (CP; purple hexagon) for Mn (upper right) (see text for details). Increased intracellular Mn(II) will ultimately be sensed by the Mn-activated repressor (DtxR, PsaR, etc.) to repress uptake, while excess Mn(II) will be effluxed from the cell by a limited number of organisms (Rosch et al., ; Veyrier et al., 2011) in order to bring the Mn/Fe ratio back into balance (Veyrier et al., 2011).

Figure 3

Superoxide disproportionation by LMW-Mn complexes (Barnese et al., 2012). (A) Plausible noncatalytic mechanism of the reaction of O₂⁻ with [Mn(II)-P₂O₇]²⁻ and Mn(II)-citrate. L, pyrophosphate or citrate. (B) Proposed catalytic mechanism of the reaction of O₂⁻ with [Mn(II)-HPO₄] and [Mn(II)-HCO₃]⁺ leading to the catalytic disproportionation of O₂⁻. L, phosphate or carbonate; Anionⁿ⁻ represents an additional bound anion to the form the intermediate. (C) Overall rate constants for the non-catalytic and catalytic disproportionation of O₂⁻, the latter of which incorporates the MnOO+ dependence of k₅. Rate law simulations reveal that 91 μM MnHPO₄ (165 μM Mn(II), 5 mM phosphate), and 25 μM MnHCO₃⁺ (formed by 36 μM Mn(II) and 5 mM carbonate) gives rise to a steady-state [O₂⁻] (3 μM) identical to 1 μM CuZn-SOD following a 25 μM burst of superoxide (Barnese et al., 2012). Thus, intracellular Mn(II) in the ≈100 μM range is expected to be sufficient to resist the effects of superoxide stress, as found previously in yeast; these studies also physically document the presence of MnHPO₄ species in whole cells (McNaughton et al., 2010). The same may well be true for manganese-centric bacterial pathogens vs. iron-centric E. coli (Aguirre et al., ; Sharma et al., 2013) (see Figure 1B). (D) Chemical structures of “layered” Mn-HPO₄, where the gray box encompasses the next layer in the crystal lattice (left) (Krishnamohan Sharma et al., 2003) and a calculated model of frozen neutral [Mn(HCO₃)₂] from ENDOR studies (right) (Potapov and Goldfarb, 2008).

Figure 4

Structural studies of Mn-specific solute binding proteins (SBPs) from S. aureus MntC (panels B-D) (Gribenko et al., 2013) and S. pneumoniae PsaA (panels E,F) (Lawrence et al., ; McDevitt et al., 2011). (A) Cartoon representation of a canonical ABC transporter, with the subunits and the direction of transport labeled. (B) Ribbon representation of the structure of Mn-bound MntC (pdb code 4K3V), with the four Mn(II)-coordinating ligands highlighted and shown in stick. This orientation is similar to that implied by the cartoon in panel (A). (C) First coordination sphere of the Mn(II) complex in MntC; this orientation differs from that in panel B in order to highlight the distorted trigonal bipyramidal Mn(II) coordination geometry. E189 tends toward monodentate ligation, while D264 is bidentate. (D) Surface representation of MntC in the same orientation as in panel B revealing that bound Mn(II) is buried from solvent. (E) Ribbon representation of a global superposition of PsaA in the Mn(II)-bound (3ZTT) and Zn(II)-bound (1PSZ) states, with metal ligands shown in stick. This orientation is from the docking surface that would interact with the transmembrane subunits (see panel A). (F) Coordination complexes of PsaA bound to Mn(II) (top) and Zn(II) (bottom) revealing that the same four protein-derived ligands are used to coordinate both cognate and noncognate metals. The Mn(II) complex is very similar to that observed for MntC, with E205 tending toward monodentate ligation (rO_e2••Mn = 2.55 Å). In contrast, the Zn(II) complex tends toward tetrahedral coordination with only one oxygen atom of each carboxylate group sufficiently close to directly coordinate the Zn(II). The overlay of these two chelates is shown in panel (E).

Figure 5

Simulated binding curves for ITC thermograms using the reported thermodynamics and injection volumes of the MnCl₂ binding to the SΔ2 mutant of CP (Damo et al., 2013). The simulated curve that corresponds to the reported K_d of 5.8 nM (at 10 μM total CP heterodimer), n = 1.0 (red vertical arrow) and ΔH_cal = 26.2 kcal mol⁻¹ (red horizontal arrow) was converted to K_a (1.7 × 10⁸M⁻¹) to create the isotherm labeled 1•K_a (black squares). Other simulated curves are shown for K_a for 10– (10 * K_a) and 100-fold (100 * K_a) higher K_a, and 10– (0.1 * K_a), 100– (0.01 * K_a) and 1000-fold (0.001 * K_a) lower K_a. These simulations reveal that binding affinities greater than ≈10⁸ M⁻¹ (K_d ≤ 10 nM) can not be reliably measured under these conditions, and are indicative of essentially stoichiometric binding as evidenced by a paucity of data points in the transition region. At 5-10-fold-higher concentration of protein, which is more typical of the SBP-Mn(II) measurements in the literature (see Table 1), a K_a > 10⁷ M⁻¹ (K_d < 100 nM) will not be reliably measured unless a chelator competitor, e.g., citrate for Mn(II), is used to measure K_a^Mn (Grossoehme and Giedroc, ; Gribenko et al., 2013).

Figure 6

Ribbon representation of the structure of the Mn(II)-bound CP heterotetramer (A) and S100A8-S100A9 heterodimer (B) (Damo et al., 2013). S100A8 chains are shaded in yellow and pale yellow, while S100A9 chains are shaded in red and salmon. The tetramer interface between the two heterodimers is marked. The two intersubunit Mn ions per heterodimer are shown as smudge spheres and the three Ca(II) ions per dimer are shown as blue spheres and labeled Ca1 bound to S100A8, Ca2 and Ca3, both bound to S100A9. The coordinating ligands to both S1 and S2 Mn(II) ions are shown in stick representation are labeled with residue number; one of the two S2 sites showed partial occupancy in the structure.