Molecular Microbiology (2014) 91(4), 834–851 ■

doi:10.1111/mmi.12504 First published online 16 January 2014

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae

Charles D. Plumptre,1† Bart A. Eijkelkamp,1† Jacqueline R. Morey,1 Felix Behr,1 Rafael M. Couñago,2,3,4 Abiodun D. Ogunniyi,1 Bostjan Kobe,2,3,4 Megan L. O’Mara,2,5 James C. Paton1 and Christopher A. McDevitt1* 1 Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia. 2 School of Chemistry and Molecular Biosciences, 3 Australian Infectious Diseases Research Centre, 4 Institute for Molecular Bioscience and 5School of Mathematics and Physics, University of Queensland, Brisbane, Queensland, Australia.

Summary Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zincbinding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxyterminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro Accepted 21 December, 2013. *For correspondence. E-mail [email protected]; Tel. (+61) 88313 0413; Fax (+61) 88303 7532. †Contributed equally.

© 2013 John Wiley & Sons Ltd

and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Introduction Streptococcus pneumoniae is one of the world’s foremost bacterial pathogens and is responsible for up to one million deaths annually, which predominantly occur in young children in developing countries (Broome, 1996; Zaidi et al., 2009). This Gram-positive bacterium asymptomatically colonizes the human nasopharynx, but is capable of spreading to the lungs and other tissues where it causes a range of serious diseases including pneumonia, meningitis, otitis media and bacteraemia. Essential to pneumococcal virulence are the transition row metal ions, such as zinc, which it acquires from the host environment (Berry and Paton, 1996; Bayle et al., 2011). Zinc, which commonly occurs as the divalent cation Zn(II), is abundant within the biosphere and is commonly employed in structural or catalytic roles in biological systems (Andreini et al., 2008). In humans, Zn(II) is estimated to interact with ∼10% of all proteins and is particularly abundant in the mucosal epithelia where it accumulates at high concentrations at the apical cell surface (Versieck, 1985; Zalewski et al., 2005). Zn(II) deficiency remains a global health problem with nearly 2 billion people lacking sufficient Zn(II), and this adversely impacts upon a range of cellular and immunological functions (Prasad, 2004). Diseases associated with Zn(II)-poor status predominantly involve acute respiratory infections, otitis media and diarrhoea, and are estimated to account for ∼800 000 deaths per year (Lonnerdal, 2000; Prasad, 2004). Zn(II) deficiency in humans mainly occurs because of issues surrounding nutritional delivery. In developing countries this is associated with the prevalence of cereal-based diets, e.g. rice, that result in the ingestion of high quantities of phytate (an organic phosphate compound) that inhibits Zn(II) absorption by the body (Lonnerdal, 2000). On the other hand, in developed countries Zn(II) deficiency predominantly occurs as a result of

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dietary lifestyle choices, e.g. veganism, that involve consumption of foods that are low in minerals (Prasad, 2004; Craig, 2009). Zn(II) is also essential for microorganisms, although at high concentrations it has been shown to be toxic (McDevitt et al., 2011). Zn(II) acquisition in S. pneumoniae is facilitated by an ATP-binding cassette (ABC) transporter, AdcCB, and two surface attached lipoproteins, AdcA and AdcAII, that belong to the Cluster A-I (formerly known as Cluster IX) family of substrate-binding proteins (SBPs) (Dintilhac and Claverys, 1997; Dintilhac et al., 1997; Berntsson et al., 2010; Couñago et al., 2012; Lewis et al., 2012). Virulence of S. pneumoniae in the host has an absolute requirement for Zn(II) acquisition, as loss of both of the SBPs has been shown to attenuate the pathogen (Bayle et al., 2011). Intriguingly, S. pneumoniae, and most other streptococci, possesses two Zn(II)-binding SBPs, in contrast to the majority of prokaryotes that have only a single copy. The cluster A-I Zn(II)-binding proteins have been studied extensively in Gram-negative organisms (Li and Jogl, 2007). The overall structure of Gramnegative cluster A-I Zn(II)-SBPs consists of two (β/α)4 domains that are connected by a rigid backbone α-helix and the interface between these domains provides the metal binding site (Li and Jogl, 2007). The amino (N)terminal domain of Zn(II)-SBPs also contains an extended flexible loop, which is rich in acidic and histidine residues (His-rich loop), that is absent from the closely related Mn(II)-binding SBPs (Chandra et al., 2007; Wei et al., 2007). The role of the loop has not been unequivocally established, but it has been implicated in aiding Zn(II) capture and/or transfer to the primary metal-binding site within the SBP (Falconi et al., 2011). Comparisons between AdcA and AdcAII of S. pneumoniae and Gram-negative Zn(II)-SBPs reveal a number of striking structural differences. AdcA contains the extended His-rich loop similar to Gram-negative Zn(II)-SBPs, while AdcAII lacks this region (Loisel et al., 2008). A further difference is that AdcA has an extended carboxy (C)terminal region of ∼200 amino acids, whose contribution to Zn(II) recruitment has been uncharacterized (David et al., 2003; Kershaw et al., 2007; Loisel et al., 2008). Notably, this region has not been observed in any Gram-negative SBPs. Consequently, how these significant structural differences affect protein function has remained unclear. Despite these differences, both proteins are transcriptionally repressed by the Zn(II)-responsive regulator AdcR under Zn(II) replete conditions (Reyes-Caballero et al., 2010; Shafeeq et al., 2011; Plumptre et al., 2012). In vitro experiments on adc mutant strains have shown an apparent functional redundancy and led to the inference that the SBPs are interchangeable (Loisel et al., 2008; 2011; Bayle et al., 2011). However, there has been a lack of any in vivo experimental evidence to support this assumption with © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

respect to host–pathogen interaction. Hence, we sought to understand the in vivo significance of the different SBPs in Zn(II) recruitment and to ascertain whether their underlying structural differences had an impact on their functional acquisition of Zn(II) and what, if any, were the consequences for colonization of the host by the pathogen. In this study we present the biochemical characterization of AdcA and elucidate the relative contributions of the two Zn(II)-binding SBPs to Zn(II) homeostasis in S. pneumoniae. This work shows that although AdcA and AdcAII are both involved in Zn(II) recruitment, they appear to employ different mechanisms to acquire the ion. Further, our data show that while the two SBPs may have overlapping functionality in vitro, they have clear additive roles in vivo to facilitate optimal colonization of the host. This work also provides the first direct evidence, to our knowledge, that the cell surface-associated pneumococcal polyhistidine triad proteins directly contribute to Zn(II) homeostasis via AdcAII. Collectively, our results allow us to propose a more accurate model for S. pneumoniae Zn(II) homeostasis and its significance in host colonization.

Results AdcA is a high affinity Zn(II)-binding protein The biochemical and biophysical properties of the S. pneumoniae cluster A-I SBPs AdcAII and PsaA and their roles in Zn(II) and Mn(II) binding have been well established (Loisel et al., 2008; McDevitt et al., 2011). By contrast, despite being clustered with the primary Zn(II) permease, AdcA has remained uncharacterized by biochemical methods. Here we recombinantly expressed dodecahistidine-tagged AdcA and purified it by immobilized metal affinity chromatography (IMAC) (Fig. 1A) and gel permeation chromatography (GPC) (Fig. 1B). GPC indicated that recombinant AdcA was isolated as a single monodisperse species with a relative molecular mass of 61.7 kDa, which matched closely with the predicted molecular mass (58.6 kDa) of monomeric dodecahistidine-tagged AdcA. GPC-purified AdcA then had its dodecahistidine tag removed prior to subsequent characterization. Endogenous metals were removed by denaturation at pH 4.0 in the presence of 20 mM ethylenediaminetetraacetic acid (EDTA) prior to refolding by dialysis in 50 mM Tris-HCl, pH 7.2, 100 mM NaCl. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of refolded tag-cleaved AdcA found that it was metal-free (apo), containing less than 0.01 mol of metal ions per mol of protein. To determine the affinity of AdcA for Zn(II), apo-AdcA was analysed by isothermal titration calorimetry (ITC). AdcA showed a complex Zn(II) binding curve as illustrated

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Fig. 1. Biochemical characterization of AdcA. A. Coomassie blue-stained 12.5% SDS polyacrylamide gel showing purified AdcA. B. Determination of the apparent molecular mass of the purified AdcA by gel-permeation chromatography on a Superdex 200 10/300 column. Inset is the linear regression of the protein molecular weight standards used to calibrate the column. AdcA eluted with a monomeric molecular mass of 61.7 kDa. C. Titration of 10 μM AdcA with 200 μM Zn(II). For each experiment the rates of heat release are shown above the corresponding plots of integrated heat. The curves were fitted to a two-site (n = 2) model and the KD calculated from replicate experiments (± SEM). D. Competitive binding experiment with AdcAC using the Mag-Fura-2-Zn(II). The normalized fluorescence emission (520 nm) of Mag-Fura-2 was monitored in response to the addition of increasing concentrations of apo-AdcAC. Data correspond to mean (± SEM) for three independent experiments. E. Metal-binding experiments of apo-AdcA with divalent metal ions analysed by ICP-MS. Columns correspond to Mn(II) (black), Fe(II) (light gray), Co(II) (dark gray), Ni(II) (white), Cu(II) (vertical lines) and Zn(II) (diagonal lines). The molar ratio of metal ion-to-protein was determined and data correspond to mean values (± SEM) of at least three independent biological experiments. © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

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by a representative binding isotherm in Fig. 1C. A two-site binding model provided the best fit for the data and affinity constants (1/KA = KD) for AdcA with Zn(II) of 2.4 ± 0.1 nM and 228 ± 88 nM were derived. Thus, the ITC data indicated that AdcA had at least two Zn(II) binding sites, but that the affinities of these sites for Zn(II) differed by nearly two orders of magnitude. By comparison with other cluster A-I Zn(II) SBPs (Table S1) it was predicted that the highaffinity site belonged to the N-terminal domain, but the paucity of information regarding the affinity of ZinT and homologous Zn(II)-binding proteins necessitated further investigation. Thus, in order to assign the observed KD values to the different Zn(II) binding sites of AdcA, we cloned and separately expressed the N- and C-terminal domains of AdcA (AdcAN and AdcAC respectively) and generated them as apo-protein variants as described for the full-length protein. The AdcAC variant, which contained the ZinT-homologous domain, was analysed using a competitive Zn(II)-binding assay with the Zn(II)-responsive fluorophore Mag-Fura-2. A titration with increasing concentrations of AdcAC revealed that the C-terminal domain had an observed KD for Zn(II) of 240 ± 19 nM (Fig. 1D). Therefore, as the affinity observed for AdcAC closely matched that observed by ITC for the second site in the full-length protein, we reasonably concluded that the lower affinity site corresponded to the C-terminal domain of AdcA, while the high affinity corresponded to the N-terminal cluster A-I domain. We then investigated the stoichiometry of Zn(II) binding by AdcA. Full-length AdcA and the truncated variants were analysed using in vitro Zn(II)-binding assays and metal associated protein was quantified by ICP-MS. This showed that apo-AdcA bound 1.82 ± 0.1 mol Zn(II)/mol protein, while the truncated apo-variants bound 0.95 ± 0.2 and 0.89 ± 0.1 mol Zn(II)/mol protein, for AdcAN and AdcAC respectively. These data were consistent with the presence of a Zn(II)-binding site in each domain of AdcA, as indicated by the ITC and Mag-Fura-2 assays. We then sought to ascertain whether Zn(II) binding by AdcA was reversible. We have previously observed that PsaA, the cluster A-I Mn(II)-recruiting SBP of S. pneumoniae, has significant similarity to AdcA (51% identity across 313 amino acids), and bound Zn(II) in an effectively irreversible manner (McDevitt et al., 2011; Couñago et al., 2014). Treatment with the chelating agent EDTA removed more than 85% of the bound Zn(II) from AdcA (0.22 ± 0.1 mol Zn(II)/mol protein), indicating that binding was reversible in contrast to PsaA (Couñago et al., 2014). Collectively, these data show that AdcA is a high affinity Zn(II)-binding SBP that possesses two Zn(II)-binding domains with submicromolar affinities, each capable of binding a single Zn(II)-atom. The higher affinity Zn(II)-binding domain corresponded with the cluster A-I domain, located in the N-terminus, while the lower affinity Zn(II)-binding domain © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

Fig. 2. Homology modelling of AdcA. Cartoon representation of the homology-based model of S. pneumoniae AdcA showing a typical cluster A-I fold in the protein’s N-terminal domain (light green) and a ZinT-like fold in the C-terminal domain (dark green). The linking region between N- and C-terminal domains is shown in magenta. Zn(II) atoms are shown as grey spheres in both metal-binding sites of AdcA. The relative orientations of the domains are arbitrary.

corresponded with the ZinT-domain located in the C-terminus.

AdcA does not bind other d-block elements Recent analysis of the Mn(II)-specific cluster A-I SBP from S. pneumoniae, PsaA, revealed that despite its physiological role in Mn(II) acquisition, the metal-binding site of the protein had the capacity to bind a broad range of different d-block metal ions (Couñago et al., 2014). As a consequence, we sought to ascertain whether AdcA also showed a similar capacity to interact with other d-block metal ions. Metal-loading assays examining d-block elements showed that, in contrast to PsaA, AdcA only specifically bound Zn(II) (Fig. 1E). Thus, these data demonstrate that metal-binding sites of AdcA are more specific for the binding of their cognate ligand, by contrast with the less stringent metal-binding properties observed in PsaA (Couñago et al., 2014).

Homology modelling of AdcA Previous studies have indicated that both AdcA and AdcAII interact with the ABC transporter AdcCB to facilitate Zn(II) import (Bayle et al., 2011; Loisel et al., 2011). Although a high-resolution structure of Zn(II)-bound AdcAII has been solved, no structural information for AdcA is yet available. In this study, we generated an energy-minimized homology model of AdcA based on the structures of ZnuA (PDB code 2OSV) and ZinT (PDB code 1OEE) (Fig. 2) in order to compare and contrast the two Zn(II)-SBPs. Based on sequence alignment and structural predictions, the highaffinity Zn(II)-binding site, located in the interdomain cleft of AdcA, would be formed by the residues His63, His140, His204 and Glu279. Similar to the high-resolution structures of AdcAII (PDB code 3CX3) and PsaA (PDB code

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Fig. 3. Phenotypic effects of adc mutations. A. In vitro growth measurements of wild-type S. pneumoniae D39 and adc mutant strains. Bacteria were grown in CDM [∼ 10 μM Zn(II)] and incubated at 37°C and growth monitored by OD600 measurements at 30 min intervals. B. Bacteria were grown in CDM supplemented with 30 μM ZnSO4 and incubated at 37°C and growth monitored by OD600 measurements at 30 min intervals. Data for A and B are representative mean (± SEM) OD600 measurements from three independent biological experiments. C. S. pneumoniae wild-type intracellular Zn(II) accumulation determined by ICP-MS. Data correspond to mean (± SEM) μg Zn(II) per g cell measurements from three independent biological experiments. D. Intracellular Co(II) accumulation determined by ICP-MS. Data correspond to mean (± SEM) ng Co(II) per g cell measurements from three independent biological experiments. E. Intracellular Mn(II) accumulation determined by ICP-MS. Data correspond to mean (± SEM) μg Mn(II) per g cell measurements from three independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). F. Analysis of PsaA expression in whole cell lysates was performed on wild-type and adc mutant strains. Equal amounts of total cell protein were loaded in each lane and PsaA quantified.

3ZTT), the binding site of AdcA would be buried ∼10–15 Å beneath the molecular surface of the protein (Lawrence et al., 1998; Loisel et al., 2008; McDevitt et al., 2011). The metal-binding residues of ZinT are absolutely conserved in the C-terminal extension of AdcA. Residues His452, His461 and His463 comprise a potential Zn(II) binding site, consistent with the recruitment of a single atom by this domain of AdcA. In contrast to the cluster A-I metal-binding site, the ZinT-like binding site would be predicted to remain solvent-accessible. AdcA and AdcAII have overlapping functionality in vitro Of the 3 cluster A-I SBPs in S. pneumoniae, both AdcA and AdcAII have a clear functional interaction with Zn(II) (Loisel et al., 2008; Bayle et al., 2011). Consequently, we sought to ascertain the relative contribution of AdcA and AdcAII to growth in cation-defined growth media (CDM) and the effect on pneumococcal transition row metal acquisition. The Zn(II) content of media can vary significantly depending on preparation, even with chemically defined formulations, and so, in contrast to earlier studies examining the S. pneumoniae adc mutants (Bayle et al., 2011; Loisel et al., 2011), the Zn(II) content of the CDM used was first determined by ICP-MS and then adjusted by the use of the specific Zn(II)-chelating agent TPEN (CDM-TPEN). The benefit of this approach was that it enabled a finer control of medium-Zn(II) content than in previous studies. Wild-type S. pneumoniae D39 and isogenic mutant strains deficient in adcA, adcAII, adcCBA, and combinations thereof, were analysed for their growth phenotypes. Wild-type S. pneumoniae growth was essentially unaffected in all media as were the isogenic deletion strains of AdcA and AdcAII. However, deletion of both SBPs or the ABC permease inhibited growth in the absence of Zn(II) supplementation (Fig. 3A and B). Cellular accumulation of transition row metal ions in the wild-type and mutant strains was examined by ICP-MS. Consistent with the growth phenotypes, Zn(II) accumulation in wild-type and the ΔadcA or the ΔadcAII deletion strains were essentially the

same. The mutant strains in which growth was impaired, the ΔadcAΔadcAII, ΔadcCBA and ΔadcAIIΔadcCBA mutants, all showed significantly reduced Zn(II) accumulation (P < 0.0001 for all mutants compared with wild-type) (Fig. 3C). Analyses of other metal ions showed that the mutations had no effect on the cellular accumulation of Ni(II) or Cu(II). Cobalt, which was detected at an order of magnitude less than most metals, showed a significant increase in cellular accumulation in the ΔadcAΔadcAII (P = 0.018), ΔadcCBA (P < 0.0001) and ΔadcAIIΔadcCBA (P < 0.0001) deletion strains. However, its increase rather than decrease in these strains indicates that AdcCB is not directly involved in its acquisition (Fig. 3D). Intriguingly, the ABC transporter deletion strains, ΔadcCBA and ΔadcAIIΔadcCBA, but not the SBP mutant strains, ΔadcA, ΔadcAII, or ΔadcAΔadcAII, showed an increase in Mn(II) accumulation (P < 0.0001) (Fig. 3E). Consequently, we sought to ascertain whether expression of the Mn(II) cluster A-I SBP (PsaA), which is directly responsible for Mn(II) uptake, was elevated in any of the mutant strains. Quantitative immunoblot analyses (Fig. 3F and Fig. S1A) showed that there was no significant change in PsaA expression at the protein level. Furthermore, qRT-PCR (Fig. S1B) indicated that there were no significant differences in psaA mRNA levels in the strains with elevated manganese content relative to wild-type. Surprisingly there was a small (twofold) but statistically significant increase in psaA mRNA in the ΔadcA strain relative to wild-type, but because this strain showed neither an elevated level of PsaA protein nor increased manganese accumulation, this result is unlikely to have consequences for the manganese uptake of this strain. These results therefore indicated that the increase in Mn(II) uptake in the ΔadcCBA and ΔadcAIIΔadcCBA strains was independent of the expression of the Mn(II) ABC permease. Taken together, these data show that AdcA and AdcAII have overlapping functionality in vitro, consistent with earlier analyses that described their roles as functionally redundant (Loisel et al., 2008; Bayle et al., 2011). The observed impact on metal accumulation also supports our biochemical observations regarding the specific role of © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

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© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

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Fig. 4. AdcR regulation of genes involved in Zn(II) homeostasis. A. The genetic organization of adcABCR, adcAII and phtE in S. pneumoniae D39 shown in arrows drawn to scale with their putative AdcR binding sites indicated in ovals. The E-values are provided above respective putative AdcR binding sites. B. The mRNA expression levels, in times-fold compared with 16S rRNA, of adcA, adcR, adcAII and phtE in S. pneumoniae D39 grown in CDM, D39 grown in CDM-TPEN (TPEN), or the ΔadcR strain. C. The protein expression levels of AdcA, AdcR, AdcAII and PhtE in S. pneumoniae D39 grown in CDM, D39 grown in CDM-TPEN (TPEN), or the ΔadcR strain. The data were normalized to those obtained for D39 grown in CDM. Data for B & C correspond to representative mean (± SEM) from at least three independent biological experiments. Statistical differences from the wild-type are indicated, as assessed by unpaired two-tailed t-tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

AdcA in Zn(II) acquisition. Intriguingly, this study revealed that while the total cellular accumulation of Zn(II) did not directly affect PsaA expression, the lack of the Zn(II)specific ABC transporter AdcCB influenced Mn(II) accumulation through an unknown mechanism. Transcriptional regulation of adcA and adcAII The MarR family metalloregulatory protein AdcR is a primary regulator of Zn(II) import and homeostasis in S. pneumoniae (Reyes-Caballero et al., 2010; Shafeeq et al., 2011). Zinc activation of homodimeric AdcR facilitates its binding to specific operator regions upstream of operons to repress gene expression (Reyes-Caballero et al., 2010; Shafeeq et al., 2011). Transcription of both adcA and adcAII are regulated by AdcR, but while adcA is clustered in an operon with the ABC permease (adcRCBA), adcAII is an ‘orphan’ SBP and is transcriptionally clustered upstream of the pneumococcal polyhistidine

triad (Pht) phtE and phtD genes, another gene family implicated in Zn(II) homeostasis (Loisel et al., 2008; Ogunniyi et al., 2009; Reyes-Caballero et al., 2010; Shafeeq et al., 2011). However, what remains unknown is whether AdcR differentially influences the transcriptional levels of any of these genes. Consequently, we investigated the transcriptional levels of candidate genes involved in Zn(II) homeostasis, i.e. adcA, adcR, adcAII and phtE, and assessed their relative repression by AdcR. An AdcR binding site-scoring matrix was generated and the S. pneumoniae D39 genome analysed using a Hidden-Markov Model approach to identify putative AdcR binding sites (Krogh et al., 1994; Panina et al., 2003). This analysis revealed that the adcRCBA operon possessed one adcR-binding site that permitted AdcR autoregulation, while two AdcR binding sites were identified upstream of adcAII (Fig. 4A). Although these findings were consistent with prior studies, we also identified three AdcR binding sites upstream of phtE in this study, © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

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whereas previously only a single binding site had been identified (Fig. 4A) (Shafeeq et al., 2011). As the different numbers of AdcR binding sites could potentially influence gene transcription we then assessed the effect of Zn(II) abundance on AdcR repression. Transcription levels of adcA, adcAII and phtE were assessed by comparison between the wild-type strain grown with Zn(II) (CDM) or without (CDM-TPEN), and contrasted against a ΔadcR mutant strain, which permitted the expression of these genes in a fully de-repressed manner (Fig. 4B). We observed that both adcA and adcR were upregulated by ∼4-fold in the wild-type strain when grown in CDM-TPEN. This degree of upregulation for adcA was not significantly different (P value = 0.7767) from that observed in the ΔadcR background, suggesting that adcA was fully de-repressed during growth in Zn(II)-restricted media. Under Zn(II)-limiting conditions adcAII demonstrated a higher level of transcriptional upregulation (∼6-fold), relative to growth in CDM, than was observed for adcA. Similarly the ΔadcR strain also showed greater de-repression of adcAII (∼14.8-fold) than was observed for adcA. Consistent with the presence of the greatest number of AdcR binding sites, phtE showed the largest increase in transcription (∼30-fold upregulated) during growth in Zn(II)restricted media, or in the absence of AdcR. Quantitative immunoblotting of whole cell S. pneumoniae lysates, from growth under the same conditions, revealed that, at equivalent concentrations of total protein, protein expression similarly correlated with the observed transcriptional data, albeit with lower fold changes (Fig. 4C). Collectively, these data provide direct evidence that the number of AdcR binding sites correlates with the extent of transcriptional repression by AdcR. Intriguingly, phtE was more tightly regulated by AdcR than either of the Zn(II)recruiting SBPs, and so we sought to further analyse the connection between the Pht proteins and Zn(II) homeostasis. The Pht proteins contribute to Zn(II) homeostasis The Pht proteins are a group of cell surface-associated proteins in S. pneumoniae that includes 4 known members, PhtA, PhtB, PhtD and PhtE. The Pht proteins contain multiple His triad motifs (HxxHxH) and have been subdivided into 3 broad groups on the basis of primary structure comparisons (Adamou et al., 2001; Loisel et al., 2011; Plumptre et al., 2012). Based on the transcriptional clustering of the pht genes with adcAII and their previously observed interactions with Zn(II), it has been postulated that the Pht proteins are involved in d-block metal ion homeostasis (Ogunniyi et al., 2009; Loisel et al., 2011; Rioux et al., 2011). However, there has been a paucity of direct evidence defining the nature of this contribution. © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

We sought to address whether deletion of the pht genes in isolation or in combination with the adc genes had an effect on growth of S. pneumoniae, and so we investigated the phenotype of the appropriate mutant strains under Zn(II)-replete and Zn(II)-restricted growth conditions. Overall our data showed that, when grown in unsupplemented CDM, loss of the Pht proteins had no effect on the phenotype of any strain (Fig. 5A). As shown in Fig. 5B, growth in CDM-TPEN caused a minor delay in growth of wild-type D39 and the individual SBP mutants (ΔadcA and ΔadcAII), due to the near complete elimination of Zn(II) from the growth medium. Furthermore, consistent with our earlier observations (Fig. 3A), the mutant strains deficient in both cluster A-I SBPs (ΔadcAΔadcAII and ΔphtABDEΔadcCBAΔadcAII) were hyper-susceptible to Zn(II) starvation, as their growth was completely attenuated. However, the pht and adc combination mutant strains demonstrated distinct phenotypes when grown in CDMTPEN. The AdcA-containing strain (ΔphtABDEΔadcAII) showed no perturbation in its growth and behaved essentially the same as the wild-type and ΔphtABDE strains, indicating that loss of the Pht proteins had a negligible effect on the efficiency of AdcA in Zn(II) recruitment. By contrast, the AdcAII-only containing strain (ΔphtABDEΔadcA) demonstrated a large and significant reduction in its growth rate. Taken together, these data suggest that the Pht proteins contribute to Zn(II) homeostasis predominantly via AdcAII. Both AdcA and AdcAII are required for full virulence Previously it has been shown that loss of both Zn(II)-SBPs attenuated the virulence of S. pneumoniae, but the phenotypes of single mutants lacking one of the SBPs were not assessed in vivo (Bayle et al., 2011). In this study we sought to determine whether loss of AdcA or AdcAII in isolation affected the virulence or invasiveness of S. pneumoniae in mouse models of disease. To investigate possible niche-specific effects on the virulence of the mutants (ΔadcA, ΔadcAII or ΔadcAΔadcAII), an intranasal challenge model was used. At 24- and 48-hour time points, mice were euthanized and the nasal wash, nasal tissue, lungs and blood were collected and the numbers of bacteria determined (Fig. 6). After 24 h (Fig. 6A), all of the mutant strains showed attenuation in the nasopharynx and the lungs compared with the wild-type strain. In the blood, only the ΔadcAΔadcAII strain was attenuated. Thus, these results show that AdcA and AdcAII are not functionally redundant in vivo as each single mutant exhibited a modest attenuation. After 48 h, the ΔadcA and ΔadcAII strains were no longer attenuated in any niches except for ΔadcA in the lungs (Fig. 6B). Hence, as the infection progresses (i.e. in the subsequent 24 h), each single mutant is able to overcome the deficiency associated with the absence of

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Fig. 5. Phenotypic effects of adc and pht mutations. A. Wild-type D39, adc and pht mutants were grown in CDM [∼ 10 μM Zn(II)] and incubated at 37°C and growth monitored by OD600 measurements at 30 min intervals. B. Wild-type D39, adc and pht mutants were grown in CDM-TPEN and incubated at 37°C and growth monitored by OD600 measurements at 30 min intervals. Data for A & B correspond to representative mean (± SEM) OD600 measurements from three independent biological experiments.

the other SBP. The double SBP mutant showed near complete attenuation of pneumococcal fitness, both in terms of colonization and invasive disease, consistent with previous findings of Bayle et al. (2011). To further assess the overall impact of the loss of the individual SBPs on virulence, we then intranasally infected mice with the same four strains of S. pneumoniae as in the previous experiment, and survival times were recorded for up to two weeks after challenge. As shown (Fig. 7A), only one mouse infected with the ΔadcAΔadcAII mutant died during the experiment, whereas none of the mice infected by wild-type survived beyond 72 h (P < 0.0001). By contrast, the ΔadcA and ΔadcAII groups showed survival curves that were not significantly different to that of the wild-type group (P values > 0.05 in both cases). Therefore, loss of AdcA or AdcAII alone does not significantly attenuate pneumococcal virulence in this model, indicating that a single Zn(II)-SBP is necessary and sufficient for full virulence despite the reduction in colonization efficiency. The Pht proteins are crucial for in vivo AdcAII function We then sought to ascertain whether there were any differences in fitness between the ΔadcA and ΔadcAII mutant strains. An in vivo competition assay was conducted using a mixed culture of the ΔadcA and ΔadcAII

mutant strains. The competitive index (defined as the proportion of ΔadcA cfu.ml−1 relative to ΔadcAII cfu.ml−1) in each niche for each mouse was determined after 24 and 48 h (Fig. 7B and C respectively). These results show that the ΔadcA mutant strain was marginally less competitive than the ΔadcAII strain after 24 h in the nasal wash, but was more competitive after 48 h in the lungs and blood. This suggests that AdcAII may be more important in facilitating pneumococcal invasive disease as infection progressed in this model. The implication that AdcA and AdcAII may have differing contributions to pneumococcal fitness could correlate with their different strategies for Zn(II) recruitment. To further analyse this hypothesis, we examined the contribution of the Pht proteins to Zn(II) acquisition via the two SBPs. As we would predict, based on our in vitro experiments, AdcAII was more dependent on their ability to aid in Zn(II) recruitment. As the Pht proteins have a role in protection of the pneumococcus against complement killing (Ogunniyi et al., 2009), an in vivo competition assay was conducted to compare the ΔphtABDEΔadcA and ΔphtABDEΔadcAII strains. The results (Fig. 8A and B) showed a huge disparity between the fitness of the two strains, with the ΔphtABDEΔadcAII strain showing a far greater in vivo fitness than the ΔphtABDEΔadcA strain. This was the case in both the nasal wash and nasal tissue at 24 h post infection and all niches examined at 48 h. Taken together, these data indi© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

AdcA and pneumococcal zinc homeostasis 843

Fig. 6. S. pneumoniae host colonization. Burden of S. pneumoniae infection was assessed by determination of the bacterial load (cfu ml−1) recovered from infected mice at 24 h (A) and 48 h (B) after challenge. The number of pneumococci in each niche at each time-point are plotted (one point represents one niche from one mouse). Solid lines denote geometric means of each group; dashed lines denote the limit of detection. Statistical differences from the wild-type are indicated, as assessed by unpaired one-tailed t-tests (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

cate that although both AdcA and AdcAII contribute to pneumococcal fitness and virulence via interaction with Zn(II), how they facilitate this role in vivo is achieved by different mechanisms. These data indicate that AdcAII is significantly more dependent upon the Pht proteins to aid in Zn(II) recruitment than AdcA.

Discussion Almost one-third of all proteins require interaction with a metal ion to facilitate their biological activity (Andreini et al., 2008). Zinc is one of the most abundant transition row metal ions and its acquisition in the host environment is an essential requirement for in vivo infection. S. pneumoniae has an absolute requirement for Zn(II) to colonize the host, but the molecular mechanisms involved in its uptake have not been fully elucidated. In this study we have shown through a combination of biochemical, in vitro, and in vivo studies that the cell-surface lipoprotein AdcA is a cluster A-I SBP that has a direct role in recruiting extracellular Zn(II), but through a different mechanism to AdcAII. These findings highlight the markedly different © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

architectures and transcriptional regulation of the two SBPs. As a consequence, this work requires that we re-examine the functional redundancy model of AdcA and AdcAII proposed in recent literature (Loisel et al., 2008; 2011; Bayle et al., 2011). Our in vitro experiments reveal that a single Zn(II)-SBP, i.e. either AdcA or AdcAII, was sufficient for Zn(II) acquisition and for in vivo systemic virulence. However, in vivo the loss of either ΔadcA or ΔadcAII had a clear, albeit modest, effect on the ability of the pneumococcus to colonize the mouse nasopharynx during the first 24 h of infection, when compared with the wild-type strain. If the SBPs were functionally redundant, then no impact on colonization would be expected upon loss of either SBP. By contrast, our data indicate that optimal colonization by S. pneumoniae required both Zn(II)-binding SBPs. Taken together, these findings indicate that in vivo AdcA and AdcAII are not redundant, but rather are complementary and aid the overall fitness of the pneumococcus. This interpretation is also consistent with distinct Zn(II)-responsive regulation and Pht-SBP interaction behaviour evidenced by AdcA and AdcAII, which will be discussed later. By comparison with other

844 C. D. Plumptre et al. ■

Fig. 7. Survival times and competitive indices of S. pneumoniae Δadc strains in a murine model. (A) Survival times of ten mice after infection with wild-type and Δadc mutant pneumococcal strains. Competitive indices between ΔadcA and ΔadcAII mutants after 24 h (B) and 48 h (C). Competitive indices were calculated from these values and the input ratio. The mean competitive index for each niche was compared with a theoretical value of 1 (dashed line) by two-tailed one-sample t-tests; significant differences are indicated where present (n.s., not significant; *, P < 0.05; **, P < 0.01).

Fig. 8. Competitive indices of S. pneumoniae ΔadcΔphtABDE strains in a murine model. Competitive indices between ΔadcAΔphtABDE and ΔadcAIIΔphtABDE mutants after 24 h (A) and 48 h (B). Competitive indices were calculated from these values and the input ratio. The mean competitive index for each niche was compared with a theoretical value of 1 (dashed line) by two-tailed one-sample t-tests; significant differences are indicated where present (n.s., not significant; ***P < 0.001; ****P < 0.0001).

microorganisms that asymptomatically colonize the nasopharynx (Vuononvirta et al., 2011), the streptococci are unique in their possession of two Zn(II)-SBPs. Despite this, Zn(II) accumulation in two other representative nasopharyngeal opportunistic pathogens, Staphylococcus aureus and Pseudomonas aeruginosa (Table S2), reached levels that were similar to or more than that accumulated by S. pneumoniae, even though these organisms possessed only a single Zn(II)-SBP. Therefore, © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

AdcA and pneumococcal zinc homeostasis 845

the possession of two Zn(II)-SBPs by S. pneumoniae, which are under different degrees of transcriptional regulation, suggests that AdcA and AdcAII act in concert to facilitate more efficient Zn(II) acquisition and that this aids in specific host niche colonization. Transcriptional regulation of both SBPs occurs via AdcR. In this work we show that there is a direct correlation between the number of AdcR binding sites and the transcriptional repression by AdcR of the respective target sites. Due to the limited data available on the binding affinity of AdcR to the sites identified, the significance of the E-values obtained in the bioinformatic analysis will not be discussed beyond what has been described in the Materials and Methods section. Despite this, the overall findings are well supported by comparison to the earlier studies, which showed that the transcriptional upregulation in the ΔadcR strain, determined in this work, were similar to those reported in earlier microarray and lacZpromoter fusion studies for adcA, adcAII and phtE (Reyes-Caballero et al., 2010; Shafeeq et al., 2011). Electrophoretic mobility shift assay analysis of AdcR binding sites has also previously shown more stringent binding of AdcR to the regulatory region of phtE compared with the regions upstream of adcR and adcAII (Shafeeq et al., 2011). Thus, we provide a mechanism by which the same transcriptional regulator can modulate the level of expression of its target genes to differing extents by varying the number of binding sites for the regulator upstream of the gene. In the context of S. pneumoniae, this potentially enables this pathogen to utilize its Zn(II) acquisition systems differently, whereby AdcA provides a ‘baseline’ level of Zn(II) uptake, while AdcAII and PhtE are able to be dramatically upregulated in response to Zn(II) restriction in the host environment. The finding that AdcAII had a more important role in the initial progression of invasive disease is also consistent with recent genome wide transposon studies of S. pneumoniae, which showed that, in in vivo models of invasive disease, the genes adcAII, adcC and adcB had significant roles in colonization (van Opijnen and Camilli, 2012). By contrast, single gene deletions of adcA and the pht proteins were not found to be essential for invasive disease (van Opijnen and Camilli, 2012). Collectively, these data indicate that Zn(II) availability in certain host niches is restricted during the initial stages colonization. This drives expression of AdcAII and the Pht proteins to increase its capacity for Zn(II) recruitment and thereby facilitate more efficient Zn(II) uptake than could occur with AdcA alone. The differences in transcriptional regulation of the two SBPs also potentially correlate with the observed structural differences. Although both SBPs share the same overall cluster A-I SBP fold, AdcAII lacks the His-rich loop, which is commonly found in those SBPs, including AdcA, that directly recruit Zn(II). Furthermore, AdcA possesses © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

an extended C-terminal region, which contains an additional Zn(II)-binding domain (Stojnev et al., 2007; Loisel et al., 2008; Berntsson et al., 2010). AdcA showed negligible transcriptional modulation in response to environmental Zn(II) abundance. Collectively, the in vivo and in vitro analyses suggest that this level of AdcA is sufficient for S. pneumoniae survival and colonization. Thus, the affinity of the Zn(II)-binding sites within AdcA would both be entirely consistent with acquisition of Zn(II) from the host environment, where the reported concentrations of Zn(II) in naïve mouse niches are ∼15 μM in blood and ∼40 μM in the nasopharynx, lung and brain (McDevitt et al., 2011). As a consequence, the structural features of AdcA provide it with the capacity to acquire Zn(II) from the extracellular environment without need for additional transfer proteins. Intriguingly, the C-terminal ZinT-domain of AdcA appears to exist for this purpose in Gramnegative organisms. Recent studies of periplasmic ZinT have shown that it transiently interacts with ZnuA via protein-protein interactions and thereby facilitates Zn(II) transfer to the cluster A-I SBP ZnuA metal-binding site (Petrarca et al., 2010; Gabbianelli et al., 2011; Ilari et al., 2013). Studies of Zn(II) homeostasis in Sa. Typhimurium showed that deletion of the zinT gene or the His-rich loop from the Zn(II)-binding SBP had little direct effect on Zn(II) homeostasis in isolation. However, a combination deletion of zinT and the His-rich loop induced a Zn(II) starvation phenotype, as if the cluster A-I Zn(II)-SBP had been deleted (Petrarca et al., 2010). By implication, these findings suggest that the His-rich loop of the SBP and/or ZinT are necessary to ensure delivery of Zn(II) to the cluster A-I SBP binding site. One speculation could be that in Grampositive organisms, cell-surface lipoproteins such as AdcA may have arisen as a fusion of the Zn(II)-binding cluster A-I SBP domain to the ZinT domain, thereby preserving the functionality of the transient Zn(II)-SBP/ZinT heterodimers that occur in Gram-negative organisms. The possible evolutionary basis of this could be the lack of an outer membrane to compartmentalize this space as is found in Gram-negative organisms. However, this raises the question as to why these additional domains are required for Zn(II)-recruitment, as cluster A-I SBPs have a high-affinity metal-binding site, which typically has an affinity binding constant in the nanomolar range. An examination of high-resolution structures of cluster A-I SBPs reveals a possible explanation for this occurrence. In cluster A-I SBPs the highaffinity metal binding site is buried up to 15 Å from the molecular surface of the protein at the bottom of the interdomain cleft (Hung et al., 1998; Loisel et al., 2008; McDevitt et al., 2011; Couñago et al., 2014). Consequently, it may be that the presence of additional His-rich regions aid in recruiting Zn(II) to the SBP such that it can be bound by the deeply buried primary metal binding site

846 C. D. Plumptre et al. ■

of the protein under in vivo conditions, an activity essential for ensuring that the SBP achieves a closed metal-bound conformation that can be recognized by the ABC permease (Davidson et al., 2008). Despite lacking additional Zn(II)-recruiting motifs, AdcAII is sufficient for Zn(II) uptake in the S. pneumoniae ΔadcA strain. The adcAII gene is encoded adjacent to phtD and phtE and although it is generally accepted that the Pht proteins play a role in Zn(II) homeostasis, the molecular basis of this role is a matter of considerable debate (Ogunniyi et al., 2009; Loisel et al., 2011; Rioux et al., 2011; Shafeeq et al., 2011; Plumptre et al., 2012). This study has shown for the first time, to our knowledge, that the loss of the Pht proteins massively reduces AdcAII functionality both in vitro and in vivo. This finding directly implicates the Pht proteins as contributing to Zn(II) homeostasis primarily in cooperation with AdcAII. Furthermore, this work implies that there is a requirement for solvent-exposed Zn(II)-recruiting regions in order to facilitate efficacious delivery of Zn(II) to the high-affinity metal binding site of cluster A-I SBPs. This is likely due to Zn(II) preferentially interacting with more accessible N- and O-ligands on cell-surface proteins of the pneumococcus that would otherwise hamper Zn(II) delivery to AdcAII. Consistent with the findings here, this effect would primarily be observable under conditions of low Zn(II), where the likelihood of a deeply buried Zn(II)-binding site interacting with Zn(II) would be quite low. Under these conditions, the Pht proteins may aid in overcoming this delivery challenge by facilitating an enrichment of the concentration of Zn(II) near the cell surface, where AdcAII is available for interaction. Alternatively, it has been suggested that PhtD could physically interact with AdcAII to transfer Zn(II) directly (Loisel et al., 2011). However, the in vitro crosslinking and colocalization experiments supporting this hypothesis are far from definitive, and it is unclear whether the membrane-bound AdcAII and cell-wall anchored Pht proteins would be able to interact due to spatial constraints imposed by their differing cellular locations (Loisel et al., 2011; Plumptre et al., 2012). Overall, at a functional level the Pht proteins appear to substitute for the roles played by the ZinT domain and the His-rich loop in AdcA. Metal accumulation studies of S. pneumoniae revealed that loss of either AdcA or AdcAII had little effect on in vitro Zn(II) accumulation, consistent with earlier findings (Bayle et al., 2011; Loisel et al., 2011). In this study, supplementation of CDM with 10 μM Zn(II) was sufficient for growth for all strains, even those with no functional high-affinity Zn(II) acquisition systems (i.e. ΔadcAΔadcAII, ΔadcCBA and ΔadcAIIΔadcCBA). A plausible explanation for this could be that that less specific secondary transporters allowed for sufficient acquisition of Zn(II) to support growth under these conditions, albeit with a significantly

diminished total accumulation of Zn(II), as shown by the ICP-MS experiments. By contrast with studies of the Mn(II) permease (McAllister et al., 2004; McDevitt et al., 2011), the cellular accumulation of Zn(II) in the ΔadcAΔadcAII, ΔadcCBA and ΔadcAIIΔadcCBA backgrounds was less diminished than might otherwise be expected. A possible explanation for this is the greater utilization of Zn(II) in biological systems. Of the transition row elements, only iron has more roles in cellular processes than Zn(II). Thus, the baseline of Zn(II) accumulation for cell viability is likely to be greater than that of most other transition row elements, i.e. Mn(II), Co(II) and Ni(II). However, Zn(II) accumulation must also be tightly regulated so as to prevent accumulation to significant excess. Although Zn(II) is not redox active, the IrvingWilliams series of thermodynamic stability constants for first row transition elements indicate that Zn(II) will outcompete lower order d-block elements for binding to proteins (Irving and Williams, 1948; 1953). Thus, the cellular concentration of Zn(II) in the pneumococcus must be carefully balanced within a narrow range such that it is sufficient for cell viability but while also being less than other ions, such as Mn(II), so as to not facilitate competition between ions for binding to proteins. Concomitant with a reduction in Zn(II) accumulation in the ΔadcAΔadcAII, ΔadcCBA and ΔadcAIIΔadcCBA strains, an increase in Co(II) accumulation occurred. As the pneumococcus does not encode a specific Co(II) transporter, the most likely explanation for this would be the Zn(II)-dependent SczA-mediated downregulation of CzcD, the cation diffusion pump responsible for Zn(II) and Co(II) efflux (Kloosterman et al., 2007). Kloosterman and co-workers have previously reported that expression of CzcD is regulated by either metal (Kloosterman et al., 2007), but under these experimental conditions we would infer that expression appears to be subordinated by the decreased intracellular Zn(II) levels, leading to the observed twofold increase in intracellular Co(II). Unrelated to the effect on Co(II) was the observation of a specific increase in intracellular Mn(II) in mutant strains deficient in the ABC transporter AdcCB. This effect was specific to the loss of AdcCB and not directly linked to the decreased ability to import Zn(II), as the ΔadcAΔadcAII strain, which had decreased Zn(II) accumulation, did not show an elevation in Mn(II) content. The increased Mn(II) accumulation was also not related to expression of PsaA, the cluster A-I SBP involved in Mn(II) binding, as quantitative immunoblotting and qRT-PCR demonstrated that PsaA expression was unchanged. The basis for this occurrence is unclear as the pneumococcus genome does not encode any other Mn(II) import pathway. Furthermore, the Mn(II) specific efflux transporter, MntE, is constitutively expressed and has not been reported to be influenced by Zn(II) abundance (Rosch et al., 2009). © 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

AdcA and pneumococcal zinc homeostasis 847

Thus, it is not readily apparent how Mn(II) accumulation increased as a result of loss of the AdcCB transport pathway. Further experiments will be required to determine the basis of the observed increase in Mn(II) accumulation. Collectively, based on the findings from this work, and others (Dintilhac and Claverys, 1997; Loisel et al., 2008; 2011; Ogunniyi et al., 2009; Reyes-Caballero et al., 2010; Bayle et al., 2011), we propose a possible mechanism for how S. pneumoniae acquires Zn(II) in host niches where Zn(II) abundance is tightly restricted (Fig. S2). We propose that, either independently or via one or more of the Pht proteins, Zn(II) is recruited to the cell wall by virtue of its ability to form stable complexes with proteins. The Pht proteins with their prevalence of His-triad regions could serve as low-affinity ‘sinks’ for Zn(II), acting to preferentially localize the Zn(II), while also reducing its association with other proteins in the cell-wall. Zinc would then move from the Pht proteins to AdcAII and/or AdcA. This could occur either via physical interaction by the SBPs to ‘extract’ the Zn(II) ion or, alternatively, it could be facilitated by other cell-surface or cell-wall proteins to aid in passage of Zn(II) towards the cell membrane where the SBPs are tethered. Irrespective of the precise mechanism, the high affinity of AdcA, and by extension AdcAII, for Zn(II) would then recruit the Zn(II) ions from lower affinity carrier molecules and facilitate its uptake into the cell via AdcCB. In summary, the bioinformatic, biochemical, biophysical and infection model data we have presented support a role for AdcA as a high affinity Zn(II)-binding cluster A-I SBP. Furthermore, while this study supports the overall conclusion that AdcA and AdcAII have overlapping functionality in an in vitro setting, this is not the case in vivo and the molecular basis of this distinct functionality is facilitated by strikingly different mechanisms. Thus, while either AdcA or AdcAII are sufficient for Zn(II) homeostasis in S. pneumoniae, as commonly occurs in most other microorganisms, the presence of both cluster A-I Zn(II)binding SBPs enables it to mount a more efficient colonization and, therefore, contributes to the virulence of this major human pathogen. The different Zn(II)-recruitment strategies potentially reflect niche-specific adaptations that aid in Zn(II) acquisition and colonization efficiency by the pneumococcus.

Homology models of the N-terminal and C-terminal domains of AdcA were constructed using the SwissModel webserver (Schwede et al., 2003), with ZnuA (PDB ID: 2OSV) and YodA (PDB ID: 1OEE) as templates for the N- and C-terminal domains respectively. The two domains are linked by an 11 residue linker region, which was manually built as an extended coil region. The resulting model of AdcA was energy-minimized in SwissPDBViewer (Guex and Peitsch, 1997) using the inbuilt 43B1 vacuum forcefield (van Gunsteren et al., 1996). It should be noted that the orientation of the N- and C-terminal domains of AdcA is unknown, thus a contact interface was not predicted.

Experimental procedures

Growth media and growth curve assays

Expression and purification of recombinant proteins

CDM corresponds to cation-defined media with no added Zn(II) or Mn(II), prepared as described previously (McDevitt et al., 2011). CDM was supplemented with 1 μM MnSO4 and varying concentrations of ZnSO4 as indicated and was used for growth of S. pneumoniae D39 and the mutant strains as described previously (McDevitt et al., 2011).To assess the effect of further Zn(II) starvation, the Zn(II)-chelating agent

Recombinant AdcA was generated by PCR amplification of the mature coding sequence of S. pneumoniae D39 adcA (residues 27–501, omitting the signal peptide and predicted lipoprotein motif) and using ligation-independent cloning to insert the gene into a C-terminal dodecahistidine tag© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

containing vector, pCAMcLIC01, to generate pCAMcLIC01AdcA. High-level protein expression and extraction was performed essentially as described in (McDevitt et al., 2011). Recombinant AdcA was isolated using a HisTrap HP column on an AKTA Purifier and was further purified on a Superdex 200 10/300 gel permeation column. Molecular mass on the Superdex column was assessed by comparison to soluble molecular weight standards (Sigma-Aldrich) of carbonic anhydrase = 29 kDa, bovine serum albumin = 66 kDa, yeast alcohol dehydrogenase = 150 kDa, sweet potato β-Amylase = 200 kDa, horse spleen apo-ferritin = 443 kDa, bovine thyroglobulin = 669 kDa, blue dextran = 2 MDa.

Apo-protein generation and metal content analysis Recombinant AdcA had the dodecahistidine tag removed by overnight enzymatic digestion by the 3C human rhinovirus protease at a cleavage site introduced between AdcA and the tag. The protein was then reverse-purified on a HisTrap HP column with the cleaved protein eluting in the absence of imidazole. Removal of the dodecahistidine tag was established by the observed reduction in molecular mass on a 15% SDS-PAGE gel and confirmed by immunoblotting. Demetallated (apo) AdcA was prepared by dialysing the protein (10 ml) in a 20 kDa MWCO membrane (Pierce) against 4 l of sodium acetate buffer, pH 3.7, with 20 mM EDTA. The sample was then dialysed against 4 l of 20 mM Tris-HCl, pH 7.2, 100 mM NaCl, at 4°C. The sample was then recovered and centrifuged at 18 000 g for 10 min to remove any insoluble material. Metal content analysis was performed by ICP-MS (Adelaide Microscopy, University of Adelaide). Ten micromolar protein was heated at 95°C for 30 min in 3.5% HNO3. The insoluble material was removed by centrifugation at 18 000 g for 20 min after which the samples were analysed on an Agilent 7500cx ICP-MS.

Homology modelling and structural analyses

848 C. D. Plumptre et al. ■

N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was supplemented at an equimolar concentration to Zn(II) determined to be in CDM by ICP-MS to provide CDM-TPEN. Addition of TPEN had no discernable effect on pneumococcal growth, except when it exceeded the prevailing Zn(II) concentration. For murine challenge experiments, bacteria were grown in serum broth [10 g l−1 peptone (Oxoid), 10 g l−1 Lab Lemco powder (Oxoid), 5 g l−1 NaCl and 10% (v/v) donor horse serum] to an OD600 of 0.16, and then diluted to the relevant challenge dose. For growth curve measurements, wild-type S. pneumoniae D39 and mutant strains were grown in CDM with 1 μM MnSO4 until they reached OD600 = 0.3. They were then subcultured into 200 μl CDM and supplemented as specified. The bacteria were incubated at 37°C and growth was monitored by measurement of the OD600 at 30 min intervals.

Biophysical analyses ITC was performed using a VP-ITC unit (GE Healthcare) with apo-AdcA, 10 μM, in 10 mM Na3-citrate buffer, pH 7.1 at 25°C. Samples were centrifuged and degassed prior to analysis. 200 μM ZnSO4 was injected at 210 s intervals with 28 injections of 10 μl with a 10 second injection time. A stir rate of 307 per min was used in both experiments. Data were analysed using the Origin 7.0 software (Microcal) and the parameters (± SEM) determined. Determination of the KD for AdcAC with Zn(II) was performed by means of a competition assay using apo-AdcAC and the Zn(II)-fluorophore MagFura-2 (Life Technologies). Excitation-emission spectra were determined on a FLUOStar Omega (BMG Labtech) at 28°C using black half-volume 384-well microtitre plates (Greiner Bio One). All experiments were performed in 20 mM MOPS (pH 6.7), 50 mM NaCl with Mag-Fura-2 at a concentration of 150 nM. Deionized water and buffers was prepared and treated with Chelex-100 (Sigma-Aldrich) to avoid contamination by metal. Filters used for Mag-Fura-2 were excitation (370 ± 10 nm) and emission (520 ± 10 nm). To determine the dissociation constant between a metal (X) and a Mag-Fura-2 (F), we considered the following equilibria: K D .X

F + X ↔ F .X

(1)

Where, for a metal that decreases the fluorescence of the probe by more than 10%, the following equation, which is an exact analytical relationship derived from the mass action equation for the formation of a 1:1 complex between probe and metal, was used to estimate the dissociation constant, KD.X

f − fmin [X ] = fmax − f K D .X

(2)

where f is the measured fluorescence intensity in the presence of metal, fmax the fluorescence in the absence of metal and fmin the fluorescence in the presence of saturating metal. In all cases, a low concentration (150 nM) of probe was used and we assumed that the free metal concentration was equal to the added metal concentration. The mean ± SEM (n = 8) KD determined for MagFura-2 with ZnSO4 in the buffer system employed in this

study was determined to be 46.7 ± 10 nM. Competition by AdcAC for Zn(II) binding was assessed by monitoring the increase in the fluorescence of 150 nM Mag-Fura-2-Zn(II) in response to increasing apo-AdcAC concentrations and analysed using log10[inhibitor] versus response model, with the experimentally derived KD for Mag-Fura-2 with Zn(II), in Graphpad Prism to determine the KD value for Zn(II) binding by AdcAC. ICP-MS of pneumococcal cultures S. pneumoniae strains were grown in CDM with 1 μM MnSO4 to an OD600 of 0.3. Staphylococcus aureus and Pseudomonas aeruginosa were grown in M9 media supplemented with 0.5% yeast extract, then treated with 5 g l−1 Chelex-100 (Sigma-Aldrich) (CxM9-Y). Metal content of the CxM9-Y medium was supplemented to 10 μM Zn(II) and 1 μM Mn(II) and confirmed by ICP-MS to be essentially identical to that of the CDM. Cells were grown in this medium to an OD600 of 0.3. The bacteria, irrespective of their growth media, were washed three times with PBS containing 5 mM EDTA, then twice with normal PBS. Bacterial pellets were desiccated by heating to 95°C overnight. The dry cell weight was measured and the pellets resuspended in 35% HNO3. Metal ion content was measured on an Agilent 7500cx ICP-MS.

Quantitative immunoblot and qRT-PCR analyses Wild-type and mutant S. pneumoniae were grown under the same conditions as for ICP-MS. After reaching an OD600 of 0.3, cells were incubated with 0.1% sodium deoxycholate (Sigma-Aldrich) at 37°C for 60 min to induce lysis. Protein concentrations were determined (Dc Bio-Rad Protein Assay, Bio-Rad) and 40 μg total protein loaded into each lane. After electrophoretic separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane using the iBlot (Invitrogen) system. Blots were incubated with murine anti-PsaA serum (McDevitt et al., 2011), followed by anti-mouse IRDye 800 (LI-COR) and were scanned using an Odyssey infrared imaging system (LI-COR). Band intensities were measured using the manufacturer’s application software. Results are representative of two independent experiments. RNA extraction and RT-PCR analysis were conducted by growing pneumococci as for the ICP-MS analyses, after which 500 μl of culture was mixed with 1 ml of RNA protect (Qiagen). RNA was extracted and purified using an RNeasy Protect Bacteria Mini Kit (Qiagen) after enzymatic lysis using lysozyme and mutanolysin, all according to the manufacturer’s instructions. The total RNA samples were treated with DNase I (Roche) and qRT-PCR was carried out using a SuperScript III OneStep RT-PCR kit (Invitrogen) on Roche LC480 Real-Time Cycler (Roche). Transcription levels of genes analysed were normalized to those obtained for 16S. Primer sequences are available in Table S3.

AdcR binding site identification The sequences of the putative AdcR binding sites from S. pneumoniae as described (Panina et al., 2003) were aligned using ClustalW2 (Krogh et al., 1994) and a subse© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 834–851

AdcA and pneumococcal zinc homeostasis 849

quent weight matrix was generated using HMMER 2.0 as an integral tool of UGENE (Okonechnikov et al., 2012). The AdcR binding sites identified in the genome of strain D39 with an E-value < 0.005 have been listed in Table S4. The E-values as displayed in Fig. 4A were obtained by iterative HMMER 2.0 analysis of the sequences listed in Table S4.

Construction of mutant strains Primers were designed to replace the adcA or adcCBA genes with a chloramphenicol acetyltransferase gene, or the adcAII gene with a spectinomycin adenyltransferase gene, by overlap extension PCR (Ogunniyi et al., 2009). For the ΔadcAII mutant strain, the spectinomycin adenyltransferase gene was then removed by back-transformation with the flanking regions of adcAII that had been joined together by overlap extension PCR (Ogunniyi et al., 2009). These gene deletions were also introduced into a ΔphtABDE mutant strain, the construction of which was described previously (Ogunniyi et al., 2009). Primer sequences are presented in Table S3.

Animal models of disease Five to six week old female CD1 (Swiss) mice were used in all experiments. For the pathogenesis experiment (in which numbers of bacteria recovered from mice were counted), and for the competition experiment, a dose of approximately 1 × 107 cfu per mouse was used; for the virulence experiment (in which survival times were measured), the dose was approximately 2 × 107 cfu per mouse. Processing of tissues and enumeration of cfu in these samples was performed as described previously (Harvey et al., 2011). All procedures performed in this study were conducted with a view to minimizing the discomfort of the animals, and used the minimum numbers to generate reproducible and statistically significant data. All experiments were approved by the University of Adelaide Animal Ethics Committee (Animal Welfare Assurance number A5491-01; project approval number S-2010001) and were performed in strict adherence to guidelines dictated by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Acknowledgements This work was supported by the Australian Research Council (ARC) grant DP120103957 to C.A.M., and the National Health & Medical Research Council (NHMRC) Project grant 1022240 to C.A.M and Program grant 565526 to J.C.P. and B.K. M.L.O. holds an ARC DECRA. B.K. is a NHMRC Senior Research Fellow. J.C.P. is a NHMRC Senior Principal Research Fellow. We thank Dr C. Adolphe, Professor A. G. McEwan and Professor A. Mark for discussions.

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