Christopher A. McDevitt

A Molecular Mechanism for Bacterial Susceptibility to Zinc

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin‐containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop‐terin‐containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron–sulphur clusters. Analysis of the N‐terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec‐dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo‐trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

The DMSO Reductase Family of Microbial Molybdenum Enzymes; Molecular Properties and Role in the Dissimilatory Reduction of Toxic Elements

The dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes is a large and diverse group that is found in bacteria and archaea. These enzymes are characterised by a bis(molybdopterin guanine dinucleotide)Mo form of the molybdenum cofactor, and they are particularly important in anaerobic respiration including the dissimilatory reduction of certain toxic oxoanions. The structural and phylogenetic relationship between the proteins of this family is discussed. High-resolution crystal structures of enzymes of the DMSO reductase family have revealed a high degree of similarity in tertiary structure. However, there is considerable variation in the structure of the molybdenum active site and it seems likely that these subtle but important differences lead to the great diversity of function seen in this family of enzymes. This diversity of catalytic capability is associated with several distinct pathways of electron transport.

Central Role of Manganese in Regulation of Stress Responses, Physiology, and Metabolism in Streptococcus pneumoniae

The importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn(2+) stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn(2+)-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn(2+) having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn(2+) transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn(2+) stress.

Imperfect coordination chemistry facilitates metal ion release in the Psa permease

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.

Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag‐coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size‐exclusion chromatography and shown to have apparent molecular masses of ∼ 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin‐arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin‐arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.

Extracellular Zinc Competitively Inhibits Manganese Uptake and Compromises Oxidative Stress Management in Streptococcus pneumoniae

Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a ‘toxic’ effect on bacterial pathogens, such as S. pneumoniae.

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

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 zinc‐binding 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 carboxy‐terminal 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 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.

The role of ATP-binding cassette transporters in bacterial pathogenicity

The ATP-binding cassette transporter superfamily is present in all three domains of life. This ubiquitous class of integral membrane proteins have diverse biological functions, but their fundamental role involves the unidirectional translocation of compounds across cellular membranes in an ATP coupled process. The importance of this class of proteins in eukaryotic systems is well established as typified by their association with genetic diseases and roles in the multi-drug resistance of cancer. In stark contrast, the ABC transporters of prokaryotes have not been exhaustively investigated due to the sheer number of different roles and organisms in which they function. In this review, we examine the breadth of functions associated with microbial ABC transporters in the context of their contribution to bacterial pathogenicity and virulence.

Dysregulation of Transition Metal Ion Homeostasis is the Molecular Basis for Cadmium Toxicity in Streptococcus Pneumoniae.

Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae, occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress.