ABSTRACT: Streptococcal solute-binding proteins (SBPs) associated with ATP-binding cassette transporters gained widespread attention first as ostensible adhesins, next as virulence determinants, and finally as metal ion transporters. In this mini-review, we will examine our current understanding of the cellular roles of these proteins, their contribution to metal ion homeostasis, and their crucial involvement in mediating streptococcal virulence. There are now more than 35 studies that have collected structural, biochemical and/or physiological data on the functions of SBPs across a broad range of bacteria. This offers a wealth of data to clarify the formerly puzzling and contentious findings regarding the metal specificity amongst this group of essential bacterial transporters. In particular we will focus on recent findings related to biological roles for manganese in streptococci. These advances will inform efforts aimed at exploiting the importance of manganese and manganese acquisition for the design of new approaches to combat serious streptococcal diseases.
Project description:Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading.
Project description:Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract.
Project description:<i>Streptococcus sanguinis</i> is a primary colonizer of teeth and is typically considered beneficial due to its antagonistic relationship with the cariogenic pathogen <i>Streptococcus mutans</i>. However, <i>S. sanguinis</i> can also act as an opportunistic pathogen should it enter the bloodstream and colonize a damaged heart valve, leading to infective endocarditis. Studies have implicated manganese acquisition as an important virulence determinant in streptococcal endocarditis. A knockout mutant lacking the primary manganese import system in <i>S. sanguinis</i>, SsaACB, is severely attenuated for virulence in an <i>in vivo</i> rabbit model. Manganese is a known cofactor for several important enzymes in <i>S. sanguinis</i>, including superoxide dismutase, SodA, and the aerobic ribonucleotide reductase, NrdEF. To determine the effect of manganese depletion on <i>S. sanguinis</i>, we performed transcriptomic analysis on a ?<i>ssaACB</i> mutant grown in aerobic fermentor conditions after the addition of the metal chelator EDTA. Despite the broad specificity of EDTA, analysis of cellular metal content revealed a decrease in manganese, but not in other metals, that coincided with a drop in growth rate. Subsequent supplementation with manganese, but not iron, zinc, or magnesium, restored growth in the fermentor post-EDTA. Reduced activity of Mn-dependent SodA and NrdEF likely contributed to the decreased growth rate post-EDTA, but did not appear entirely responsible. With the exception of the Dps-like peroxide resistance gene, <i>dpr</i>, manganese depletion did not induce stress response systems. By comparing the transcriptome of ?<i>ssaACB</i> cells pre- and post-EDTA, we determined that manganese deprivation led to altered expression of diverse systems. Manganese depletion also led to an apparent induction of carbon catabolite repression in a glucose-independent manner. The combined results suggest that manganese limitation produces effects in <i>S. sanguinis</i> that are diverse and complex, with no single protein or system appearing entirely responsible for the observed growth rate decrease. This study provides further evidence for the importance of this trace element in streptococcal biology. Future studies will focus on determining mechanisms for regulation, as the multitude of changes observed in this study indicate that multiple regulators may respond to manganese levels.
Project description:ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn(2+) or Mn(2+) specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn(2+) over Mn(2+) specificity, the Zn(2+) ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn(2+)-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn(2+)-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn(2+)-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn(2+) specificity.
Project description:Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens. Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to Escherichia coli YiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the ND-DD A-site motif of MntE and the most N-terminal His in the HD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity.Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogen Streptococcus pneumoniae expresses two metal CDF transporters, CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.
Project description:Secondary active transporters of the SLC11/NRAMP family catalyse the uptake of iron and manganese into cells. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Here we describe the structural and functional properties of the prokaryotic SLC11 transporter EcoDMT. Its crystal structure reveals a previously unknown outward-facing state of the protein family. In proteoliposomes EcoDMT mediates proton-coupled uptake of manganese at low micromolar concentrations. Mutants of residues in the transition-metal ion-binding site severely affect transport, whereas a mutation of a conserved histidine located near this site results in metal ion transport that appears uncoupled to proton transport. Combined with previous results, our study defines the conformational changes underlying transition-metal ion transport in the SLC11 family and it provides molecular insight to its coupling to protons.
Project description:Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448?mntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448?mntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448?mntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448?mntE-?perR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence.Manganese is traditionally viewed as a beneficial metal ion to bacteria, and it is also established that most bacteria can tolerate high concentrations of this transition metal. In this work, we show that in group A Streptococcus, mutation of the mntE locus, which encodes a transport protein of the cation diffusion facilitator (CDF) family, results in accumulation of manganese and sensitivity to this transition metal ion. The toxicity of manganese is indirect and is the result of a failure of the PerR regulator to respond to oxidative stress in the presence of high intracellular manganese concentrations. These results highlight the importance of MntE in manganese homeostasis and maintenance of an optimal manganese/iron ratio in GAS and the impact of manganese on resistance to oxidative stress and virulence.
Project description:The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis.
Project description:SLC30 proteins belong to the cation diffusion facilitator (CDF) superfamily of metal transporters. SLC30A10 mediates manganese efflux, while other SLC30 members transport zinc. Metal specificity of CDFs may be conferred by amino acids that form a transmembrane metal binding site (Site A). Site A of zinc-transporting CDFs, such as SLC30A1/ZnT1, have a HXXXD motif, but manganese transporters, such as SLC30A10, harbor a NXXXD motif. This critical histidine-to-asparagine substitution, at residue 43, was proposed to underlie manganese transport specificity of SLC30A10. However, we recently discovered that asparagine-43 was dispensable for manganese efflux in HeLa cells; instead, glutamate-25, aspartate-40, asparagine-127, and aspartate-248 were required. In contrast, another group reported that asparagine-43 was required in a chicken cell line. The goal of this study was to resolve the divergent results about the requirement of the crucial asparagine-43 residue. For this, we compared the manganese efflux activity of four cell types that stably over-expressed SLC30A10wild-type (WT), SLC30A10N43A or SLC30A10E25A: physiologically-relevant hepatic HepG2 and neuronal AF5 cells, HEK cells, and embryonic fibroblasts from Slc30a10-/- mice. In all cell types, manganese efflux activity of SLC30A10N43A was comparable to WT, while SLC30A10E25A lacked activity. Importantly, unlike SLC30A10, the histidine residue of the HXXXD motif of SLC30A1/ZnT1 was required for zinc transport. These results imply that the mechanisms of ion coordination within the transmembrane domain of SLC30A10 substantially differ from previously-studied CDFs, suggest that factors beyond Site A residues may confer metal specificity to CDFs, and improve understanding of the pathobiology of manganese toxicity due to mutations in SLC30A10.
Project description:Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics.