Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages.
ABSTRACT: Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.
Project description:Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-122]
Project description:Transition metals are central for bacterial virulence and host defense. P(1B)-ATPases are responsible for cytoplasmic metal efflux and play roles either in limiting cytosolic metal concentrations or in the maturation of secreted metalloproteins. The P(1B)-ATPase, CtpC, is required for Mycobacterium tuberculosis survival in a mouse model (Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989-12994). CtpC prevents Zn(2+) toxicity, suggesting a role in Zn(2+) export from the cytosol (Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M., Waddell, S. J., Foti, M., Lugo-Villarino, G., Gao, Q., Maridonneau-Parini, I., Butcher, P. D., Castagnoli, P. R., Gicquel, B., de Chastellièr, C., and Neyrolles, O. (2011) Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10, 248-259). However, key metal-coordinating residues and the overall structure of CtpC are distinct from Zn(2+)-ATPases. We found that isolated CtpC has metal-dependent ATPase activity with a strong preference for Mn(2+) over Zn(2+). In vivo, CtpC is unable to complement Escherichia coli lacking a functional Zn(2+)-ATPase. Deletion of M. tuberculosis or Mycobacterium smegmatis ctpC leads to cytosolic Mn(2+) accumulation but no alterations in other metals levels. Whereas ctpC-deficient M. tuberculosis is sensitive to extracellular Zn(2+), the M. smegmatis mutant is not. Both ctpC mutants are sensitive to oxidative stress, which might explain the Zn(2+)-sensitive phenotype of the M. tuberculosis ctpC mutant. CtpC is a high affinity/slow turnover ATPase, suggesting a role in protein metallation. Consistent with this hypothesis, mutation of CtpC leads to a decrease of Mn(2+) bound to secreted proteins and of the activity of secreted Fe/Mn-superoxide dismutase, particularly in M. smegmatis. Alterations in the assembly of metalloenzymes involved in redox stress response might explain the sensitivity of M. tuberculosis ctpC mutants to oxidative stress and growth and persistence defects in mice infection models.
Project description:BACKGROUND:The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS:In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19?±?0.04 µM and Vmax 2.29?±?0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS:The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.
Project description:BACKGROUND:P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. RESULTS:In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. CONCLUSIONS:The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.
Project description:An integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted in Thlaspi caerulescens, a Zn/Cd-hyperaccumulating plant species. A heavy metal transporter cDNA, ZNT1, was cloned from T. caerulescens through functional complementation in yeast and was shown to mediate high-affinity Zn(2+) uptake as well as low-affinity Cd(2+) uptake. It was found that this transporter is expressed at very high levels in roots and shoots of the hyperaccumulator. A study of ZNT1 expression and high-affinity Zn(2+) uptake in roots of T. caerulescens and in a related nonaccumulator, Thlaspi arvense, showed that alteration in the regulation of ZNT1 gene expression by plant Zn status results in the overexpression of this transporter and in increased Zn influx in roots of the hyperaccumulating Thlaspi species. These findings yield insights into the molecular regulation and control of plant heavy metal and micronutrient accumulation and homeostasis, as well as provide information that will contribute to the advancement of phytoremediation by the future engineering of plants with improved heavy metal uptake and tolerance.
Project description:Zinc transporter 1 (ZNT1) is the only zinc transporter predominantly located on the plasma membrane, where it plays a pivotal role exporting cytosolic zinc to the extracellular space. Numerous studies have focused on the physiological and pathological functions of ZNT1. However, its biochemical features remain poorly understood. Here, we investigated the regulation of ZNT1 expression in human and vertebrate cells, and found that ZNT1 expression is posttranslationally regulated by cellular zinc status. We observed that under zinc-sufficient conditions, ZNT1 accumulates on the plasma membrane, consistent with its zinc efflux function. In contrast, under zinc-deficient conditions, ZNT1 molecules on the plasma membrane were endocytosed and degraded through both the proteasomal and lysosomal pathways. Zinc-responsive ZNT1 expression corresponded with that of metallothionein, supporting the idea that ZNT1 and metallothionein cooperatively regulate cellular zinc homeostasis. ZNT1 is N-glycosylated on Asn299 in the extracellular loop between transmembrane domains V and VI, and this appears to be involved in the regulation of ZNT1 stability, as nonglycosylated ZNT1 is more stable. However, this posttranslational modification had no effect on ZNT1's ability to confer cellular resistance against high zinc levels or its subcellular localization. Our results provide molecular insights into ZNT1-mediated regulation of cellular zinc homeostasis, and indicate that the control of cellular and systemic zinc homeostasis via dynamic regulation of ZNT1 expression is more sophisticated than previously thought.
Project description:Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.
Project description:P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (PIB-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, PIB-ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting PIB-ATPase ZntA from Shigella sonnei (SsZntA), aiming at developing tools to assist the characterization of the structure and function of this class of transporters. We identify six different Nbs that bind detergent stabilized SsZntA. We further assess the effect of the Nbs on the catalytic function of SsZntA, and find that five nanobodies associate without affecting the function, while one nanobody significantly reduces the ATPase activity. This study paves the way for more refined mechanistical and structural studies of zinc-transporting PIB-ATPases.
Project description:The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1(-/-) MT(-/-) ZnT4(-/-) cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1(-/-) MT(-/-) ZnT4(-/-) cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1(-/-) MT(-/-) ZnT4(-/-) cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
Project description:BACKGROUND: Lead is a heavy toxic metal element in biological systems and is one of the major pollutants as a result of its widespread use in industries. In spite of its negative roles the coordination chemistry of Pb(II) complexes is a matter of interest. The N,N'-bidentate aromatic bases such as BPY,4-BPY and PHEN (BPY?=?2,2'bipyridine, 4-BPY?=?4,4'-dimethyl-2,2'-bipyridine, PHEN?=?1,10-Phenanthroline) are widely used to build supramolecular architectures because of their excellent coordinating ability and large conjugated system that can easily form ?-? interactions among their aromatic moieties. A series of novel Pb(II) complexes in concert with 5-CTPC, 5-BTPC (5-CTPC?=?5-chlorothiophen-2-carboxylate, 5-BTPC?=?5-bromothiophen-2-carboxylate) and corresponding bidentate chelating N.N' ligands have been synthesized and characterized. RESULTS: Five new Pb (II) complexes [Pb(BPY)(5-CTPC)2] (1), [Pb(4-BPY)(5-CTPC)2] (2), [Pb2(PHEN)2(5-CTPC)4] (3), [Pb(4-BPY)(5-BTPC)2] (4) and [Pb2(PHEN)2(5-BTPC)2(ACE)2] (5) have been synthesized. Even though in all these complexes the molar ratio of Pb, carboxylate, N,N-chelating ligand are the same (1:2:1), there is a significant structural diversity. These complexes have been characterised and investigated by elemental analysis, IR, 1H-NMR,13C-NMR, TGA, and photoluminescence studies. Single crystal X-ray diffraction studies reveal that complexes (1, 2) and (4) are mononuclear while (3 and 5) are dinuclear in nature which may result from the chelating nature of the ligands, various coordination modes of the carboxylates, and the coordination geometry of the Pb(II) ions. CONCLUSIONS: The observation of structures 2,4 and 3,5 show the structural changes made just chloro/bromo substituent of the thiophene ring. A detailed packing analysis has been undertaken to delineate the role of valuable non covalent interactions like X…?, H…X, (X?=?Cl/Br). A quadruple hydrogen bond linking the monomeric units and generating a supramolecular architecture is observed in (1). The metal bite unit comprised of PbN2C2 (i.e. Pb-N-C-C-N-Pb) is the repeating unit in all the five complexes and they have almost same geometrical parameters. This metal bite has been identified as the self assembly unit in complexes.