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Emerging Microbes & Infections 2019Transition metals are nutrients essential for life. However, an excess of metals can be toxic to cells, and host-imposed metal toxicity is an important mechanism for...
Transition metals are nutrients essential for life. However, an excess of metals can be toxic to cells, and host-imposed metal toxicity is an important mechanism for controlling bacterial infection. Accordingly, bacteria have evolved metal efflux systems to maintain metal homeostasis. Here, we established that PmtA functions as a ferrous iron [Fe(II)] and cobalt [Co(II)] efflux pump in , an emerging zoonotic pathogen responsible for severe infections in both humans and pigs. expression is induced by Fe(II), Co(II), and nickel [Ni(II)], whereas PmtA protects against Fe(II) and ferric iron [Fe(III)]-induced bactericidal effect, as well as Co(II) and zinc [Zn(II)]-induced bacteriostatic effect. In the presence of elevated concentrations of Fe(II) and Co(II), Δ accumulates high levels of intracellular iron and cobalt, respectively. Δ is also more sensitive to streptonigrin, a Fe(II)-activated antibiotic. Furthermore, growth defects of Δ under Fe(II) or Co(II) excess conditions can be alleviated by manganese [Mn(II)] supplementation. Finally, PmtA plays a role in tolerance to HO-induced oxidative stress, yet is not involved in the virulence of in mice. Together, these data demonstrate that PmtA acts as a Fe(II) and Co(II) efflux pump, and contributes to oxidative stress resistance.
Topics: Bacterial Proteins; Biological Transport, Active; Cobalt; Gene Deletion; Iron; Methyltransferases; Streptococcus suis; Trace Elements
PubMed: 31469035
DOI: 10.1080/22221751.2019.1660233 -
Molecular Carcinogenesis Aug 2005NAD(P)H:quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive...
NAD(P)H:quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species (ROS). The aim of our study was to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated increased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage-independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months.
Topics: Agar; Blotting, Western; Cell Proliferation; Clone Cells; Humans; Immunohistochemistry; NAD(P)H Dehydrogenase (Quinone); Pancreatic Neoplasms; Phenotype; Streptonigrin; Tissue Array Analysis; Tumor Cells, Cultured
PubMed: 16003741
DOI: 10.1002/mc.20107 -
Journal of Bacteriology May 1995The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a...
The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.
Topics: Aeromonas; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacteriophages; Calcium; Cell Membrane; Hemin; Immunoglobulins; Lipopolysaccharides; Macromolecular Substances; Models, Biological; Morphogenesis; Protein Binding; Protoporphyrins
PubMed: 7751277
DOI: 10.1128/jb.177.10.2684-2694.1995 -
American Journal of Physiology. Renal... Nov 2014Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein...
Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.
Topics: Acute Kidney Injury; Amphetamines; Animals; Humans; Hydrolases; Male; Mice, Inbred C57BL; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Reperfusion Injury
PubMed: 25164081
DOI: 10.1152/ajprenal.00243.2014 -
Cell Death & Disease Apr 2013Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on...
Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca(2+) signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca(2+) concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca(2+) or a Ca(2+)-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca(2+) increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells.
Topics: Apoptosis; Calcium; Caspase 7; Cell Communication; Connexin 43; Connexins; Fas-Associated Death Domain Protein; Gap Junctions; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; HeLa Cells; Humans; Poly(ADP-ribose) Polymerases; Signal Transduction; Streptonigrin; Gap Junction alpha-4 Protein
PubMed: 23579271
DOI: 10.1038/cddis.2013.105 -
Scientific Reports Feb 2016Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on...
Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on (2S,3R)-β-methyltryptophan as an intermediate. In this study, the biosynthesis of (2S,3R)-β-methyltryptophan and its isomer (2S,3S)-β-methyltryptophan by enzymes from the streptonigrin biosynthetic pathway is demonstrated. StnR is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase that catalyzes a transamination between L-tryptophan and β-methyl indolepyruvate. StnQ1 is an S-adenosylmethionine (SAM)-dependent C-methyltransferase and catalyzes β-methylation of indolepyruvate to generate (R)-β-methyl indolepyruvate. Although StnR exhibited a significant preference for (S)-β-methyl indolepyruvate over the (R)-epimer, StnQ1 and StnR together catalyze (2S,3R)-β-methyltryptophan formation from L-tryptophan. StnK3 is a cupin superfamily protein responsible for conversion of (R)-β-methyl indolepyruvate to its (S)-epimer and enables (2S,3S)-β-methyltryptophan biosynthesis from L-tryptophan when combined with StnQ1 and StnR. Most importantly, (2S,3S)-β-methyltryptophan was established as the biosynthetic intermediate of the streptonigrin pathway by feeding experiments with a knockout mutant, contradicting the previous proposal that stated (2S,3R)-β-methyltryptophan as the intermediate. These data set the stage for the complete elucidation of the streptonigrin biosynthetic pathway, which would unlock the potential of creating new streptonigrin analogues by genetic manipulation of the biosynthetic machinery.
Topics: Antineoplastic Agents; Bacterial Proteins; Chromatography, High Pressure Liquid; Escherichia coli; Mass Spectrometry; Methyltransferases; Plasmids; S-Adenosylmethionine; Stereoisomerism; Streptomyces; Streptonigrin; Tryptophan
PubMed: 26847951
DOI: 10.1038/srep20273 -
Infection and Immunity Jul 2018Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP)...
Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression Here, we determined whether 1E2 affects ST3 gene expression during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the operon, which encodes an iron uptake system, and decreased the expression of , which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. , 1E2 induced dose-dependent ST3 growth arrest and altered and expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding , suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.
Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Capsules; Female; Gene Expression; Mice; Mice, Inbred C57BL; Nasopharynx; Oxidative Stress; Pneumococcal Vaccines; Streptococcus pneumoniae
PubMed: 29735523
DOI: 10.1128/IAI.00300-18 -
Applied and Environmental Microbiology Aug 2007In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur...
In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.
Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Bacterial Proteins; Gene Expression Regulation, Bacterial; Heat-Shock Response; Homeostasis; Iron; Manganese; Molecular Sequence Data; Oxidative Stress; Plant Diseases; Repressor Proteins; Nicotiana; Virulence
PubMed: 17545320
DOI: 10.1128/AEM.00531-07 -
Microbiology (Reading, England) May 2012Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616...
Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H(2)O(2)), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H(2)O(2)-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur-oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H(2)O(2), superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur-oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.
Topics: Bacterial Proteins; Burkholderia; Catalase; DNA, Bacterial; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Iron; Mutation; Nitrites; Oxidative Stress; Peroxidases; Peroxiredoxins; Phenotype; Repressor Proteins; Sequence Analysis, DNA; Suppression, Genetic
PubMed: 22361941
DOI: 10.1099/mic.0.057372-0 -
Scientific Reports Jun 2020Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung...
Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung cancer cell lines with high (H1975) and very low (H661) baseline expression of SERPINA1 gene encoding AAT protein. Comparison of cells grown for 3 weeks in a regular medium versus medium supplemented with 2 mg/ml of AAT revealed that in the presence of AAT cells acquire better proliferative properties, resistance to staurosporine (STS)-induced apoptosis, and show higher expression of CLU, a pro-tumorigenic gene coding clusterin protein. Similarly, the co-administration of STS with AAT or addition of AAT to the cells pre-treated with STS abrogated effects of STS in both cell lines. Following experiments with H1975 cells have shown that AAT blocks critical steps in STS-induced cell death: inhibition of AKT/MAPK pathways, and activation of caspase 3 and autophagy. AAT does not inhibit apoptosis-triggered by chloroquine (inhibitor of autophagy) or streptonigrin (inducer of p53 pathway). The anti-apoptotic effects of AAT were unaffected by lipopolysaccharide (LPS). However, AAT induced TLR4 levels and enhanced LPS effects on the production of IL-6, a tumor-promoting cytokine. Our data provide further evidence that AAT plays a significant role in the tumorigenesis.
Topics: Apoptosis; Autophagy; Cell Movement; Cell Proliferation; Cytokines; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Lipopolysaccharides; Lung Neoplasms; Protective Agents; Serine Proteinase Inhibitors; Staurosporine; Tumor Cells, Cultured; alpha 1-Antitrypsin
PubMed: 32533048
DOI: 10.1038/s41598-020-66825-w