scholarly journals Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism

2020 ◽  
Vol 6 (9) ◽  
pp. eaaz0260 ◽  
Author(s):  
Jessica Jones-Carson ◽  
Atsushi Yahashiri ◽  
Ju-Sim Kim ◽  
Lin Liu ◽  
Liam F. Fitzsimmons ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
De Novo ◽  
Purine Metabolism ◽  
Bacterial Cell Division ◽  
Nucleotide Biosynthesis ◽  
Nucleoside Triphosphates ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Quinol Oxidases ◽  
Monophosphate Dehydrogenase

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5′-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5′-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

scholarly journals Interaction of Temporin-L Analogues with the E. coli FtsZ Protein

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 704
Author(s):  
Angela Di Somma ◽  
Carolina Canè ◽  
Antonio Moretta ◽  
Angela Duilio
Keyword(s):  
Protein Interactions ◽  
Bacterial Infections ◽  
Antibacterial Properties ◽  
Bacterial Cell Division ◽  
Structural Determinants ◽  
E Coli ◽  
Functional Studies ◽  
Division Process ◽  
Z Ring ◽  
Peptide Complexes

The research of new therapeutic agents to fight bacterial infections has recently focused on the investigation of antimicrobial peptides (AMPs), the most common weapon that all organisms produce to prevent invasion by external pathogens. Among AMPs, the amphibian Temporins constitute a well-known family with high antibacterial properties against Gram-positive and Gram-negative bacteria. In particular, Temporin-L was shown to affect bacterial cell division by inhibiting FtsZ, a tubulin-like protein involved in the crucial step of Z-ring formation at the beginning of the division process. As FtsZ represents a leading target for new antibacterial compounds, in this paper we investigated in detail the interaction of Temporin L with Escherichia coli FtsZ and designed two TL analogues in an attempt to increase peptide-protein interactions and to better understand the structural determinants leading to FtsZ inhibition. The results demonstrated that the TL analogues improved their binding to FtsZ, originating stable protein-peptide complexes. Functional studies showed that both peptides were endowed with a high capability of inhibiting both the enzymatic and polymerization activities of the protein. Moreover, the TL analogues were able to inhibit bacterial growth at low micromolar concentrations. These observations may open up the way to the development of novel peptide or peptidomimetic drugs tailored to bind FtsZ, hampering a crucial process of bacterial life that might be proposed for future pharmaceutical applications.

scholarly journals Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

2013 ◽  
Vol 210 (7) ◽  
pp. 1433-1445 ◽  
Author(s):  
Nataša Obermajer ◽  
Jeffrey L. Wong ◽  
Robert P. Edwards ◽  
Kong Chen ◽  
Melanie Scott ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
T Cells ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
De Novo ◽  
Inflammatory Diseases ◽  
Suppressor Cells ◽  
Guanosine Monophosphate ◽  
No Production ◽  
No Signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)+IL-23R+IL-17+ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4+ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4+ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

scholarly journals Induction of nitric oxide synthesis in J774 cells lowers intracellular glutathione: effect of modulated glutathione redox status on nitric oxide synthase induction

1997 ◽  
Vol 322 (2) ◽  
pp. 477-481 ◽  
Author(s):  
John S. HOTHERSALL ◽  
Fernando Q. CUNHA ◽  
Guy H. NEILD ◽  
Alberto A. NOROHNA-DUTRA
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
De Novo ◽  
Redox Status ◽  
No Production ◽  
Duration Of Treatment ◽  
Intracellular Glutathione ◽  
J774 Cells ◽  
Oxide Synthase ◽  
Glutathione Redox

Under pathological conditions, the induction of nitric oxide synthase (NOS) in macrophages is responsible for NO production to a cytotoxic concentration. We have investigated changes to, and the role of, intracellular glutathione in NO production by the activated murine macrophage cell line J774. Total glutathione concentrations (reduced, GSH, plus the disulphide, GSSG) were decreased to 45% of the control 48 h after cells were activated with bacterial lipopolysaccharide plus interferon γ. This was accompanied by a decrease in the GSH/GSSG ratio from 12:1 to 2:1. The intracellular decrease was not accounted for by either GSH or GSSG efflux; on the contrary, rapid export of glutathione in control cells was abrogated during activation. The loss of intra- and extracellular glutathione indicates either a decrease in synthesis de novo, or an increase in utilization, rather than competition for available NADPH. All changes in activated cells were prevented by pretreatment with the NOS inhibitor l-N-(1-iminoethyl)ornithine. Basal glutathione levels in J774 cells were manipulated by pretreatment with (1) buthionine sulphoximine (glutathione synthase inhibitor), (2) acivicin (γ-glutamyltranspeptidase inhibitor), (3) bromo-octane (glutathione S-transferase substrate) and (4) diamide/zinc (thiol oxidant and glutathione reductase inhibitor). All treatments significantly decreased the output of NO following activation. The degree of inhibition was dependent on (i) duration of treatment prior to activation, (ii) rate of depletion or subsequent recovery and (iii) thiol end product. The level of GSH did not significantly affect the production of NO, after induction of NOS. Thus, glutathione redox status appears to plays an important role in NOS induction during macrophage activation.

scholarly journals Effect of hypoxia on purine metabolism of human skeletal muscle cells

Biotecnia ◽  
2021 ◽  
Vol 23 (2) ◽  
Author(s):  
Tania Zenteno-Savín ◽  
Crisalejandra Rivera-Pérez ◽  
Ramón Gaxiola-Robles ◽  
Norma Olguín-Monroy ◽  
Orlando Lugo-Lugo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Muscle Cells ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Purine Metabolism ◽  
Inosine Monophosphate Dehydrogenase ◽  
Inosine Monophosphate ◽  
Hypoxic Conditions ◽  
Human Skeletal Muscle Cells

Mammals experience some degree of hypoxia during their lifetime. In response to hypoxic challenge, mammalian cells orchestrate specific responses at transcriptional and posttranslational level which lead to changes in the purine metabolites in order to cope with threatening conditions. The aim of this study was to evaluate the response of the enzymes involved in the purine metabolism of human muscle cells to hypoxic conditions. Muscle cells in culture were exposed to hypoxia and the enzymatic activity of inosine monophosphate dehydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) as well as their transcript expression were quantified under normoxic and hypoxic conditions. Purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine diphosphate (GDP) and guanosine triphosphate (GTP)) concentrations were also quantified. Significant reduction of IMPDH activity and HX and IMP concentrations (p < 0.05) were observed after hypoxia, suggesting a decrease of de novo synthesis of purines. After hypoxia a global reduction of transcripts was observed, suggesting a reduction of the metabolic machinery of purine metabolism to new steady states that balance ATP demand and ATP supply pathways.

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