The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCEClostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)33. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)33 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV-visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV-visible spectrophotometric studies. UV-visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M-1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ±â€¯5.2 µM when compared to IC50 = 13.75 ±â€¯1.08 µM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes.

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100µM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. IMPORTANCE: Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly.

Kinetics and mechanism of the oxidation of a cobaloxime by sodium hypochlorite in aqueous solution: Is it an outer-sphere mechanism?

The kinetics and mechanism of the oxidation of [Co(dmgBF2)2(OH2)2] (where dmgBF2 = difluoroboryldimethylglyoximato) by sodium hypochlorite (NaOCl) were investigated by stopped-flow spectrophotometry at 450 nm over the temperature range of 10 °C ≤ θ ≤ 25 °C, pH range of 5.0 ≤ pH ≤ 7.8, and at an ionic strength of 0.60 M (NaCl). The pKa1 value for [Co(dmgBF2)2(H2O)2] was calculated as 5.27 ± 0.14 at I = 0.60 (NaCl). The redox process was dependent on pH and oxidant concentration in a complex manner, that is, kobs = ((k2[H+] + k1Ka)/([H+] + Ka))[OCl-]T, where at 25.3 °C, k1 was calculated as 3.54 × 104 M-1 s-1, and k2 as 2.51 × 104 M-1 cm-1. At a constant pH value, while varying the concentration of sodium hypochlorite two rate constants were calculated, viz., k'1 = 7.56 s-1 (which corresponded to a reaction pathway independent of the NaOCl concentration) and k'2 = 2.26 × 104 M-1 s-1, which was dependent on the concentration of NaOCl. From the variation in pH, [Formula: see text], and [Formula: see text] were calculated as 58 ± 16 kJ mol-1, 46 ± 1 kJ mol-1, 34 ± 55 J mol-1 K-1, and -6 ± 4 Jmol-1 K-1, respectively. The self-exchange rate constant, k11, for sodium hypochlorite (as ClO-) was calculated to be 1.2 × 103 M-1 s-1, where an outer-sphere electron transfer mechanism was assumed. A green product, [Co(dmgBF2)2(OH2)(OH)]·1.75NaOCl, which can react with DMSO, was isolated from the reaction at pH 8.04 with a yield of 13%.

Computational, electrochemical, and spectroscopic studies of two mononuclear cobaloximes: the influence of an axial pyridine and solvent on the redox behaviour and evidence for pyridine coordination to cobalt(i) and cobalt(ii) metal centres.

[Co(dmgBF2)2(H2O)2] (where dmgBF2 = difluoroboryldimethylglyoximato) was used to synthesize [Co(dmgBF2)2(H2O)(py)]·0.5(CH3)2CO (where py = pyridine) in acetone. The formulation of complex was confirmed by elemental analysis, high resolution MS, and various spectroscopic techniques. The complex [Co(dmgBF2)2(solv)(py)] (where solv = solvent) was readily formed in situ upon the addition of pyridine to complex . A spectrophotometric titration involving complex and pyridine proved the formation of such a species, with formation constants, log K = 5.5, 5.1, 5.0, 4.4, and 3.1 in 2-butanone, dichloromethane, acetone, 1,2-difluorobenzene/acetone (4 : 1, v/v), and acetonitrile, respectively, at 20 °C. In strongly coordinating solvents, such as acetonitrile, the lower magnitude of K along with cyclic voltammetry, NMR, and UV-visible spectroscopic measurements indicated extensive dissociation of the axial pyridine. In strongly coordinating solvents, [Co(dmgBF2)2(solv)(py)] can only be distinguished from [Co(dmgBF2)2(solv)2] upon addition of an excess of pyridine, however, in weakly coordinating solvents the distinctions were apparent without the need for excess pyridine. The coordination of pyridine to the cobalt(ii) centre diminished the peak current at the Epc value of the Co(I/0) redox couple, which was indicative of the relative position of the reaction equilibrium. Herein we report the first experimental and theoretical (59)Co NMR spectroscopic data for the formation of Co(i) species of reduced cobaloximes in the presence and absence of py (and its derivatives) in CD3CN. From spectroelectrochemical studies, it was found that pyridine coordination to a cobalt(i) metal centre is more favourable than coordination to a cobalt(ii) metal centre as evident by the larger formation constant, log K = 4.6 versus 3.1, respectively, in acetonitrile at 20 °C. The electrosynthesis of hydrogen by complexes and in various solvents demonstrated the dramatic effects of the axial ligand and the solvent on the turnover number of the respective catalyst.