scholarly journals Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase inStaphylococcus aureus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Thanh Nguyen ◽  
Truc Kim ◽  
Hai Minh Ta ◽  
Won Sik Yeo ◽  
Jongkeun Choi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Viability ◽  
Structure Function ◽  
Active Site ◽  
Bacterial Cell ◽  
Content Type ◽  
Bacterial Cell Death ◽  
Mannitol Metabolism ◽  
Key Enzyme ◽  
Antibacterial Target

ABSTRACTMannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme inStaphylococcus aureusmannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH fromS. aureusUSA300, a strain of community-associated methicillin-resistantS. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target.In vitrocell viability assessment of ΔmtlDknockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor ofS. aureusM1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions ofSaM1PDH at the atomic level, the crystal structure ofSaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to theSaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we proposeSaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors ofSaM1PDH based on structure-function studies.IMPORTANCEDue to the shortage of effective antibiotics against drug-resistantStaphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase ofS. aureusUSA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of usingSaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with aSaM1PDH inhibitor or knockout of the gene encodingSaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism ofSaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses ofSaM1PDH. Our results strongly support the concept that targeting ofSaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

scholarly journals Novel Quorum-Sensing Peptides Mediating Interspecies Bacterial Cell Death

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Sathish Kumar ◽  
Ilana Kolodkin-Gal ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Bacterial Cell ◽  
Content Type ◽  
E Coli ◽  
New Class ◽  
Bacterial Cell Death ◽  
Induced Module

ABSTRACTEscherichia colimazEFis a toxin-antitoxin stress-induced module mediating cell death. It requires the quorum-sensing signal (QS) “extracellular death factor” (EDF), the penta-peptide NNWNN (EcEDF), enhancing the endoribonucleolytic activity ofE. colitoxin MazF. Here we discovered thatE. coli mazEF-mediated cell death could be triggered by QS peptides from the supernatants (SN) of the Gram-positive bacteriumBacillus subtilisand the Gram-negative bacteriumPseudomonas aeruginosa. In the SN ofB. subtilis, we found one EDF, the hexapeptide RGQQNE, calledBsEDF. In the SN ofP. aeruginosa, we found three EDFs: the nonapeptide INEQTVVTK, calledPaEDF-1, and two hexadecapeptides, VEVSDDGSGGNTSLSQ, calledPaEDF-2, and APKLSDGAAAGYVTKA, calledPaEDF-3. When added to a dilutedE. colicultures, each of these peptides acted as an interspecies EDF that triggeredmazEF-mediated death. Furthermore, though their sequences are very different, each of these EDFs amplified the endoribonucleolytic activity ofE. coliMazF, probably by interacting with different sites onE. coliMazF. Finally, we suggest that EDFs may become the basis for a new class of antibiotics that trigger death from outside the bacterial cells.IMPORTANCEBacteria communicate with one another via quorum-sensing signal (QS) molecules. QS provides a mechanism for bacteria to monitor each other’s presence and to modulate gene expression in response to population density. Previously, we addedE. coliEDF (EcEDF), the peptide NNWNN, to this list of QS molecules. Here we extended the group of QS peptides to several additional different peptides. The new EDFs are produced by two other bacteria,Bacillus subtilisandPseudomonas aeruginosa. Thus, in this study we established a “new family of EDFs.” This family provides the first example of quorum-sensing molecules participating in interspecies bacterial cell death. Furthermore, each of these peptides provides the basis of a new class of antibiotics triggering death by acting from outside the cell.

scholarly journals In Staphylococcus aureus, the Particulate State of the Cell Envelope Is Required for the Efficient Induction of Host Defense Responses

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
ByungHyun Kim ◽  
TingTing Jiang ◽  
Jun-Hyun Bae ◽  
Hye Su Yun ◽  
Seong Han Jang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Peritoneal Cavity ◽  
Immune Activation ◽  
Bacterial Cell ◽  
Physical State ◽  
Cell Envelope ◽  
Innate Immune ◽  
Soluble Form ◽  
Content Type ◽  
Bacterial Cell Envelope

ABSTRACT Upon microbial infection, host immune cells recognize bacterial cell envelope components through cognate receptors. Although bacterial cell envelope components function as innate immune molecules, the role of the physical state of the bacterial cell envelope (i.e., particulate versus soluble) in host immune activation has not been clearly defined. Here, using two different forms of the staphylococcal cell envelope of Staphylococcus aureus RN4220 and USA300 LAC strains, we provide biochemical and immunological evidence that the particulate state is required for the effective activation of host innate immune responses. In a murine model of peritoneal infection, the particulate form of the staphylococcal cell envelope (PCE) induced the production of chemokine (C-X-C motif) ligand 1 (CXCL1) and CC chemokine ligand 2 (CCL2), the chemotactic cytokines for neutrophils and monocytes, respectively, resulting in a strong influx of the phagocytes into the peritoneal cavity. In contrast, compared with PCE, the soluble form of cell envelope (SCE), which was derived from PCE by treatment with cell wall-hydrolyzing enzymes, showed minimal activity. PCE also induced the secretion of calprotectin (myeloid-related protein 8/14 [MRP8/14] complex), a phagocyte-derived antimicrobial protein, into the peritoneal cavity at a much higher level than did SCE. The injected PCE particles were phagocytosed by the infiltrated neutrophils and monocytes and then delivered to mediastinal draining lymph nodes. More importantly, intraperitoneally (i.p.) injected PCE efficiently protected mice from S. aureus infection, which was abolished by the depletion of either monocytes/macrophages or neutrophils. This study demonstrated that the physical state of bacterial cells is a critical factor for efficient host immune activation and the protection of hosts from staphylococcal infections.

scholarly journals Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

2012 ◽  
Vol 56 (8) ◽  
pp. 4095-4102 ◽  
Author(s):  
Steven D. Podos ◽  
Jane A. Thanassi ◽  
Michael J. Pucci
Keyword(s):  
Staphylococcus Aureus ◽  
Competitive Inhibition ◽  
Resistance Mechanism ◽  
Dna Ligase ◽  
Adenylation Domain ◽  
Content Type ◽  
Antistaphylococcal Activity ◽  
Ob Fold ◽  
Antibacterial Target

ABSTRACTWe report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD+-dependent DNA ligase (LigA) as an antibacterial target inStaphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [Ki] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC50] = 28 nM) or its isolated adenylation domain (IC50= 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistantS. aureus(MSSA and MRSA) strains (MIC = 1.0 μg/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 × 10−7) of high-level resistant mutants (MIC > 64) with associatedligAlesions. There were no observable effects on growth rate in these mutants. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains.In vitrocharacterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrateKmand a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy.

Comparison of performances of flexible sensors on foil and paper for efficient bacterial concentration measurement

Sensor Review ◽  
2020 ◽  
Vol 40 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Tijana Kojic ◽  
Milan Radovanovic ◽  
Goran M. Stojanovic ◽  
Bojana Pivas ◽  
Deana Medic ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Resonant Frequency ◽  
Bacterial Cell ◽  
Physiological Saline ◽  
Bacterial Count ◽  
Cell Counting ◽  
Bacterial Cells ◽  
Content Type ◽  
Flexible Sensors

Purpose The purpose of this study was to develop flexible sensors for detection of different concentrations of bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus, in saline. Design/methodology/approach The sensors were fabricated using ink-jet printing technology and they consist of a pair of silver interdigitated electrodes printed on mechanically flexible substrates – foil and paper. In house measurement setup for testing and characterization of sensors has been developed. Structural, electrical and mechanical properties of flexible sensors have been determined and compared. Findings The characteristics of sensor – the resonant frequency as a function of different concentrations of each bacteria – are presented. The obtained results demonstrate different resonant frequencies for each dilution of Pseudomonas aeruginosa and Staphylococcus aureus in physiological saline. Research limitations/implications Both sensors showed accurate measurements of bacterial count, which can be achieved with detection of resonant frequency, and this is reflective of the number of bacterial cells within a sample. Practical implications The findings suggest that the newly developed method based on measuring resonant frequency corresponds well with bacterial cell count, thus establishing a new proof-of-concept that such method can have significant applications in bacterial cell counting that are economic and easily maintained. Social implications Fast, cost-effective, accurate and non-invasive method for detection of different bacteria from saline was developed. Originality/value For the first time, comparison between performances of flexible sensors on foil and paper for bacteria detection is demonstrated. Almost linear dependence between shift of resonant frequency of developed sensors and concentration of bacteria has been obtained.

scholarly journals Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

BMC Genomics ◽  
2008 ◽  
Vol 9 (1) ◽  
pp. 590 ◽  
Author(s):  
Remco Kort ◽  
Bart J Keijser ◽  
Martien PM Caspers ◽  
Frank H Schuren ◽  
Roy Montijn
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Bacterial Cell ◽  
Transcriptional Activity ◽  
Molecular Biomarkers ◽  
Bacterial Cell Death

scholarly journals Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b

Antibiotics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 373
Author(s):  
Adrien Boes ◽  
Jean Michel Brunel ◽  
Adeline Derouaux ◽  
Frédéric Kerff ◽  
Ahmed Bouhss ◽  
...  
Keyword(s):  
Fluorescence Anisotropy ◽  
Bacterial Cell ◽  
Chain Elongation ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Lipid Ii ◽  
Bacterial Cell Death ◽  
Glycan Chain ◽  
Competitive Inhibitors ◽  
Antibacterial Target

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

scholarly journals Antisense Growth Inhibition of Methicillin-Resistant Staphylococcus aureus by Locked Nucleic Acid Conjugated with Cell-Penetrating Peptide as a Novel FtsZ Inhibitor

2014 ◽  
Vol 59 (2) ◽  
pp. 914-922 ◽  
Author(s):  
Jingru Meng ◽  
Fei Da ◽  
Xue Ma ◽  
Ning Wang ◽  
Yukun Wang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Division ◽  
Nucleic Acid ◽  
Bacterial Cell ◽  
Locked Nucleic Acid ◽  
Bacterial Cell Division ◽  
Inhibitory Effects ◽  
Content Type ◽  
Division Process

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) infections are becoming increasingly difficult to treat, owing to acquired antibiotic resistance. The emergence and spread of MRSA limit therapeutic options and require new therapeutic strategies, including novel MRSA-active antibiotics. Filamentous temperature-sensitive protein Z (FtsZ) is a highly conserved bacterial tubulin homologue that is essential for controlling the bacterial cell division process in different species ofS. aureus. We conjugated a locked nucleic acid (LNA) that targetedftsZmRNA with the peptide (KFF)3K, to generate peptide-LNA (PLNA). The present study aimed to investigate whether PLNA could be used as a novel antibacterial agent. PLNA787, the most active agent synthesized, exhibited promising inhibitory effects on four pathogenicS. aureusstrainsin vitro. PLNA787 inhibited bacterial growth and resolved lethal Mu50 infections in epithelial cell cultures. PLNA787 also improved the survival rates of Mu50-infected mice and was associated with reductions of bacterial titers in several tissue types. The inhibitory effects onftsZmRNA and FtsZ protein expression and inhibition of the bacterial cell division process are considered to be the major mechanisms of PLNA. PLNA787 demonstrated activity against MRSA infectionsin vitroandin vivo. Our findings suggest thatftsZmRNA is a promising new target for developing novel antisense antibiotics.

scholarly journals Gallium Nitrate Is Efficacious in Murine Models of Tuberculosis and Inhibits Key Bacterial Fe-Dependent Enzymes

2013 ◽  
Vol 57 (12) ◽  
pp. 6074-6080 ◽  
Author(s):  
Oyebode Olakanmi ◽  
Banurekha Kesavalu ◽  
Rajamouli Pasula ◽  
Maher Y. Abdalla ◽  
Larry S. Schlesinger ◽  
...  
Keyword(s):  
Active Site ◽  
Scid Mice ◽  
Infection Model ◽  
Gallium Nitrate ◽  
Murine Models ◽  
Antituberculous Therapy ◽  
Content Type ◽  
Therapeutic Uses ◽  
Key Enzyme ◽  
Mycobacterial Growth

ABSTRACTAcquiring iron (Fe) is critical to the metabolism and growth ofMycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga3+from Fe3+. Unlike Fe3+, Ga3+cannot be physiologically reduced to Ga2+. Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth ofM. tuberculosisin broth and within cultured human macrophages. We now report that Ga(NO3)3shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally withM. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3died. All Ga(NO3)3-treated mice survived.M. tuberculosisCFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga onM. tuberculosisribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreasedM. tuberculosisRR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3shows efficacy in murineM. tuberculosisinfection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Ga's mechanism of action and to optimize delivery forms for possible therapeutic uses in humans.

scholarly journals Potent Sub-MIC Effect of GSK1322322 and Other Peptide Deformylase Inhibitors onIn VitroGrowth of Staphylococcus aureus

2013 ◽  
Vol 58 (1) ◽  
pp. 290-296 ◽  
Author(s):  
Deborah Butler ◽  
Dongzhao Chen ◽  
Karen O'Dwyer ◽  
Thomas Lewandowski ◽  
Kelly Aubart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Alternative Therapy ◽  
Multidrug Resistant ◽  
Peptide Deformylase ◽  
Content Type ◽  
Inhibition Of Growth ◽  
Serious Infections ◽  
Antibacterial Target

ABSTRACTPeptide deformylase (PDF), a clinically unexploited antibacterial target, plays an essential role in protein maturation. PDF inhibitors, therefore, represent a new antibiotic class with a unique mode of action that provides an alternative therapy for the treatment of infections caused by drug-resistant pathogens, including methicillin-resistantStaphylococcus aureus(MRSA). GSK1322322 is a novel PDF inhibitor that is in phase II clinical development for the treatment of lower respiratory tract and skin infections. We have discovered that PDF inhibitors can preventS. aureusin vitrogrowth for up to 6 h at concentrations 8- to 32-fold below their MICs. This phenomenon seems specific to PDF inhibitors, as none of the antimicrobial agents with alternative mechanisms of action tested show such a potent and widespread effect. It also appears limited toS. aureus, as PDF inhibitors do not show such an inhibition of growth at sub-MIC levels inStreptococcus pneumoniaeorHaemophilus influenzae. Analysis of the effect of GSK1322322 on the early growth of 100 randomly selectedS. aureusstrains showed that concentrations equal to or below 1/8× MIC inhibited growth of 91% of the strains tested for 6 h, while the corresponding amount of moxifloxacin or linezolid only affected the growth of 1% and 6% of strains, respectively. Furthermore, the sub-MIC effect demonstrated by GSK1322322 appears more substantial on those strains at the higher end of the MIC spectrum. These effects may impact the clinical efficacy of GSK1322322 in serious infections caused by multidrug-resistantS. aureus.

scholarly journals Impact of Granulocytes on the Antimicrobial Effect of Tedizolid in a Mouse Thigh Infection Model

2011 ◽  
Vol 55 (11) ◽  
pp. 5300-5305 ◽  
Author(s):  
G. L. Drusano ◽  
Weiguo Liu ◽  
Robert Kulawy ◽  
Arnold Louie
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Cell ◽  
Antimicrobial Effect ◽  
Cell Killing ◽  
Infection Model ◽  
Killing Effect ◽  
Content Type ◽  
Exposure Response ◽  
Therapy Initiation ◽  
Antistaphylococcal Activity

ABSTRACTTedizolid (TR-700, formerly torezolid) is the active component of the new oxazolidinone prodrug tedizolid phosphate (TR-701). We had previously demonstrated that tedizolid possessed potent antistaphylococcal activity superior to that of linezolid in a neutropenic mouse thigh infection model (A. Louie, W. Liu, R. Kulawy, and G. L. Drusano, Antimicrob. Agents Chemother. 55:3453-3460, 2011). In the current investigation, we used a mouse thigh infection model to delineate the effect of an interaction of TR-700 and granulocytes on staphylococcal cell killing. We compared the antistaphylococcal killing effect of doses of TR-701 equivalent to human exposures ranging from 200 to 3,200 mg/day in both granulocytopenic and normal mice. The mice were evaluated at 24, 48, and 72 h after therapy initiation. In granulocytopenic mice, a clear exposure response in which, depending on the time point of evaluation, stasis was achieved at “human-equivalent” doses of slightly below 2,300 mg/day (at 24 h) to slightly below 2,000 mg/day (at 72 h) was observed. In immune-normal animals, stasis was achieved at human-equivalent doses of slightly greater than 100 mg/day or less. The variance in bacterial cell killing results was attributable to the presence of granulocytes (without drug), the direct effect of TR-700 onStaphylococcus aureus, and the effect of the drug onStaphylococcus aureusmediated through granulocytes. The majority of the bacterial cell killing in normal animals was attributable to the effect of TR-700 mediated through granulocytes. Additional studies need to be undertaken to elucidate the mechanism underlying this observation.

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