scholarly journals Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jolanta Krucinska ◽  
Michael N. Lombardo ◽  
Heidi Erlandsen ◽  
Akram Hazeen ◽  
Searle S. Duay ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Antibacterial Properties ◽  
High Energy ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Promising Target ◽  
Antibiotic Discovery ◽  
Growth And Reproduction

AbstractMany years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

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.

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

scholarly journals Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

1971 ◽  
Vol 123 (4) ◽  
pp. 501-505 ◽  
Author(s):  
J. W. Dale
Keyword(s):  
Escherichia Coli ◽  
Host Species ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
R Factor ◽  
Relationship Of ◽  
The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

Synthesis, Characterization, and Antibacterial Evaluation of Curcumin-Sulfanilamide Compound

2020 ◽  
Vol 840 ◽  
pp. 265-269
Author(s):  
Nurjanah Nurjanah ◽  
Endang Saepudin
Keyword(s):  
Antibacterial Activity ◽  
Functional Group ◽  
Biological Activities ◽  
Curcuma Longa ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Ftir Spectrophotometry

Curcumin, a diarylheptanoids compound which isolated primary from Curcuma longa, exhibits a variety of exciting biological activities, including as an antibacterial agent. In the present study, a sulfanilamide-contained curcumin compound was synthesized and characterized to investigate the antibacterial activity against gram-positive bacteria S. aureus, B. subtilis and gram-negative bacteria E. coli. The characterization of the synthesized compound was determined by analysing peak absorbance, functional group, and molecular weight using mass spectroscopy, UV/Vis and FTIR spectrophotometry. Curcumin-sulfanilamide compound exhibited the best antibacterial activity against gram-negative bacteria compared to curcumin and the curcumin-derived compound containing isoxazole with inhibitory zone of 11 mm.

scholarly journals Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

2021 ◽  
Author(s):  
Valérie Biou ◽  
Ricardo Jorge D Adaixo ◽  
Mohamed Chami ◽  
Pierre-Damien Coureux ◽  
Benoist Laurent ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Molecular Motor ◽  
Opportunistic Pathogen ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Class ◽  
Heme Acquisition ◽  
Shed Light

ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens. ExbBSm alone behaves as a stable pentamer, and the complex displays the ExbB5-ExbD2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbBSm and ExbBEc that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbBSm has a ca. 40 residues long periplasmic extension absent in E. coli. Such long ExbBs are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens. We also show that it is involved in heme acquisition via the Has system from S. marcescens. ExbBSm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

scholarly journals Mode of action of the 2-phenylquinoline efflux inhibitor PQQ4R againstEscherichia coli

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3168 ◽  
Author(s):  
Diana Machado ◽  
Laura Fernandes ◽  
Sofia S. Costa ◽  
Rolando Cannalire ◽  
Giuseppe Manfroni ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Gram Negative ◽  
E Coli ◽  
Efflux Pump Inhibitors

Efflux pump inhibitors are of great interest since their use as adjuvants of bacterial chemotherapy can increase the intracellular concentrations of the antibiotics and assist in the battle against the rising of antibiotic-resistant bacteria. In this work, we have described the mode of action of the 2-phenylquinoline efflux inhibitor (4-(2-(piperazin-1-yl)ethoxy)-2-(4-propoxyphenyl) quinolone – PQQ4R), againstEscherichia coli,by studding its efflux inhibitory ability, its synergistic activity in combination with antibiotics, and compared its effects with the inhibitors phenyl-arginine-β-naphthylamide (PAβN) and chlorpromazine (CPZ). The results showed that PQQ4R acts synergistically, in a concentration dependent manner, with antibiotics known to be subject to efflux inE. colireducing their MIC in correlation with the inhibition of their efflux. Real-time fluorometry assays demonstrated that PQQ4R at sub-inhibitory concentrations promote the intracellular accumulation of ethidium bromide inhibiting its efflux similarly to PAβN or CPZ, well-known and described efflux pump inhibitors for Gram-negative bacteria and whose clinical usage is limited by their levels of toxicity at clinical and bacteriological effective concentrations. The time-kill studies showed that PQQ4R, at bactericidal concentrations, has a rapid antimicrobial activity associated with a fast decrease of the intracellular ATP levels. The results also indicated that the mode of action of PQQ4R involves the destabilization of theE. coliinner membrane potential and ATP production impairment, ultimately leading to efflux pump inhibition by interference with the energy required by the efflux systems. At bactericidal concentrations, membrane permeabilization increases and finally ATP is totally depleted leading to cell death. Since drug resistance mediated by the activity of efflux pumps depends largely on the proton motive force (PMF), dissipaters of PMF such as PQQ4R, can be regarded as future adjuvants of conventional therapy againstE. coliand other Gram-negative bacteria, especially their multidrug resistant forms. Their major limitation is the high toxicity for human cells at the concentrations needed to be effective against bacteria. Their future molecular optimization to improve the efflux inhibitory properties and reduce relative toxicity will optimize their potential for clinical usage against multi-drug resistant bacterial infections due to efflux.

scholarly journals Phylogenomics, Epigenomics, Virulome, and Mobilome of Gram-negative Bacteria Co-resistant to Carbapenems and Polymyxins: A One-Health Systematic Review and Meta-analyses

2021 ◽  
Author(s):  
Winnie Thabisa Ramaloko ◽  
John Osei Sekyere
Keyword(s):  
Bacterial Infections ◽  
One Health ◽  
Phylogenetic Analyses ◽  
Restriction Enzymes ◽  
Type I ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Meta Analyses ◽  
Restriction Modification

Gram-negative bacteria (GNB) continue to develop resistance against important antibiotics including last-resort ones such as carbapenems and polymyxins. An analysis of GNB with co-resistance to carbapenems and polymyxins from a One Health perspective is presented. Data of species name, country, source of isolation, resistance genes (ARGs), plasmid type, clones, and mobile genetic elements (MGEs) were deduced from 129 articles from January 2016 to March 2021. Available genomes and plasmids were obtained from PATRIC and NCBI. Resistomes and methylomes were analysed using BAcWGSTdb and REBASE whilst Kaptive was used to predict capsule typing. Plasmids and other MEGs were identified using MGE Finder and ResFinder. Phylogenetic analyses were done using RAxML and annotated with MEGA 7. A total of 877 isolates, 32 genomes and 44 plasmid sequences were analysed. Most of these isolates were reported in Asian countries and were isolated from clinical, animal, and environmental sources. Colistin resistance was mostly mediated by mgrB inactivation, while OXA-48/181 was the most reported carbapenemase. IncX and IncI were the most common plasmids hosting carbapenemases and mcr genes. The isolates were co-resistant to other antibiotics, with floR (chloramphenicol) and fosA3 (fosfomycin) being common; E. coli ST156 and K. pneumoniae ST258 strains were common globally. Virulence genes and capsular KL-types were also detected. Type I, II, III and IV restriction modification systems were detected, comprising various MTases and restriction enzymes. The escalation of highly resistant isolates drains the economy due to untreatable bacterial infections, which leads to increasing global mortality rates and healthcare costs.

scholarly journals Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from Enterobacter cloacae

1999 ◽  
Vol 43 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Yoshimi Matsumoto ◽  
Matsuhisa Inoue
Keyword(s):  
Klebsiella Oxytoca ◽  
Enterobacter Cloacae ◽  
Gram Negative Bacteria ◽  
Phenotypic Characteristics ◽  
Gene Encoding ◽  
E Coli ◽  
Class A ◽  
Citrobacter Diversus ◽  
High Degree

ABSTRACT Enterobacter cloacae 8009 produced an inducible class A β-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other β-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. Thebla gene was transferable to Escherichia coliby electroporation of plasmid DNA. The molecular mass of the β-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A β-lactamases like FEC-1. The gene encoding this β-lactamase was cloned and sequenced. The deduced amino acid sequence of the β-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A β-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the β-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal β-lactamases from Citrobacter diversus(80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intactampR produced a small amount of β-lactamase constitutively, suggesting that AmpR works as an activator ofampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A β-lactamase in gram-negative bacteria.

scholarly journals Characterization of Escherichia coliD-arabinose 5-phosphate isomerase encoded by kpsF: implications for group 2 capsule biosynthesis

2006 ◽  
Vol 395 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Timothy C. Meredith ◽  
Ronald W. Woodard
Keyword(s):  
Biochemical Properties ◽  
Coli Strain ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Catalytic Constant ◽  
Group 2 ◽  
Strain Cft073 ◽  
K Antigen ◽  
Pentose Pathway

In Escherichia coli, there are multiple paralogous copies of the enzyme API [A5P (D-arabinose 5-phosphate) isomerase], which catalyses the conversion of the pentose pathway intermediate Ru5P (D-ribulose 5-phosphate) into A5P. A5P is a precursor of Kdo (3-deoxy-D-manno-octulosonate), an integral carbohydrate component of various glycolipids coating the surface of the OM (outer membrane) of Gram-negative bacteria, including LPS (lipopolysaccharide) and many group 2 K-antigen capsules. The K-antigen-specific API KpsF has been cloned from the uropathogenic E. coli strain CFT073 and its biochemical properties characterized. Purified recombinant KpsF [K-API (K-antigen API)] is tetrameric and has optimal activity at pH 7.8. The enzyme is specific for A5P and Ru5P, with Km (app) values of 0.57 mM for A5P and 0.3 mM for Ru5P. The apparent kcat in the A5P to Ru5P direction is 15 and 19 s−1 in the Ru5P to A5P direction. While most of the properties are quite similar to its LPS API counterpart KdsD, the catalytic constant is nearly 10-fold lower. K-API is now the second Kdo biosynthetic related gene that has been characterized from the kps group 2 capsule cluster.

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