scholarly journals Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260023
Author(s):  
Ankit Pandeya ◽  
Ling Yang ◽  
Olaniyi Alegun ◽  
Chamikara Karunasena ◽  
Chad Risko ◽  
...  
Keyword(s):  
Density Functional ◽  
Gene Knockout ◽  
Cell Envelope ◽  
Three Dimensional ◽  
Health Concern ◽  
Gram Negative Bacteria ◽  
Cell Fractionation ◽  
Gram Negative ◽  
Biotin Conjugation ◽  
Dynamics Simulations

Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.

scholarly journals Super-Cationic Peptide Dendrimers—Synthesis and Evaluation as Antimicrobial Agents

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 695
Author(s):  
Estelle J. Ramchuran ◽  
Isabel Pérez-Guillén ◽  
Linda A. Bester ◽  
René Khan ◽  
Fernando Albericio ◽  
...  
Keyword(s):  
Antibacterial Agent ◽  
Antimicrobial Agents ◽  
Health Concern ◽  
Gram Negative Bacteria ◽  
Cationic Peptide ◽  
Public Health Concern ◽  
Gram Negative ◽  
Peptide Dendrimers ◽  
Microbial Infections ◽  
Lead Candidate

Microbial infections are a major public health concern. Antimicrobial peptides (AMPs) have been demonstrated to be a plausible alternative to the current arsenal of drugs that has become inefficient due to multidrug resistance. Herein we describe a new AMP family, namely the super-cationic peptide dendrimers (SCPDs). Although all members of the series exert some antibacterial activity, we propose that special attention should be given to (KLK)2KLLKLL-NH2 (G1KLK-L2KL2), which shows selectivity for Gram-negative bacteria and virtually no cytotoxicity in HepG2 and HEK293. These results reinforce the validity of the SCPD family as a valuable class of AMP and support G1KLK-L2KL2 as a strong lead candidate for the future development of an antibacterial agent against Gram-negative bacteria.

Fine structural observations of Desulfovibrio desulfuricans

1973 ◽  
Vol 19 (6) ◽  
pp. 753-756
Author(s):  
Terrence M. Hammill ◽  
Geno J. Germano
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Cell Envelope ◽  
Desulfovibrio Desulfuricans ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Ultrathin Sections ◽  
Basal Apparatus ◽  
Specialized Region ◽  
Whole Mounts

Glutaraldehyde-fixed, platinum-carbon-shadowed whole mounts, and ultrathin sections of glutaraldehyde-OsO4-fixed cells of Desulfovibrio desulfuricans were observed by electron microscopy. The preparations demonstrated a typical Vibrio form with a single polar flagellum. The cell envelope and the formation of external blebs were shown to be similar to other gram-negative bacteria. The protoplast, apparently devoid of mesosomes or other membranous structures, was densely packed with ribosomes and contained a fibrous nucleoid. A specialized region near the flagellar end of the cell was commonly observed and termed the basal apparatus. Cell division appeared to be by constriction.

scholarly journals Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling

2019 ◽  
Author(s):  
Jehangir Cama ◽  
Margaritis Voliotis ◽  
Jeremy Metz ◽  
Ashley Smith ◽  
Jari Iannucci ◽  
...  
Keyword(s):  
Rational Design ◽  
Cell Envelope ◽  
Time Lapse ◽  
Drug Accumulation ◽  
Cell Uptake ◽  
Gram Negative Bacteria ◽  
Label Free ◽  
Transport Pathways ◽  
Gram Negative ◽  
Accumulation Kinetics

AbstractThe double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to the complexity of the problem and a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to quantify antibiotic uptake label-free in hundreds of individual Escherichia coli cells. By manipulating the microenvironment, we showed that drug (ofloxacin) accumulation is higher in growing versus non-growing cells. Using genetic knockouts, we provide the first direct evidence that growth phase is more important for drug accumulation than the presence or absence of individual transport pathways. We use our experimental results to inform a mathematical model that predicts drug accumulation kinetics in subcellular compartments. These novel experimental and theoretical results pave the way for the rational design of new Gram-negative antibiotics.

Diacylglycerol kinase A is essential for polymyxin resistance provided by EptA, MCR-1 and other lipid A phosphoethanolamine transferases.

2021 ◽  
Author(s):  
Alexandria B. Purcell ◽  
Bradley J. Voss ◽  
M. Stephen Trent
Keyword(s):  
Lipid A ◽  
Cell Envelope ◽  
Diacylglycerol Kinase ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Severe Reduction ◽  
Bacterial Fitness ◽  
Phosphoethanolamine Transferases ◽  
Kinase A

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

scholarly journals How the assembly and protection of the bacterial cell envelope depend on cysteine residues

2020 ◽  
Vol 295 (34) ◽  
pp. 11984-11994 ◽  
Author(s):  
Jean-François Collet ◽  
Seung-Hyun Cho ◽  
Bogdan I. Iorga ◽  
Camille V. Goemans
Keyword(s):  
Amino Acid ◽  
Cell Envelope ◽  
Multilayered Structure ◽  
Cysteine Residues ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Amino Acid Residues ◽  
Gram Negative ◽  
Oxidative Inactivation ◽  
Peptidoglycan Cell Wall

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria.

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Four Reasons to Consider a Novel Class of Innate Immune Molecules in the Oral Epithelium

2003 ◽  
Vol 82 (12) ◽  
pp. 944-950 ◽  
Author(s):  
E.E. LeClair
Keyword(s):  
Host Defense ◽  
Innate Immune System ◽  
Physiological Response ◽  
Three Dimensional ◽  
Innate Immune ◽  
Oral Epithelium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Defense Protein ◽  
Lines Of Evidence

An expanding number of innate immune molecules occupy the “epithelial frontier”. This review introduces a recently recognized class of mammalian proteins with similarity to PLUNC (palate, lung and nasal epithelium clone), which is itself related to the host defense protein BPI (bactericidal/permeability-increasing protein). Four emerging lines of evidence unite the PLUNC-like proteins: conserved genetic structure, epithelial expression, three-dimensional protein similarity, and a physiological response to injury or inflammation. By analogy to known proteins of the innate immune system, an emerging hypothesis for this family is that they act as sensors of Gram-negative bacteria in the oral cavity, among other areas.

scholarly journals The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

2008 ◽  
Vol 190 (6) ◽  
pp. 2065-2074 ◽  
Author(s):  
Mary E. Laubacher ◽  
Sarah E. Ades
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Stress Responses ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Envelope Stress ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

scholarly journals Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

10.3791/57169 ◽  
2018 ◽  
Author(s):  
Christopher D. Radka ◽  
Lauren L. Radford ◽  
Adriana V.F. Massicano ◽  
Lawrence J. DeLucas ◽  
Suzanne E. Lapi ◽  
...  
Keyword(s):  
Metal Uptake ◽  
Gram Negative Bacteria ◽  
Cell Fractionation ◽  
Gram Negative ◽  
Essential Metal ◽  
X Ray

Catalyzed β scission of a carbenium ion III — Scission observed in ab initio molecular dynamics simulations

2009 ◽  
Vol 87 (10) ◽  
pp. 1512-1520 ◽  
Author(s):  
Greg M. Berner ◽  
Allan L. L. East
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Density Functional ◽  
Time Windows ◽  
Three Dimensional ◽  
Ab Initio Molecular Dynamics ◽  
Catalytic Systems ◽  
Carbenium Ions ◽  
Carbenium Ion ◽  
Dynamics Simulations

The β scission (cracking) of branched carbenium ions have been observed in molecular dynamics simulations, possibly for the first time. Simulations were performed with molecular dynamics based on PW91 density functional theory, and which included three-dimensional periodic boundary replication of the unit cell to mimic long-range bulk effects. A rising-temperature algorithm was used to encourage reaction within the narrow time windows (∼10 ps) of the simulations. Twenty-eight simulations were performed, featuring alkyl ions in three different catalytic systems: the ionic liquid, [(C5H5NH+)5(Al2Cl7−)6]−, the chabazite zeolite, [AlSi23O48]−, and the chabazite zeolite, [Al4Si20O45(OH)3]−. Twenty-four runs began with unbranched sec-n-alkyl ions, but only one exhibited β scission, and only after branching to a tertiary ion and under extreme heating. In contrast, the four simulations that began with branched alkyl ions were all successful in demonstrating β scission at lower temperatures: 2,4,4-trimethyl-2-pentyl ion and 2,4-dimethyl-2-hexyl ion in each of the first two catalysts. The lifetimes of desorbed alkyl ions in the chabazite models were < 5 ps at 1000–1500 K. The β scission results support the classical Weitkamp et al. ( Appl. Catal. 1983, 8, 123 ) mechanism over the nonclassical Sie ( Ind. Eng. Chem. Res. 1992, 31, 1881 ) and the chemisorping Kazansky et al. ( J. Catal. 1989, 119, 108 ) mechanisms.

Sign in / Sign up

Export Citation Format

Share Document