scholarly journals Molecular Validation of LpxC as an Antibacterial Drug Target in Pseudomonas aeruginosa

2006 ◽  
Vol 50 (6) ◽  
pp. 2178-2184 ◽  
Author(s):  
Khisimuzi E. Mdluli ◽  
Pamela R. Witte ◽  
Toni Kline ◽  
Adam W. Barb ◽  
Alice L. Erwin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Bacterial Species ◽  
Inducible Promoter ◽  
Antibacterial Drug ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
E Coli ◽  
Antibiotic Drugs

ABSTRACT LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeruginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxC gene of P. aeruginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeruginosa. It was found that compound L-161,240, the most potent inhibitor from the previous study, was active against a P. aeruginosa construct in which the endogenous lpxC gene was inactivated and in which LpxC activity was supplied by the lpxC gene from E. coli. Conversely, an E. coli construct in which growth was dependent on the P. aeruginosa lpxC gene was resistant to the compound. The differential activities of L-161,240 against the two bacterial species are thus the result primarily of greater potency toward the E. coli enzyme rather than of differences in the intrinsic resistance of the bacteria toward antibacterial compounds due to permeability or efflux. These data validate P. aeruginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

2018 ◽  
Vol 10 (464) ◽  
pp. eaal0033 ◽  
Author(s):  
Ahsan R. Akram ◽  
Sunay V. Chankeshwara ◽  
Emma Scholefield ◽  
Tashfeen Aslam ◽  
Neil McDonald ◽  
...  
Keyword(s):  
Lipid A ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Water Soluble ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Human Lungs ◽  
Fluorescent Peptide ◽  
Bacterial Lipid

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

scholarly journals Antimicrobial-Resistant Enteric Bacteria from Dairy Cattle

2006 ◽  
Vol 73 (1) ◽  
pp. 156-163 ◽  
Author(s):  
Ashish A. Sawant ◽  
Narasimha V. Hegde ◽  
Beth A. Straley ◽  
Sarah C. Donaldson ◽  
Brenda C. Love ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dna Hybridization ◽  
Bacterial Species ◽  
Dairy Herd ◽  
Enteric Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Predominant Species ◽  
Lactating Dairy Cattle

ABSTRACT A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which Escherichia coli (87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in E. coli isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (≥3 to 6 antimicrobials) was seen in 40% of E. coli isolates from healthy lactating cattle. Of 113 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the tet(A) determinant. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant E. coli isolates (n = 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions, E. coli subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric E. coli from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.

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 National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Po-Yu Liu ◽  
Yu-Lin Lee ◽  
Min-Chi Lu ◽  
Pei-Lan Shao ◽  
Po-Liang Lu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Gram Negative Bacteria ◽  
National Surveillance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

scholarly journals Antibacterial Activityof Aqueous And Ethanolic Leaf Extracts Of Vernonia Amygdalina On Selected Species Of Gram Positive And Gram Negative Bacteria

2013 ◽  
Vol 2 (1) ◽  
pp. 147-152 ◽  
Author(s):  
AM Bukar ◽  
MA Isa ◽  
HS Bello ◽  
AS Abdullahi
Keyword(s):  
Antibacterial Activity ◽  
Antibacterial Drug ◽  
Gram Negative Bacteria ◽  
Phytochemical Screening ◽  
Vernonia Amygdalina ◽  
Leaf Extracts ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Pseudomonas Species

The phytochemical screening and antibacterial activity of ethanolic and Methanolic leaves extract of Vernonia amygdalina against five clinical isolates (Staphylococcus aureus, E. coli, Pseudomonas species, Salmonella species and Proteus species) was determined using standard method of analysis. The results of the antibacterial activity of ethanol, methanol and aqueous extract of leaves of V. amygdalina have diameters ranging between 0.4 to 10mm. The plant extracts from the plants had profound activities against gram-positive than gram negative bacteria. From the above studies, it has clearly indicated that V. amygdalina extract may represent new sources of antibacterial drug, if the phytoactive components are purified and proper dosage are determined for administration. International Journal of Environment, Volume-2, Issue-1, Sep-Nov 2013, Pages 147-152 DOI: http://dx.doi.org/10.3126/ije.v2i1.9217

scholarly journals Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Axel B. Janssen ◽  
Toby L. Bartholomew ◽  
Natalia P. Marciszewska ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Anionic Lipid ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Two Component ◽  
Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

scholarly journals LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Tian ◽  
Guillaume Manat ◽  
Elise Gasiorowski ◽  
Rodolphe Auger ◽  
Samia Hicham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Lipid A ◽  
Negative Charge ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

scholarly journals Regulatory Conservation and Divergence of ς32 Homologs from Gram-Negative Bacteria: Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens

1998 ◽  
Vol 180 (9) ◽  
pp. 2402-2408 ◽  
Author(s):  
Kenji Nakahigashi ◽  
Hideki Yanagi ◽  
Takashi Yura
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Serratia Marcescens ◽  
Proteus Mirabilis ◽  
Heat Shock Response ◽  
Gram Negative Bacteria ◽  
Heat Shock Genes ◽  
Gram Negative ◽  
E Coli ◽  
Shock Response

ABSTRACT The heat shock response in Escherichia coli is mediated primarily by the rpoH gene, encoding ς32, which is specifically required for transcription of heat shock genes. A number of ς32 homologs have recently been cloned from gram-negative bacteria that belong to the gamma or alpha subdivisions of the proteobacteria. We report here some of the regulatory features of several such homologs (RpoH) expressed in E. coli as well as in respective cognate bacteria. When expressed in an E. coli ΔrpoH strain lacking its own ς32, these homologs activated the transcription of heat shock genes (groE and dnaK) from the start sites normally used in E. coli. The level of RpoH inSerratia marcescens and Pseudomonas aeruginosacells was very low at 30°C but was elevated markedly upon a shift to 42°C, as found previously with E. coli. The increased RpoH levels upon heat shock resulted from both increased synthesis and stabilization of the normally unstable RpoH protein. In contrast, the RpoH level in Proteus mirabilis was relatively high at 30°C and increased less markedly upon heat shock, mostly by increased synthesis; this ς32 homolog was already stable at 30°C, and little further stabilization occurred upon the shift to 42°C. The increased synthesis of RpoH homologs in all these gamma proteobacteria was observed even in the presence of rifampin, suggesting that the induction occurred at the level of translation. Thus, the basic regulatory strategy of the heat shock response by enhancing the RpoH level is well conserved in the gamma proteobacteria, but some divergence in the actual mechanisms used occurred during evolution.

scholarly journals Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

2005 ◽  
Vol 71 (4) ◽  
pp. 1850-1855 ◽  
Author(s):  
Jennifer R. Walker ◽  
Elliot Altman
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Gram Negative Bacteria ◽  
Cell Fractionation ◽  
Peptide Antibiotics ◽  
Small Peptides ◽  
Gram Negative ◽  
E Coli ◽  
Serovar Typhimurium

ABSTRACT Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.

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