scholarly journals Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa : Characterization, structure, and development as a screening platform

2019 ◽  
Vol 29 (4) ◽  
pp. 905-918
Author(s):  
Yaritza Escamilla ◽  
Casey A. Hughes ◽  
Jan Abendroth ◽  
David M. Dranow ◽  
Samantha Balboa ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trna Synthetase ◽  
Screening Platform

scholarly journals Characterization and structure determination of prolyl‐tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform

2019 ◽  
Vol 28 (4) ◽  
pp. 727-737 ◽  
Author(s):  
Noah Pena ◽  
David M. Dranow ◽  
Yanmei Hu ◽  
Yaritza Escamilla ◽  
James M. Bullard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structure Determination ◽  
Trna Synthetase ◽  
Screening Platform

scholarly journals Identification and Characterization of Chemical Compounds that Inhibit Leucyl-tRNA Synthetase from Pseudomonas aeruginosa

2020 ◽  
Vol 17 (1) ◽  
pp. 119-130 ◽  
Author(s):  
Regina Zamacona ◽  
Pamela N. Chavero ◽  
Eduardo Medellin ◽  
Yanmei Hu ◽  
Casey A. Hughes ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Cell ◽  
Bacterial Infections ◽  
Chemical Compounds ◽  
Human Cell Line ◽  
Trna Synthetase ◽  
Multi Drug Resistance ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Screening Platform

Background: Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen implicated as the causative agent in a high-percentage of nosocomial and community acquired bacterial infections. The gene encoding leucyl-tRNA synthetase (LeuRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. Methods: LeuRS was kinetically evaluated and the KM values for interactions with leucine, ATP and tRNA were 6.5, 330, and 3.0 μM, respectively. LeuRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen over 2000 synthetic and natural chemical compounds. Results: The initial screen resulted in the identification of two inhibitory compounds, BT03C09 and BT03E07. IC50s against LeuRS observed for BT03C09 and BT03E07 were 23 and 15 μM, respectively. The minimum inhibitory concentrations (MIC) were determined against nine clinically relevant bacterial strains. In time-kill kinetic analysis, BT03C09 was observed to inhibit bacterial growth in a bacteriostatic manner, while BT03E07 acted as a bactericidal agent. Neither compound competed with leucine or ATP for binding LeuRS. Limited inhibition was observed in aminoacylation assays with the human mitochondrial form of LeuRS, however when tested in cultures of human cell line, BT03C09 was toxic at all concentration whereas BT03E07 only showed toxic effects at elevated concentrations. Conclusion: Two compounds were identified as inhibitors of LeuRS in a screen of over 2000 natural and synthetic compounds. After characterization one compound (BT03E07) exhibited broad spectrum antibacterial activity while maintaining low toxicity against human mitochondrial LeuRS as well as against human cell cultures.

scholarly journals Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

2020 ◽  
Vol 21 (9) ◽  
pp. 3034 ◽  
Author(s):  
Shella Gilbert-Girard ◽  
Kirsi Savijoki ◽  
Jari Yli-Kauhaluoma ◽  
Adyary Fallarero
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Rapid Screening ◽  
Bacterial Biofilms ◽  
Main Concern ◽  
Assay Optimization ◽  
Screening Platform

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

scholarly journals Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Yaritza Escamilla ◽  
Casey A Hughes ◽  
James M Bullard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trna Synthetase

scholarly journals Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

2020 ◽  
Vol 25 (9) ◽  
pp. 1072-1086
Author(s):  
Casey A. Hughes ◽  
Varesh Gorabi ◽  
Yaritza Escamilla ◽  
Frank B. Dean ◽  
James M. Bullard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Trna Synthetase ◽  
Gram Negative Bacteria ◽  
Compound Library ◽  
Mechanism Of Inhibition ◽  
Inhibitory Compounds ◽  
Human Cell Cultures ◽  
Inhibition Studies ◽  
Substrate Competition

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes ( tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s−1 and 3.1, 3.8, and 1.9 s−1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.

scholarly journals Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

2014 ◽  
Vol 112 (2) ◽  
pp. 382-387 ◽  
Author(s):  
Tateki Suzuki ◽  
Akiyoshi Nakamura ◽  
Koji Kato ◽  
Dieter Söll ◽  
Isao Tanaka ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Complex Assembly ◽  
Evolutionary Pathway ◽  
Bacterial Type ◽  
Direct Attachment ◽  
Cognate Trna

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNAAsn, and instead synthesize asparagine from tRNAAsn-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNAAsn, and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNAAsn to form Asn-tRNAAsn for use in protein synthesis. ND-AspRS, GatCAB, and tRNAAsn may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNAAsn. We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNAAsn molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNAAsn formation evolutionarily preceded the direct attachment of Asn to tRNAAsn, our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

scholarly journals Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Sidra Irum ◽  
Saadia Andleeb ◽  
Amjad Ali ◽  
Muhammad Ibrahim Rashid ◽  
Mahnoor Majid
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Drug Targets ◽  
Interaction Analysis ◽  
Hypothetical Protein ◽  
Resistance Mechanisms ◽  
Trna Synthetase ◽  
Therapeutic Options ◽  
Aconitate Hydratase ◽  
Semialdehyde Dehydrogenase

Pseudomonas aeruginosa is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. Here, we designed a chimeric vaccine against P. aeruginosa using a subtractive proteomics approach and identified nine unique enzymes as novel drug targets in PAO1 proteome. A total of five unique proteins were selected as potential vaccine candidates based on essentiality, extracellular localization, virulence, antigenicity, pathway association, protein-protein interaction analysis, hydrophilicity, and low molecular weight. These include two outer membrane porins OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These proteins were further analyzed using a reverse vaccinology approach to identify immunogenic and antigenic T cell and B cell epitopes. The best scoring epitopes qualifying for all set criteria were then further subjected to the construction of a polypeptide multi-epitope vaccine construct with cholera toxin B (CtxB) subunit as an adjuvant. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa.

scholarly journals Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa Are Individually Implicated in the Recognition of tRNAAsn

2006 ◽  
Vol 188 (1) ◽  
pp. 269-274 ◽  
Author(s):  
Dominic Bernard ◽  
Pierre-Marie Akochy ◽  
David Beaulieu ◽  
Jacques Lapointe ◽  
Paul H. Roy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aspartic Acid ◽  
Trna Synthetase ◽  
Crystallographic Data ◽  
Sequence Comparisons ◽  
First Time

ABSTRACT In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNAAsn but by specific tRNA-dependent transamidation of aspartyl-tRNAAsn. This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNAAsp and tRNAAsn with aspartic acid. Recently, it has been shown for the first time in an organism (Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNAAsn but does not participate in Gln-tRNAGln formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNAAsn. Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNAAsp charging over that of tRNAAsn by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNAAsp and tRNAAsn. Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.

scholarly journals Comprehensive Host Cell-Based Screening Assays for Identification of Anti-Virulence Drugs Targeting Pseudomonas aeruginosa and Salmonella Typhimurium

2020 ◽  
Vol 8 (8) ◽  
pp. 1096
Author(s):  
Julia von Ambüren ◽  
Fynn Schreiber ◽  
Julia Fischer ◽  
Sandra Winter ◽  
Edeltraud van Gumpel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Cell ◽  
Bacterial Infections ◽  
Host Cells ◽  
Infection Model ◽  
Serovar Typhimurium ◽  
New Antibiotics ◽  
Screening Assays ◽  
Screening Platform

The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.

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