scholarly journals 1829. The Paradox of KPC Bearing Strains of Klebsiella pneumoniae with the D179Y Substitution: Resistance to Ceftazidine/Avibactam (CZA) and Susceptibility to Meropenem (MEM)

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S41-S42
Author(s):  
Melissa D Barnes ◽  
Magdalena A Taracilla ◽  
Joseph D Rutter ◽  
Minh-Hong Nguyen ◽  
Ryan K Shields ◽  
...  
Keyword(s):  
Rational Design ◽  
Carbapenem Resistance ◽  
Single Amino Acid ◽  
Molar Ratio ◽  
E Coli ◽  
Acyl Complexes ◽  
Omega Loop ◽  
Catalytic Mechanisms ◽  
Mechanistic Basis ◽  
Enterobacteriaceae Infections

Abstract Background Resistance to CZA is a serious limitation of treatment for KPC bearing Enterobacteriaceae infections. Recently, a single amino acid substitution (D179Y) was described in KPC-2 and KPC-3 bearing CZA-resistant K. pneumoniae recovered from patients failing treatment. In class A β-lactamases the D179 residue is located at the neck of the omega loop and is critical for KPC catalytic activity. In attempts to understand the evolution of substrate specificity in KPC-2, the D179Y variant of KPC-2 was shown to be resistant to CZA (ceftazidime forms a long-lived acyl enzyme with in KPC-2), but susceptible to MEM. A similar observation was made in clinical and laboratory-generated K. pneumoniae and E. coli strains bearing D179Y KPC-3. We were compelled to explore the catalytic mechanisms of susceptibility to MEM of the D179Y variants in KPC-2 vs. KPC-3. Methods KPC-2, KPC-3, and D179Y in the respective KPC were cloned into an expression vector and the β-lactamase proteins were purified. 5 mg of each β-lactamase with and without MEM (1:1 molar ratio) was incubated for the time indicated and analyzed using the Quadrupole Time-of-Flight (QTOF) timed mass spectrometry for the reaction intermediates. To assess thermal stability, denaturation melting curves were run for 2 hours using 12 µM β-lactamase. Results The D179Y variant forms prolonged acyl-complexes with meropenem in KPC-3 and KPC-2, which can be detected up to 24 hours (Figure 1). This prolonged trapping of meropenem by D179Y variants is not evident with the respective KPCs. Further, the tyrosine substitution at the D179 position (Tm = 48–52°C) destabilizes the KPC β-lactamases (TmKPC-2/3 = 52–56°C). Conclusion These data suggest that MEM acts as a covalent β-lactamase inhibitor more than as a substrate for KPC-2 and -3. The mechanistic basis of paradoxical susceptibility to carbapenems provides an impetus to develop better therapeutic approaches to the increasing threat of carbapenem resistance and highlights how the rational design of novel β-lactam/β-lactamase inhibitors must consider mechanistic bases of resistance. Disclosures All Authors: No reported Disclosures.

scholarly journals Systematic detection of amino acid substitutions in proteome reveals a mechanistic basis of ribosome errors

2018 ◽  
Author(s):  
Ernest Mordret ◽  
Avia Yehonadav ◽  
Georgina D Barnabas ◽  
Jürgen Cox ◽  
Orna Dahan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Error Rates ◽  
Mass Spectrometry Data ◽  
Single Amino Acid ◽  
Post Translational Modification ◽  
E Coli ◽  
Wide Range ◽  
Proteome Level ◽  
Mechanistic Basis ◽  
Translation Errors

Translation errors limit the accuracy of information transmission from DNA to proteins. Selective pressures shape the way cells produce their proteins: the translation machinery and the mRNA sequences it decodes co-evolved to ensure that translation proceeds fast and accurately in a wide range of environmental conditions. Our understanding of the causes of amino acid misincorporations and of their effect on the evolution of protein sequences is largely hindered by the lack of experimental methods to observe errors at the full proteome level. Here, we systematically detect and quantify errors in entire proteomes from mass spectrometry data. Following HPLC MS-MS data acquisition, we identify E. coli and S. cerevisiae peptides whose mass and fragment ion spectrum are consistent with that of a peptide bearing a single amino acid substitution, and verify that such spectrum cannot result from a post-translational modification. Our analyses confirm that most substitutions occur due to codon-to-anticodon mispairing within the ribosome. Patterns of errors due to mispairing were similar in bacteria and yeast, suggesting that the error spectrum is chemically constrained. Treating E. coli cells with a drug known to affect ribosomal proofreading increased the error rates due to mispairing at the wobble codon position. Starving bacteria for serine resulted in specific patterns of substitutions reflecting the amino acid deficiency. Overall, translation errors tend to occur at positions that are less evolutionarily conserved, and that minimally affect protein energetic stability, indicating that they are selected against. Genome wide ribosome density data suggest that errors occur at sites where ribosome velocity is relatively high, supporting the notion of a trade-off between speed and accuracy as predicted by proofreading theories. Together our results reveal a mechanistic basis for ribosome errors in translation.

scholarly journals CTX-M-Type Extended-Spectrum β-Lactamase That Hydrolyzes Ceftazidime through a Single Amino Acid Substitution in the Omega Loop

2001 ◽  
Vol 45 (12) ◽  
pp. 3355-3361 ◽  
Author(s):  
Laurent Poirel ◽  
Thierry Naas ◽  
Isabelle Le Thomas ◽  
Amal Karim ◽  
Edouard Bingen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Rectal Swab ◽  
Single Amino Acid ◽  
E Coli ◽  
Class A ◽  
Omega Loop ◽  
Similar Restriction ◽  
Biochemical Analyses ◽  
Hydrolysis Of

ABSTRACT Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniaeILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 β-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 β-lactamase. The amino acid sequence of the CTX-M-18 β-lactamase differed from that of the CTX-M-9 β-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. Thebla CTX-M-18 andbla CTX-M-19 genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.

scholarly journals Substrate Spectrum Extension of PenA in Burkholderia thailandensis with a Single Amino Acid Deletion, Glu168del

2012 ◽  
Vol 56 (7) ◽  
pp. 4005-4008 ◽  
Author(s):  
Hyojeong Yi ◽  
Karan Kim ◽  
Kwang-Hwi Cho ◽  
Oksung Jung ◽  
Heenam Stanley Kim
Keyword(s):  
Deletion Mutation ◽  
Single Amino Acid ◽  
Functional Domain ◽  
Amino Acid Deletion ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Class A ◽  
Omega Loop ◽  
Substitution Mutations ◽  
Substrate Spectrum

ABSTRACTWe describe a deletion mutation in a class A β-lactamase, PenA, ofBurkholderia thailandensisthat extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations.

scholarly journals ESBL Activity, MDR, and Carbapenem Resistance among Predominant Enterobacterales Isolated in 2019

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 744
Author(s):  
Altaf Bandy ◽  
Bilal Tantry
Keyword(s):  
Escherichia Coli ◽  
Saudi Arabia ◽  
Antimicrobial Resistance ◽  
Carbapenem Resistance ◽  
Resistant Bacteria ◽  
Sociodemographic Characteristics ◽  
Chi Square ◽  
E Coli ◽  
Chi Square Test ◽  
Resistance Rates

Antimicrobial-resistance in Enterobacterales is a serious concern in Saudi Arabia. The present study retrospectively analyzed the antibiograms of Enterobacterales identified from 1 January 2019 to 31 December 2019 from a referral hospital in the Aljouf region of Saudi Arabia. The revised document of the Centers for Disease Control (CDC) CR-2015 and Magiorakos et al.’s document were used to define carbapenem resistance and classify resistant bacteria, respectively. The association of carbapenem resistance, MDR, and ESBL with various sociodemographic characteristics was assessed by the chi-square test and odds ratios. In total, 617 Enterobacterales were identified. The predominant (n = 533 (86.4%)) isolates consisted of 232 (37.6%), 200 (32.4%), and 101 (16.4%) Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, respectively. In general, 432 (81.0%) and 128 (24.0%) isolates were of MDR and ESBL, respectively. The MDR strains were recovered in higher frequency from intensive care units (OR = 3.24 (1.78–5.91); p < 0.01). E. coli and K. pneumoniae resistance rates to imipenem (2.55 (1.21–5.37); p < 0.01) and meropenem (2.18 (1.01–4.67); p < 0.04), respectively, were significantly higher in winter. The data emphasize that MDR isolates among Enterobacterales are highly prevalent. The studied Enterobacterales exhibited seasonal variation in antimicrobial resistance rates towards carbapenems and ESBL activity.

scholarly journals Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiwei Wang ◽  
Yan Gao ◽  
Yanting Tang ◽  
Xiaoting Zhou ◽  
Yuezheng Lai ◽  
...  
Keyword(s):  
Rational Design ◽  
Pathogenic Bacteria ◽  
Structural Topology ◽  
Antimicrobial Drugs ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cryo Electron Microscopy ◽  
Functional Mechanisms ◽  
The Relationship ◽  
Potential Oxygen

AbstractCytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

scholarly journals A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance

Microbiology ◽  
2015 ◽  
Vol 161 (4) ◽  
pp. 895-902 ◽  
Author(s):  
Mouparna Dutta ◽  
Debasish Kar ◽  
Ankita Bansal ◽  
Sandeep Chakraborty ◽  
Anindya S. Ghosh
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Cell Shape ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Beta Lactam ◽  
E Coli

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Ultrafast excited state dynamics and light-switching of [Ru(phen)2(dppz)]2+ in G-quadruplex DNA

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Chunfan Yang ◽  
Qian Zhou ◽  
Zeqing Jiao ◽  
Hongmei Zhao ◽  
Chun-Hua Huang ◽  
...  
Keyword(s):  
Excited State ◽  
State Formation ◽  
Rational Design ◽  
Bound Water ◽  
Binding Modes ◽  
Relaxation Dynamics ◽  
Dark State ◽  
Excited State Dynamics ◽  
G Quadruplex ◽  
Mechanistic Basis

AbstractThe triplet metal to ligand charge transfer (3MLCT) luminescence of ruthenium (II) polypyridyl complexes offers attractive imaging properties, specifically towards the development of sensitive and structure-specific DNA probes. However, rapidly-deactivating dark state formation may compete with 3MLCT luminescence depending on different DNA structures. In this work, by combining femtosecond and nanosecond pump-probe spectroscopy, the 3MLCT relaxation dynamics of [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) in two iconic G-quadruplexes has been scrutinized. The binding modes of stacking of dppz ligand on the terminal G-quartet fully and partially are clearly identified based on the biexponential decay dynamics of the 3MLCT luminescence at 620 nm. Interestingly, the inhibited dark state channel in ds-DNA is open in G-quadruplex, featuring an ultrafast picosecond depopulation process from 3MLCT to a dark state. The dark state formation rates are found to be sensitive to the content of water molecules in local G-quadruplex structures, indicating different patterns of bound water. The unique excited state dynamics of [Ru(phen)2(dppz)]2+ in G-quadruplex is deciphered, providing mechanistic basis for the rational design of photoactive ruthenium metal complexes in biological applications.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1599-1610 ◽  
Author(s):  
Bradley T Smith ◽  
Graham C Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Cellular Response ◽  
Sos Response ◽  
Posttranslational Regulation ◽  
E Coli ◽  
Sos Mutagenesis ◽  
Induced Mutagenesis ◽  
Mechanistic Basis ◽  
Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

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