scholarly journals MapB Protein is the Essential Methionine Aminopeptidase in Mycobacterium tuberculosis

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 393 ◽  
Author(s):  
Miriam Vanunu ◽  
Patrick Schall ◽  
Tali-Haviv Reingewertz ◽  
Pradip K. Chakraborti ◽  
Bernhard Grimm ◽  
...  
Keyword(s):  
Drug Targets ◽  
Point Mutations ◽  
Life Forms ◽  
Growth Defect ◽  
Resistant Bacteria ◽  
Correct Identification ◽  
Double Deletion ◽  
Methionine Aminopeptidases ◽  
Weak Growth ◽  
Partial Activity

M. tuberculosis (Mtb), which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6–9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the cotranslational control of proteins as they determine their half life (N-terminal end rule) and facilitate further modifications such as acetylation and others. Mtb (and M. bovis) possess two MetAP isoforms, MetAP1a and MetAP1c, encoded by the mapA and mapB genes, respectively. Conflicting evidence was reported in the literature on which of the two variants is essential. To resolve this question, we performed a targeted genetic deletion of each of these two genes. We show that a deletion mutant of mapA is viable with only a weak growth defect. In contrast, we provide two lines of genetic evidence that mapB is indispensable. Furthermore, construction of double-deletion mutants as well as the introduction of point mutations into mapB resulting in proteins with partial activity showed partial, but not full, redundancy between mapB and mapA. We propose that it is MetAP1c (mapB) that is essentially required for mycobacteria and discuss potential reasons for its vitality.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

Antibiotic adjuvants: a promising approach to combat multidrug resistant bacteria

2021 ◽  
Vol 22 ◽  
Author(s):  
Namita Sharma ◽  
Anil K. Chhillar ◽  
Sweety Dahiya ◽  
Pooja Choudhary ◽  
Aruna Punia ◽  
...  
Keyword(s):  
Drug Targets ◽  
Pathogenic Bacteria ◽  
Antibacterial Agents ◽  
Bacterial Resistance ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Antibacterial Drugs ◽  
Effective Strategies ◽  
The Impact ◽  
Emergence Of Resistance

The escalating emergence and prevalence of infections caused by multi-drug resistant (MDR) pathogenic bacteria accentuate the crucial need to develop novel and effectual therapeutic strategies to control this threat. Recent past surprisingly indicates a staggering decline in effective strategies against MDR. Different approaches have been employed to minimize the effect of resistance but the question still lingers over the astounding number of drugs already tried and tested to no avail, furthermore, the detection of new drug targets and the action of new antibacterial agents against already existing drug targets also complicate the condition. Antibiotic adjuvants are considered as one such promising approach for overcoming the bacterial resistance. Adjuvants can potentiate the action of generally adopted antibacterial drugs against MDR bacterial pathogens either by minimizing the impact and emergence of resistance or improving the action of antibacterial drugs. This review provides an overview of mechanism of antibiotic resistance, main types of adjuvants and their mode of action, achievements and progression.

scholarly journals Blocking the Trigger: Inhibition of the Initiation of Bacterial Chromosome Replication as an Antimicrobial Strategy

Antibiotics ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 111
Author(s):  
Julia E. Grimwade ◽  
Alan C. Leonard
Keyword(s):  
Drug Targets ◽  
Antimicrobial Agents ◽  
Current Knowledge ◽  
Chromosome Replication ◽  
Bacterial Chromosome ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Drug Resistant Bacteria ◽  
New Antibiotics ◽  
Novel Drug

All bacterial cells must duplicate their genomes prior to dividing into two identical daughter cells. Chromosome replication is triggered when a nucleoprotein complex, termed the orisome, assembles, unwinds the duplex DNA, and recruits the proteins required to establish new replication forks. Obviously, the initiation of chromosome replication is essential to bacterial reproduction, but this process is not inhibited by any of the currently-used antimicrobial agents. Given the urgent need for new antibiotics to combat drug-resistant bacteria, it is logical to evaluate whether or not unexploited bacterial processes, such as orisome assembly, should be more closely examined for sources of novel drug targets. This review will summarize current knowledge about the proteins required for bacterial chromosome initiation, as well as how orisomes assemble and are regulated. Based upon this information, we discuss current efforts and potential strategies and challenges for inhibiting this initiation pharmacologically.

scholarly journals A Common Cross-species Function for the Double Epidermal Growth Factor-like Modules of the Highly DivergentPlasmodiumSurface Proteins MSP-1 and MSP-8

2004 ◽  
Vol 279 (19) ◽  
pp. 20147-20153 ◽  
Author(s):  
Damien R. Drew ◽  
Rebecca A. O'Donnell ◽  
Brian J. Smith ◽  
Brendan S. Crabb
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Drug Targets ◽  
Point Mutations ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Parasite Line ◽  
Asexual Blood Stage ◽  
Epidermal Growth

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show thatPlasmodium falciparumparasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module fromP. bergheiMSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least somePlasmodiumsurface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains fromP. falciparumandP. chabaudiMSP-1 were also not capable of replacing theP. falciparumMSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.

scholarly journals Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

2015 ◽  
Vol 60 (2) ◽  
pp. 990-1002 ◽  
Author(s):  
Susann Skagseth ◽  
Trine Josefine Carlsen ◽  
Gro Elin Kjæreng Bjerga ◽  
James Spencer ◽  
Ørjan Samuelsen ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Point Mutations ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Hydrophilic Residues

ABSTRACTMetallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeledin silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

scholarly journals Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

2018 ◽  
Author(s):  
Jiayun Liu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Gail Cassell ◽  
Dmitry A. Maslov ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Drug Targets ◽  
Drug Exposure ◽  
Efflux Pump ◽  
Active Form ◽  
Point Mutations ◽  
Drug Susceptibility ◽  
Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

scholarly journals Rap1p Requires Gcr1p and Gcr2p Homodimers to Activate Ribosomal Protein and Glycolytic Genes, Respectively

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 133-143 ◽  
Author(s):  
Stephen J Deminoff ◽  
George M Santangelo
Keyword(s):  
Ribosomal Protein ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Growth Defect ◽  
Phosphorylation State ◽  
Glycolytic Gene ◽  
Activation Mechanisms ◽  
Regulatory Complex ◽  
Activator Complex

Abstract Efficient transcription of ribosomal protein (RP) and glycolytic genes requires the Rap1p/Gcr1p regulatory complex. A third factor, Gcr2p, is required for only the glycolytic (specialized) mode of transcriptional activation. It is recruited to the complex by Gcr1p and likely mediates a change in the phosphorylation state and/or conformation of the latter. We show here that leucine zipper motifs in Gcr1p and Gcr2p (1LZ and 2LZ) are each specific to one of the two activation mechanisms—mutations in 1LZ and 2LZ impair transcription of RP and glycolytic genes, respectively. Although neither class of mutations causes more than a mild growth defect, simultaneous impairment of 1LZ and 2LZ results in a severe synthetic defect and a reduction in the expression of both sets of genes. Intracistronic complementation by point mutations in the charged e and g positions confirmed that Gcr1p/Gcr1p and Gcr2p/Gcr2p homodimers are the forms required for the different roles of the activator complex. Direct heterodimerization between 1LZ and 2LZ apparently does not occur. Dichotomous Rap1p activation and its striking requirement for distinct homodimeric subunits give cells the capacity to switch between coordinated and uncoupled RP and glycolytic gene regulation.

scholarly journals PATH-24. DETECTION OF POINT MUTATIONS AND GENE FUSIONS FROM CIRCULATING CELL-FREE DNA (CFDNA) OF GLIOBLASTOMA (GBM) PATIENTS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii169-ii169
Author(s):  
Milana Frenkel-Morgenstern ◽  
Vikrant Palande ◽  
Rajesh Detroja ◽  
Alessandro Gorohovski ◽  
Rainer Glass ◽  
...  
Keyword(s):  
Drug Targets ◽  
Gene Fusion ◽  
Fusion Gene ◽  
Point Mutations ◽  
Invasive Procedure ◽  
Clonal Diversity ◽  
Healthy Controls ◽  
Gene Fusions ◽  
Non Invasive ◽  
Tumor Dna

Abstract BACKGROUND GBM is characterized by intratumoral heterogeneity. Tumor heterogeneity, clonal diversity and mutation acquisition hamper the ability to tailor personalized therapy for GBM. Tumor sampling has limited ability to accurately capture the molecular landscape of the tumor and to disclose acquired molecular aberrations. Mutation analysis of cfDNA is a non-invasive procedure which may overcome these limitations as it may reflect the real composition of the tumor and track the molecular evolution. We sequenced cfDNA of GBM patients and assessed mutation patterns and fusion genes. METHODS We collected blood and respective tumor samples from 27 GBM patients and blood samples from 14 healthy controls. Tumor DNA, cfDNA and WBC DNA were sequenced using deep sequencing procedures. The data were analyzed for detection of single nucleotide polymorphism (SNPs) and gene-gene fusions. RESULTS GBM cfDNA concentrations were significantly elevated (median: 23.63 ng/mL; range 12.6–137) compared to healthy controls (median 2.06; range 1.68–7.62) (p &lt; 0.0001). We identified unique SNPs in each glioma patient’s cfDNA and the corresponding tumor DNA including the top-10 most frequently mutated genes in GBM. For example, mutation of TP53 was detected in18.75%; EGFR in 37.5%; NF1-12.5%; LRP1B-25% and IRS4 in 25%. For gene-gene fusion we used the in-house fusion gene database, ChiTaRS 5.0, and identified fusions in cfDNA and tumor DNA. Thus, KMT2A-FLNA was the most frequent fusion found in 16.4% of samples. BCR-ABL1 in 8.82% and FGFR1-BCR in 2.94%. Other fusions included COL1A1-PDGFB (5.88%), NIN-PDGFRB (5.88%), KIF5B-RET (5.88%) and also TPM3-ROS1(2.94%), TFG-ALK(2.94%), MSN-ALK (2.94%) and NPM1-ALK (2.94%) which may be targeted by brain penetrating drugs that are ROS1 and ALK inhibitors. CONCLUSIONS Our study suggests that plasma cfDNA analysis may help to uncover real time mutational and gene fusion status of GBM by a non-invasive procedure. It may identify drug targets based on personalized gene-gene fusions.

scholarly journals Defeating Antibiotic-Resistant Bacteria: Exploring Alternative Therapies for a Post-Antibiotic Era

2020 ◽  
Vol 21 (3) ◽  
pp. 1061 ◽  
Author(s):  
Chih-Hung Wang ◽  
Yi-Hsien Hsieh ◽  
Zachary M. Powers ◽  
Cheng-Yen Kao
Keyword(s):  
Drug Targets ◽  
Resistant Bacteria ◽  
Combination Therapies ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Alternative Drug ◽  
Novel Approaches ◽  
Antibiotic Discovery ◽  
Medical Advances ◽  
Common Infections

Antibiotics are one of the greatest medical advances of the 20th century, however, they are quickly becoming useless due to antibiotic resistance that has been augmented by poor antibiotic stewardship and a void in novel antibiotic discovery. Few novel classes of antibiotics have been discovered since 1960, and the pipeline of antibiotics under development is limited. We therefore are heading for a post-antibiotic era in which common infections become untreatable and once again deadly. There is thus an emergent need for both novel classes of antibiotics and novel approaches to treatment, including the repurposing of existing drugs or preclinical compounds and expanded implementation of combination therapies. In this review, we highlight to utilize alternative drug targets/therapies such as combinational therapy, anti-regulator, anti-signal transduction, anti-virulence, anti-toxin, engineered bacteriophages, and microbiome, to defeat antibiotic-resistant bacteria.

scholarly journals A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents

2019 ◽  
Vol 47 (15) ◽  
pp. 8163-8179 ◽  
Author(s):  
Tim R Blower ◽  
Afif Bandak ◽  
Amy S Y Lee ◽  
Caroline A Austin ◽  
John L Nitiss ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Drug Targets ◽  
Topoisomerase Ii ◽  
Point Mutations ◽  
Amino Acid Position ◽  
Drug Binding ◽  
Type Ii ◽  
Resistance Mutations ◽  
Selective Sensitivity

AbstractType II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational ‘landscape’ of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2β) is additionally revealed to be significantly regulated by the enzymes’ adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.

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