Single Substitution Research Articles

Ab Initio Investigation of the Auger Spectra of Methane, Ethane, Ethylene, and Acetylene.

We present an ab initio computational study of the Auger spectra of methane, ethane, ethylene, and acetylene. Auger spectroscopy is an established technique to probe the electronic structure of molecules and exploits the Auger-Meitner effect that core-ionized states undergo. We compute partial decay widths using coupled-cluster theory with single and double substitutions (CCSD) and equation-of-motion CCSD theory combined with complex-scaled basis functions and Feshbach-Fano projection. We generate Auger spectra from these partial widths and draw conclusions about the strength of particular decay channels and trends among the four molecules. A connection to experimental results about fragmentation pathways of the electronic states produced by Auger decay is also made.

SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional controls of submergence tolerance in rice.

Most rice (Oryza sativa) cultivars cannot survive under prolonged submergence. However, some O. sativa ssp. indica cultivars, such as FR13A, are highly tolerant owing to the SUBMERGENCE 1A-1 (SUB1A-1) allele, which encodes a Group VII ethylene-responsive factor (ERFVII) protein; other submergence-intolerant cultivars contain a SUB1A-2 allele. The two alleles differ only by a single substitution at the 186th amino acid position from serine in SUB1A-1 to proline in SUB1A-2 resulting in only SUB1A-1 being able to be phosphorylated. Two other ERFVIIs, ERF66 and ERF67, function downstream of SUB1A-1 to form a regulatory cascade in response to submergence stress. Here, we show that SUB1A-1, but not SUB1A-2, interacts with ADA2b of the ADA2b-GCN5 acetyltransferase complex, in which GCN5 functions as a histone acetyltransferase. Phosphorylation of SUB1A-1 at serine 186 enhances the interaction of SUB1A-1 with ADA2b. ADA2b and GCN5 expression was induced under submergence, suggesting that these two genes might play roles in response to submergence stress. In transient assays, binding of SUB1A-1 to the ERF67 promoter and ERF67 transcription were highly induced when SUB1A-1 was expressed together with the ADA2b-GCN5 acetyltransferase complex. Taken together, these results suggest that phospho-SUB1A-1 recruits the ADA2-GCN5 acetyltransferase complex to modify the chromatin structure of the ERF66/ERF67 promoter regions and activate gene expression, which in turn enhances rice submergence tolerance.

A single F153Sβ3 mutation causes constitutive integrin αIIbβ3 activation in a variant form of Glanzmann thrombasthenia.

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate exvivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate exvivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbβ3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report β3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sβ3 substitution within the βI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153β3. F153 is completely conserved among β3 of several species and all human β-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sβ3 also displays reduced levels of a constitutively activated αIIb-S153β3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153β3 is critical for maintaining the resting conformation of α2- and α1-helices of the βI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the βI-domain toward the constitutively active αIIbβ3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbβ3 activation. The data collectively demonstrate that disruption of F153β3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153β3 may be compensated by a hyperactive conformation that promotes viable hemostasis.

N-terminal lid swapping contributes to the substrate specificity and activity of thermophilic lipase TrLipE.

TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from Thermomicrobium roseum has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low Km and high kcat value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest kcat/Km value of 363.88 ± 15.83 L⋅min-1⋅mmol-1. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.

Ultraviolet photodissociation spectra of singly protonated hexapeptides DYYVVR, DYFVVR, and DFYVVR at room temperature

AbstractDYYVVR is a tryptic peptide of Janus kinase 3 and has the two active tyrosine residues needed for the regulation of the protein's activity by phosphorylation. As a proof‐of‐concept experiment for localization of phosphorylation site, two‐dimensional ultraviolet‐mass spectrometry of singly protonated DYYVVR has been investigated at room temperature. Comparing the absorption spectrum with those of peptides with single phenylalanine substitution, DYFVVR and DFYVVR, it is confirmed that the ultraviolet (UV) absorption below 35 100 cm−1 is due to the first tyrosine residue. Combined with the fact that the absorption of a phosphorylated tyrosine is shifted above 37 500 cm−1, two‐dimensional analysis employing mass spectrometry and ultraviolet spectroscopy can be used as a tool for localization of phosphorylation site in a peptide.

Repartitioned Brillouin-Wigner perturbation theory with a size-consistent second-order correlation energy.

Second-order Møller-Plesset perturbation theory (MP2) often breaks down catastrophically in small-gap systems, leaving much to be desired in its performance for myriad chemical applications such as noncovalent interactions, thermochemistry, and dative bonding in transition metal complexes. This divergence problem has reignited interest in Brillouin-Wigner perturbation theory (BWPT), which is regular at all orders but lacks size consistency and extensivity, severely limiting its application to chemistry. In this work, we propose an alternative partitioning of the Hamiltonian that leads to a regular BWPT perturbation series that, through the second order, is size-extensive, size-consistent (provided its Hartree-Fock reference is also), and orbital invariant. Our second-order size-consistent Brillouin-Wigner (BW-s2) approach can describe the exact dissociation limit of H2 in a minimal basis set, regardless of the spin polarization of the reference orbitals. More broadly, we find that BW-s2 offers improvements relative to MP2 for covalent bond breaking, noncovalent interaction energies, and metal/organic reaction energies, although rivaling coupled-cluster with single and double substitutions for thermochemical properties.

PA-E18G substitution in influenza A virus confers resistance to ZX-7101, a cap-dependent endonuclease inhibitor

Cap-dependent endonuclease (CEN) in the polymerase acidic protein (PA) of influenza A virus (IAV) represents a promising drug target due to its critical role in viral gene transcription. The CEN inhibitor, baloxavir marboxil (BXM), was approved in Japan and the US in 2018 and several other countries subsequently. Along with the clinical use of BXM, the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern. Herein, we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A, an analogue of BXM. The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes, including pH1N1, H3N2, H7N9 and H9N2, in MDCK cells, and the 50% effective concentration (EC50) was calculated to nanomole level and comparable to that of baloxavir acid (BXA), the active form of BXM. Furthermore, in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice, with reduced viral RNA loads and alleviated pulmonary damage. Importantly, serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage. Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA. Taken together, our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance, which provides critical clues for future drug development and drug resistance surveillance.

Identification of the novel HLA-DQA1*05:75 allele by next-generation sequencing in a Korean cord blood donor.

HLA-DQA1*05:75 differs from DQA1*05:05:01:01 by a single substitution at nucleotide 701 (G/A) in exon 4.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Preparation and characterization of a laccase-like enzyme from Thermomicrobium roseum

In this study, a laccase-like gene from Thermomicrobium roseum DSM 5159 (TrLac-like) (NCBI: WP_012642205.1) was recombinantly expressed in Bacillus subtilis WB600. The optimum temperature and pH for TrLac-like were 50 °C and 6.0, respectively. TrLac-like showed high tolerance to mixed systems of water and organic solvents, indicating its potential for large-scale application in various industries. It showed 36.81 % similarity with YlmD from Geobacillus stearothermophilus (PDB:6T1B) in sequence alignment; therefore, 6T1B was employed as the template for homology modeling. To improve catalytic efficiency, amino acid substitutions within 5 Å of the inosine ligand were simulated to reduce the binding energy and promote substrate affinity. Single and double substitutions (44 and 18, respectively) were prepared, and the catalytic efficiency of the mutant A248D was increased to approximately 110-fold that of the wild type, while the thermal stability was maintained. Bioinformatics analysis revealed that the significant improvement in catalytic efficiency could be attributed to the formation of new hydrogen bonds between the enzyme and substrate. With a further decrease in the binding energy, the catalytic efficiency of the multiple mutant H129N/A248D was approximately 14-fold higher than that of the wild type but lower than that of the single mutant A248D. This is possibly because kcat also decreased with the decrease of Km; consequently, the substrate could not be released in time owing to the enzyme with the combination mutation not being able to release the substrate at a high rate.

Mechanistic and kinetic study of the oxidation of trifluoroacetonitrile by hydroxyl and oxygen

AbstractTrifluoroacetonitrile (CF3CN) is one of the perfluoronitriles to be used as replacements for electrical insulation, working fluids, and biomedical applications. Potential energy surfaces for the reactions of CF3CN with OH radicals in the presence of molecular O2 have been calculated in details using the second‐order Møller−Plesset perturbation theory (MP2) and coupled‐cluster theory with single and double substitutions (CCSD) for geometrical optimization, and the restricted open‐shell complete basis set quadratic ROCBS‐QB3, multireference second‐order Rayleigh–Schrodinger perturbation theory extrapolated to complete basis set limit (RS2/CBS), and the explicitly‐correlated RS2 theory (RS2‐F12) for energetics. The CF3CN + OH reaction undergoes predominantly via the CO addition/elimination mechanism, leading to CF3C(OH)N radical adduct followed by the formation of CF3 and HOCN. The OH radicals could be recycled through the interception of CF3C(OH)N by O2 to form the orientation‐dependent CF3C(OH)NO2 radicals, leading to CF3C(O)NO + OH via the successive H‐migration and OO bond fission. Under the typical atmospheric conditions, the OH‐recycling mechanism is dominant for the oxidation of CF3CN. The production of CF3 and HOCN can be significant at high temperatures and low pressures. Theoretical rate coefficients are in excellent agreement with the experimental data. The effect of substitution on the reactivity of the perfluoronitriles toward OH was revealed. The present work provides a fundamental understanding on the degradation of CF3CN and serves as a theoretical basis for its secondary chemistry.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

Relating Molecular Dynamics Simulations to Functional Activity for Gly-Rich Membranolytic Helical Kiadin Peptides.

Kiadins are in silico designed peptides with a strong similarity to diPGLa-H, a tandem sequence of PGLa-H (KIAKVALKAL) and with single, double or quadruple glycine substitutions. They were found to show high variability in their activity and selectivity against Gram-negative and Gram-positive bacteria, as well as cytotoxicity against host cells, which are influenced by the number and placing of glycine residues along the sequence. The conformational flexibility introduced by these substitutions contributes differently peptide structuring and to their interactions with the model membranes, as observed by molecular dynamics simulations. We relate these results to experimentally determined data on the structure of kiadins and their interactions with liposomes having a phospholipid membrane composition similar to simulation membrane models, as well as to their antibacterial and cytotoxic activities, and also discuss the challenges in interpreting these multiscale experiments and understanding why the presence of glycine residues in the sequence affected the antibacterial potency and toxicity towards host cells in a different manner.

Coronavirus XBB.1.5 as an Indicator of the Long-Term Continuation of the Covid-19 pandemic. What Next for Vaccination?

The article discusses the properties of the pandemic strain XBB.1.5 S protein in comparison with the 1918 and 2009 pandemic strains hemagglutinin H1. The S-protein XBB.1.5 already contains more than 40 mutations realized by substituting different amino acids through single and dinucleotide substitutions, deletions and the use of predominantly transversions. The variability of H1N1 influenza virus hemagglutinin is associated with single nucleotide substitutions at a constant length. Conditional extrapolation of influenza virus hemagglutinin variability data on coronavirus S-protein sizes suggests that new pandemic strains will emerge in the next 2-3 years, avoiding the immune defense formed by vaccination against the strains preceding them. The inability to create through the adaptive immune system a long-term immunity to pandemic coronaviruses, as well as to other respiratory viruses with a short incubation cycle, puts on the agenda the need to find new vaccine designs that provide a combination of long-term adaptive and trained immunity. The problem in the search for such vaccines is associated with the regulation of the activity of the innate immune system and ensuring the stability of trained immunity.

Structural insights into a regulatory mechanism of FIR RRM1-FUSE interaction.

FUBP-interacting repressor (FIR) is a suppressor of transcription of the proto-oncogene MYC. FIR binds to the far upstream element (FUSE) of the MYC promoter. Competition of FIR with FUSE-binding protein 1 (FUBP1) is a key mechanism of MYC transcriptional regulation. To gain insights into the structural mechanisms regulating FIR DNA interaction, we determined the crystal structure of two FIR RRM domains (RRM1-2) with single-stranded FUSE DNA sequences. These structures revealed an ability of the RRM domain to recognize diverse FUSE regions through distinct intermolecular interactions and binding modes. Comparative structural analyses against available RRM-ssDNA/RNA complexes showed that the nucleotide configurations in FIR were similar to those in other RRMs that harbour a tyrosine at the conserved aromatic position in the RNP2 motif (Y-type RRM), but not those with a phenylalanine (F-type RRM). Site-directed mutagenesis experiments demonstrated that a single substitution, Y115F, altered the binding affinities of oligonucleotides to FIR RRM, suggesting an important role of this conserved aromatic residue in ssDNA/RNA interactions. Our study provides the structural basis for further mechanistic studies on this important protein-DNA interaction.

Exploiting Molecular Symmetry to Quantitatively Map the Excited-State Landscape of Iron-Sulfur Clusters.

Cuboidal [Fe4S4] clusters are ubiquitous cofactors in biological redox chemistry. In the [Fe4S4]1+ state, pairwise spin coupling gives rise to six arrangements of the Fe valences ("valence isomers") among the four Fe centers. Because of the magnetic complexity of these systems, it has been challenging to understand how a protein's active site dictates both the arrangement of the valences in the ground state as well as the population of excited-state valence isomers. Here, we show that the ground-state valence isomer landscape can be simplified from a six-level system in an asymmetric protein environment to a two-level system by studying the problem in synthetic [Fe4S4]1+ clusters with solution C3v symmetry. This simplification allows for the energy differences between valence isomers to be quantified (in some cases with a resolution of <0.1 kcal/mol) by simultaneously fitting the VT NMR and solution magnetic moment data. Using this fitting protocol, we map the excited-state landscape for a range of clusters of the form [(SIMes)3Fe4S4-X/L]n, (SIMes = 1,3-dimesityl-imidazol-4,5-dihydro-2-ylidene; n = 0 for anionic, X-type ligands and n = +1 for neutral, L-type ligands) and find that a single ligand substitution can alter the relative ground-state energies of valence isomers by at least 103 cm-1. On this basis, we suggest that one result of "non-canonical" amino acid ligation in Fe-S proteins is the redistribution of the valence electrons in the manifold of thermally populated excited states.

A Single Nonsynonymous Substitution in the RNA-Dependent RNA Polymerase of Potato virus Y Allows the Simultaneous Breakdown of Two Different Forms of Antiviral Resistance in Capsicum annuum.

The dominant Pvr4 gene in pepper (Capsicum annuum) confers resistance to members of six potyvirus species, all of which belong to the Potato virus Y (PVY) phylogenetic group. The corresponding avirulence factor in the PVY genome is the NIb cistron (i.e., RNA-dependent RNA polymerase). Here, we describe a new source of potyvirus resistance in the Guatemalan accession C. annuum cv. PM949. PM949 is resistant to members of at least three potyvirus species, a subset of those controlled by Pvr4. The F1 progeny between PM949 and the susceptible cultivar Yolo Wonder was susceptible to PVY, indicating that the resistance is recessive. The segregation ratio between resistant and susceptible plants observed in the F2 progeny matched preferably with resistance being determined by two unlinked recessive genes independently conferring resistance to PVY. Inoculations by grafting resulted in the selection of PVY mutants breaking PM949 resistance and, less efficiently, Pvr4-mediated resistance. The codon substitution E472K in the NIb cistron of PVY, which was shown previously to be sufficient to break Pvr4 resistance, was also sufficient to break PM949 resistance, a rare example of cross-pathogenicity effect. In contrast, the other selected NIb mutants showed specific infectivity in PM949 or Pvr4 plants. Comparison of Pvr4 and PM949 resistance, which share the same target in PVY, provides interesting insights into the determinants of resistance durability.

Excited-State Dynamics during Primary C-I Homolysis in Acetyl Iodide Revealed by Ultrafast Core-Level Spectroscopy.

In typical carbonyl-containing molecules, bond dissociation events follow initial excitation to nπC═O* states. However, in acetyl iodide, the iodine atom gives rise to electronic states with mixed nπC═O* and nσC-I* character, leading to complex excited-state dynamics, ultimately resulting in dissociation. Using ultrafast extreme ultraviolet (XUV) transient absorption spectroscopy and quantum chemical calculations, we present an investigation of the primary photodissociation dynamics of acetyl iodide via time-resolved spectroscopy of core-to-valence transitions of the I atom after 266 nm excitation. The probed I 4d-to-valence transitions show features that evolve on sub-100-fs time scales, reporting on excited-state wavepacket evolution during dissociation. These features subsequently evolve to yield spectral signatures corresponding to free iodine atoms in their spin-orbit ground and excited states with a branching ratio of 1.1:1 following dissociation of the C-I bond. Calculations of the valence excitation spectrum via equation-of-motion coupled cluster with single and double substitutions (EOM-CCSD) show that initial excited states are of spin-mixed character. From the initially pumped spin-mixed state, we use a combination of time-dependent density functional theory (TDDFT)-driven nonadiabatic ab initio molecular dynamics and EOM-CCSD calculations of the N4,5 edge to reveal a sharp inflection point in the transient XUV signal that corresponds to rapid C-I homolysis. By examining the molecular orbitals involved in the core-level excitations at and around this inflection point, we are able to piece together a detailed picture of C-I bond photolysis in which d → σ* transitions give way to d → p excitations as the bond dissociates. We also report theoretical predictions of short-lived, weak 4d → 5d transitions in acetyl iodide, validated by weak bleaching in the experimental transient XUV spectra. This joint experimental-theoretical effort has thus unraveled the detailed electronic structure and dynamics of a strongly spin-orbit coupled system.

Polyclonal Spread of Fosfomycin Resistance among Carbapenemase-Producing Members of the Enterobacterales in the Czech Republic.

Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.

Role of KatG in Francisella tularensis Susceptibility to Resazurin.

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the risk of potential release of antibiotic-resistant F. tularensis strains, new therapeutics against F. tularensis must be developed. Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The mode of action of resazomycins is not understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The FTL_1504 gene was identified as being mutated in all of the Rz-resistant (Rzr) strains sequenced. FTL_1504 encodes for the catalase KatG which catalyzes the decomposition of hydrogen peroxide to water and oxygen. Moreover, KatG has also been shown to play an essential role in the activity of a different antibiotic, isonazid. Four different coding mutations were characterized in katG that included single base pair substitutions and a deletion with all resulting in the introduction of a premature stop codon. All Rzr mutants tested had reduced catalase activity compared to wild-type F. tularensis LVS with three of the four mutations resulting in loss of KatG expression. Based on this data, we hypothesize KatG plays a role in the susceptibility of F. tularensis to resazurin. To address this, we are generating a katG deletion mutant in F. tularensis LVS and measuring its Rz susceptibility via agar dilution assays. Investigation of the contribution of KatG may unveil a unique role for the oxidative stress response in the mode of action of this family of antibiotics