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Sökning: WFRF:(Hughes Diarmaid 1956 )

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1.
  • Abdulkarim, Farhad, et al. (författare)
  • Homologous recombination between the tuf genes of Salmonella typhimurium
  • 1996
  • Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 260:4, s. 506-522
  • Tidskriftsartikel (refereegranskat)abstract
    • The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.
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2.
  • Abdulkarim, Farhad, et al. (författare)
  • Missense substitutions lethal to essential functions of EF-Tu
  • 1991
  • Ingår i: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 73:12, s. 1457-1464
  • Tidskriftsartikel (refereegranskat)abstract
    • We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.
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3.
  • Abdulkarim, Farhad, et al. (författare)
  • Mutants of EF-Tu defective in binding aminoacyl-tRNA
  • 1996
  • Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 382:3, s. 297-303
  • Tidskriftsartikel (refereegranskat)abstract
    • Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120Gln, Gln124Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu · GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k(cat)/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120Gln, Gln124Arg and Tyr160Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu · GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30-fold lower than in the mild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (k(d)) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.
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4.
  • Abdulkarim, Farhad, et al. (författare)
  • Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP
  • 1994
  • Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 352, s. 118-122
  • Tidskriftsartikel (refereegranskat)abstract
    • The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis.We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at sevendifferent sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu’GTP and EF-Tu.GDP. In the active GTP form of EF-Tu themutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.
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5.
  • Andersson, Dan, et al. (författare)
  • Antibiotikaresistens: är den reversibel?
  • 1998
  • Ingår i: Smittskydd: Smittskyddsinstitutets tidskrift. - 1401-0690. ; 4:1, s. 3-5
  • Recension (övrigt vetenskapligt/konstnärligt)
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6.
  • Andersson, Dan I., et al. (författare)
  • Antibiotic resistance and its cost : is it possible to reverse resistance?
  • 2010
  • Ingår i: Nature Reviews Microbiology. - : Springer Science and Business Media LLC. - 1740-1526 .- 1740-1534. ; 8:4, s. 260-271
  • Forskningsöversikt (refereegranskat)abstract
    • Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.
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7.
  • Andersson, D.I, et al. (författare)
  • Antibiotikaresistens här för att stanna?
  • 1998
  • Ingår i: Läkartidningen. - 0023-7205 .- 1652-7518. ; 95:37, s. 3940-3944
  • Forskningsöversikt (övrigt vetenskapligt/konstnärligt)
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8.
  • Andersson, Dan I., et al. (författare)
  • Biological roles of translesion synthesis DNA polymerases in eubacteria
  • 2010
  • Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:3, s. 540-548
  • Forskningsöversikt (refereegranskat)abstract
    • Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.
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9.
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10.
  • Andersson, Dan I, et al. (författare)
  • Muller's ratchet decreases fitness of a DNA-based microbe
  • 1996
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 93:2, s. 906-907
  • Tidskriftsartikel (refereegranskat)abstract
    • Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.
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11.
  • Andersson, Dan I, et al. (författare)
  • Selection and Transmission of Antibiotic-Resistant Bacteria
  • 2017
  • Ingår i: Microbiology Spectrum. - 2165-0497. ; 5:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Ever since antibiotics were introduced into human and veterinary medicine to treat and prevent bacterial infections there has been a steady selection and increase in the frequency of antibiotic resistant bacteria. To be able to reduce the rate of resistance evolution, we need to understand how various biotic and abiotic factors interact to drive the complex processes of resistance emergence and transmission. We describe several of the fundamental factors that underlay resistance evolution, including rates and niches of emergence and persistence of resistant bacteria, time- and space-gradients of various selective agents, and rates and routes of transmission of resistant bacteria between humans, animals and other environments. Furthermore, we discuss the options available to reduce the rate of resistance evolution and/or transmission and their advantages and disadvantages.
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12.
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13.
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14.
  • Aranzana-Climent, Vincent, et al. (författare)
  • Translational in vitro and in vivo PKPD modelling for apramycin against Gram-negative lung pathogens to facilitate prediction of human efficacious dose in pneumonia
  • 2022
  • Ingår i: Clinical Microbiology and Infection. - : Elsevier B.V.. - 1198-743X .- 1469-0691. ; 28:10, s. 1367-1374
  • Tidskriftsartikel (refereegranskat)abstract
    • Objectives: New drugs and methods to efficiently fight carbapenem-resistant gram-negative pathogens are sorely needed. In this study, we characterized the preclinical pharmacokinetics (PK) and pharmacodynamics of the clinical stage drug candidate apramycin in time kill and mouse lung infection models. Based on in vitro and in vivo data, we developed a mathematical model to predict human efficacy. Methods: Three pneumonia-inducing gram-negative species Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were studied. Bactericidal kinetics were evaluated with time-kill curves; in vivo PK were studied in healthy and infected mice, with sampling in plasma and epithelial lining fluid after subcutaneous administration; in vivo efficacy was measured in a neutropenic mouse pneumonia model. A pharmacokinetic-pharmacodynamic model, integrating all the data, was developed and simulations were performed. Results: Good lung penetration of apramycin in epithelial lining fluid (ELF) was shown (area under the curve (AUC)ELF/AUCplasma = 88%). Plasma clearance was 48% lower in lung infected mice compared to healthy mice. For two out of five strains studied, a delay in growth (∼5 h) was observed in vivo but not in vitro. The mathematical model enabled integration of lung PK to drive mouse PK and pharmacodynamics. Simulations predicted that 30 mg/kg of apramycin once daily would result in bacteriostasis in patients. Discussion: Apramycin is a candidate for treatment of carbapenem-resistant gram-negative pneumonia as demonstrated in an integrated modeling framework for three bacterial species. We show that mathematical modelling is a useful tool for simultaneous inclusion of multiple data sources, notably plasma and lung in vivo PK and simulation of expected scenarios in a clinical setting, notably lung infections. © 2022 The Author(s)
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15.
  • Arrazuria, Rakel, et al. (författare)
  • Expert workshop summary : Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections
  • 2022
  • Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 13
  • Tidskriftsartikel (refereegranskat)abstract
    • The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop "Advancing toward a standardized murine model to evaluate treatments for AMR lung infections". This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (beta -lactam/beta -lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model.
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16.
  • Arrazuria, Rakel, et al. (författare)
  • Variability of murine bacterial pneumonia models used to evaluate antimicrobial agents
  • 2022
  • Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 13
  • Forskningsöversikt (refereegranskat)abstract
    • Antimicrobial resistance has become one of the greatest threats to human health, and new antibacterial treatments are urgently needed. As a tool to develop novel therapies, animal models are essential to bridge the gap between preclinical and clinical research. However, despite common usage of in vivo models that mimic clinical infection, translational challenges remain high. Standardization of in vivo models is deemed necessary to improve the robustness and reproducibility of preclinical studies and thus translational research. The European Innovative Medicines Initiative (IMI)-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium, aims to develop a standardized, quality-controlled murine pneumonia model for preclinical efficacy testing of novel anti-infective candidates and to improve tools for the translation of preclinical data to the clinic. In this review of murine pneumonia model data published in the last 10 years, we present our findings of considerable variability in the protocols employed for testing the efficacy of antimicrobial compounds using this in vivo model. Based on specific inclusion criteria, fifty-three studies focusing on antimicrobial assessment against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were reviewed in detail. The data revealed marked differences in the experimental design of the murine pneumonia models employed in the literature. Notably, several differences were observed in variables that are expected to impact the obtained results, such as the immune status of the animals, the age, infection route and sample processing, highlighting the necessity of a standardized model.
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17.
  • Arwidsson, Ola, et al. (författare)
  • Evidence against reciprocal recombination as the basis for tuf gene conversion in Salmonella enterica serovar Typhimurium
  • 2004
  • Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 338:3, s. 463-467
  • Tidskriftsartikel (refereegranskat)abstract
    • The duplicate tuf genes on the Salmonella enterica serovar Typhimurium chromosome co-evolve by a RecA-, RecB-dependent gene conversion mechanism. Gene conversion is defined as a non-reciprocal transfer of genetic information. However, in a replicating bacterial chromosome there is a possibility that a reciprocal genetic exchange between different tuf genes sitting on sister chromosomes could result in "apparent" gene conversion. We asked whether the major mechanism of tuf gene conversion was classical or apparent. We devised a genetic selection that allowed us to isolate and examine both expected products from a reciprocal recombination event between the tuf genes. Using this selection we tested within individual cultures for a correlation in the frequency of jackpots as expected if recombination were reciprocal. We found no correlation, either in the frequency of each type of recombinant product, or in the DNA sequences of the products resulting from each recombination event. We conclude that the evidence argues in favor of a non-reciprocal gene conversion mechanism as the basis for tuf gene co-evolution.
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18.
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19.
  • Bartke, Katrin, et al. (författare)
  • Evolution of Bacterial Interspecies Hybrids with Enlarged Chromosomes
  • 2022
  • Ingår i: Genome Biology and Evolution. - : Oxford University Press. - 1759-6653. ; 14:10
  • Tidskriftsartikel (refereegranskat)abstract
    • Conjugation driven by a chromosomally integrated F-plasmid (high frequency of recombination strain) can create bacteria with hybrid chromosomes. Previous studies of interspecies hybrids have focused on hybrids in which a region of donor chromosome replaces an orthologous region of recipient chromosome leaving chromosome size unchanged. Very little is known about hybrids with enlarged chromosomes, the mechanisms of their creation, or their subsequent trajectories of adaptative evolution. We addressed this by selecting 11 interspecies hybrids between Escherichia coli and Salmonella Typhimurium in which genome size was enlarged. In three cases, this occurred by the creation of an F '-plasmid while in the remaining eight, it was due to recombination of donor DNA into the recipient chromosome. Chromosome length increased by up to 33% and was associated in most cases with reduced growth fitness. Two hybrids, in which chromosome length was increased by the addition of 0.97 and 1.3 Mb, respectively, were evolved to study genetic pathways of fitness cost amelioration. In each case, relative fitness rapidly approached one and this was associated with large deletions involving recombination between repetitive DNA sequences. The locations of these repetitive sequences played a major role in determining the architecture of the evolved genotypes. Notably, in ten out of ten independent evolution experiments, deletions removed DNA of both species, creating high-fitness strains with hybrid chromosomes. In conclusion, we found that enlargement of a bacterial chromosome by acquisition of diverged orthologous DNA is followed by a period of rapid evolutionary adjustment frequently creating irreversibly hybrid chromosomes.
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20.
  • Bartke, Katrin, et al. (författare)
  • Genetic Architecture and Fitness of Bacterial Interspecies Hybrids
  • 2021
  • Ingår i: Molecular biology and evolution. - : Oxford University Press. - 0737-4038 .- 1537-1719. ; 38:4, s. 1472-1481
  • Tidskriftsartikel (refereegranskat)abstract
    • Integration of a conjugative plasmid into a bacterial chromosome can promote the transfer of chromosomal DNA to other bacteria. Intraspecies chromosomal conjugation is believed responsible for creating the global pathogens Klebsiella pneumoniae ST258 and Escherichia coli ST1193. Interspecies conjugation is also possible but little is known about the genetic architecture or fitness of such hybrids. To study this, we generated by conjugation 14 hybrids of E. coli and Salmonella enterica. These species belong to different genera, diverged from a common ancestor >100 Ma, and share a conserved order of orthologous genes with similar to 15% nucleotide divergence. Genomic analysis revealed that all but one hybrid had acquired a contiguous segment of donor E. coli DNA, replacing a homologous region of recipient Salmonella chromosome, and ranging in size from similar to 100 to >4,000 kb. Recombination joints occurred in sequences with higher-than-average nucleotide identity. Most hybrid strains suffered a large reduction in growth rate, but the magnitude of this cost did not correlate with the length of foreign DNA. Compensatory evolution to ameliorate the cost of low-fitness hybrids pointed towards disruption of complex genetic networks as a cause. Most interestingly, 4 of the 14 hybrids, in which from 45% to 90% of the Salmonella chromosome was replaced with E. coli DNA, showed no significant reduction in growth fitness. These data suggest that the barriers to creating high-fitness interspecies hybrids may be significantly lower than generally appreciated with implications for the creation of novel species.
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21.
  • Becker, K., et al. (författare)
  • Antibacterial activity of apramycin at acidic pH warrants wide therapeutic window in the treatment of complicated urinary tract infections and acute pyelonephritis
  • 2021
  • Ingår i: EBioMedicine. - : Elsevier B.V.. - 2352-3964. ; 73
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section. © 2021 The Author(s)
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22.
  • Becker, K., et al. (författare)
  • Efficacy of EBL-1003 (apramycin) against Acinetobacter baumannii lung infections in mice
  • 2021
  • Ingår i: Clinical Microbiology and Infection. - : Elsevier B.V.. - 1198-743X .- 1469-0691. ; 27:9, s. 1315-
  • Tidskriftsartikel (refereegranskat)abstract
    • Objectives: Novel therapeutics are urgently required for the treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) causing critical infections with high mortality. Here we assessed the therapeutic potential of the clinical-stage drug candidate EBL-1003 (crystalline free base of apramycin) in the treatment of CRAB lung infections. Methods: The genotypic and phenotypic susceptibility of CRAB clinical isolates to aminoglycosides and colistin was assessed by database mining and broth microdilution. The therapeutic potential was assessed by target attainment simulations on the basis of time–kill kinetics, a murine lung infection model, comparative pharmacokinetic analysis in plasma, epithelial lining fluid (ELF) and lung tissue, and pharmacokinetic/pharmacodynamic (PKPD) modelling. Results: Resistance gene annotations of 5451 CRAB genomes deposited in the National Database of Antibiotic Resistant Organisms (NDARO) suggested >99.9% of genotypic susceptibility to apramycin. Low susceptibility to standard-of-care aminoglycosides and high susceptibility to EBL-1003 were confirmed by antimicrobial susceptibility testing of 100 A. baumannii isolates. Time–kill experiments and a mouse lung infection model with the extremely drug-resistant CRAB strain AR Bank #0282 resulted in rapid 4-log CFU reduction both in vitro and in vivo. A single dose of 125 mg/kg EBL-1003 in CRAB-infected mice resulted in an AUC of 339 h × μg/mL in plasma and 299 h × μg/mL in ELF, suggesting a lung penetration of 88%. PKPD simulations suggested a previously predicted dose of 30 mg/kg in patients (creatinine clearance (CLCr) = 80 mL/min) to result in >99% probability of –2 log target attainment for MICs up to 16 μg/mL. Conclusions: This study provides proof of concept for the efficacy of EBL-1003 in the treatment of CRAB lung infections. Broad in vitro coverage, rapid killing, potent in vivo efficacy, and a high probability of target attainment render EBL-1003 a strong therapeutic candidate for a priority pathogen for which treatment options are very limited. © 2020 The Author(s)
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23.
  • Benediktsdottir, Andrea, 1990-, et al. (författare)
  • Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors : Synthesis and biological evaluation
  • 2021
  • Ingår i: European Journal of Medicinal Chemistry. - : Elsevier. - 0223-5234 .- 1768-3254. ; 224
  • Tidskriftsartikel (refereegranskat)abstract
    • Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.
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24.
  • Benediktsdottir, Andrea, et al. (författare)
  • Design, synthesis, and in vitro biological evaluation of meta-sulfonamidobenzamide-based antibacterial LpxH inhibitors
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it an ideal antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates N-methyl removal when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter hERG blocking. Furthermore, a LpxH-bound X-ray structure of the meta-sulfonamidobenzamide analog facilitated comparison with complexes with ortho-N-methyl-sulfonamidobenzamide analogs, and with the natural enzymatic reaction product lipid X, providing new insights into the enzyme-ligand interactions. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH-targeting hits with the potential for optimization in future antibacterial hit-to-lead programs.  
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25.
  • Berryhill, Brandon A., et al. (författare)
  • Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections
  • 2021
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 118:10
  • Tidskriftsartikel (refereegranskat)abstract
    • In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. Because of the mode of action of PYOSa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear populations of S. aureus. Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.
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26.
  • Bjorkman, J, et al. (författare)
  • Virulence of antibiotic-resistant Salmonella typhimurium
  • 1998
  • Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - : NATL ACAD SCIENCES. - 0027-8424. ; 95:7, s. 3949-3953
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice, Of seven resistant mutants examined, sis were avirulent and one was similar to the wild type In competition experiments in mi
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27.
  • Björkman, J, et al. (författare)
  • Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance
  • 2000
  • Ingår i: Science. - : AMER ASSOC ADVANCEMENT SCIENCE. - 0036-8075 .- 1095-9203. ; 287:5457, s. 1479-1482
  • Tidskriftsartikel (refereegranskat)abstract
    • Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.
  •  
28.
  • Björkman, Johanna, et al. (författare)
  • Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium
  • 1999
  • Ingår i: Molecular Microbiology. - : BLACKWELL SCIENCE LTD. - 0950-382X .- 1365-2958. ; 31:1, s. 53-58
  • Tidskriftsartikel (populärvet., debatt m.m.)abstract
    • Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.
  •  
29.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Autoregulation of the tufB operon in Salmonella
  • 2016
  • Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 100:6, s. 1004-1016
  • Tidskriftsartikel (refereegranskat)abstract
    • In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3′-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5′-end of the tufB mRNA. Formation of the ‘closed’ structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.
  •  
30.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella
  • 2018
  • Ingår i: Molecular Microbiology. - : WILEY. - 0950-382X .- 1365-2958. ; 108:6, s. 697-710
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.
  •  
31.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis
  • 2015
  • Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 70:3, s. 680-685
  • Tidskriftsartikel (refereegranskat)abstract
    • ObjectivesMutations in the β-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (RifR). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst RifR clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of RifR mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of RifR mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms.MethodsWe constructed 122 different RifR mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of RifRM. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates.Results(i) RifR mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent RifR mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a RifR mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates.ConclusionsThis suggests that the success of RifR mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.
  •  
32.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Expression of the qepA1 gene is induced under antibiotic exposure
  • 2021
  • Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 76:6, s. 1433-1440
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundThe qepA1 gene encodes an efflux pump that reduces susceptibility to ciprofloxacin. Little is known about the regulation of qepA1 expression.ObjectivesTo assess the potential role of ciprofloxacin and other antibiotics in the regulation of qepA1 gene expression. To identify the promoter that drives qepA1 expression and other factors involved in expression regulation. To assess whether the identified features are universal among qepA alleles.MethodsA translational qepA1-yfp fusion under the control of the qepA1 upstream region was cloned into the Escherichia coli chromosome. Expression of the fusion protein was measured in the presence of various antibiotics. Deletions within the upstream region were introduced to identify regions involved in gene expression and regulation. The qepA1 coding sequence and upstream region were compared with all available qepA sequences.ResultsCellular stress caused by the presence of various antibiotics can induce qepA1 expression. The qepA1 gene is fused to a class I integron and gene expression is driven by the Pc promoter within the integrase gene. A segment within the integron belonging to a truncated dfrB4 gene is essential for the regulation of qepA1 expression. This genetic context is universal among all sequenced qepA alleles.ConclusionsThe fusion of the qepA1 gene to a class I integron has created a novel regulatory unit that enables qepA1 expression to be under the control of antibiotic exposure. This setup mitigates potential negative effects of QepA1 production on bacterial fitness by restricting high-level expression to environmental conditions in which QepA1 is beneficial.
  •  
33.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Fitness-compensatory mutations in rifampicin-resistant RNA polymerase
  • 2012
  • Ingår i: Molecular Microbiology. - : Blackwell Publishing. - 0950-382X .- 1365-2958. ; 85:1, s. 142-151
  • Tidskriftsartikel (refereegranskat)abstract
    • Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant.
  •  
34.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates
  • 2013
  • Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 68:11, s. 2493-2497
  • Tidskriftsartikel (refereegranskat)abstract
    • ObjectivesThe evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism.MethodsWe created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains.ResultsCompensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation.ConclusionsThe compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.
  •  
35.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance
  • 2017
  • Ingår i: MICROBIAL CELL. - : Shared Science Publishers OG. - 2311-2638. ; 4:8, s. 275-277
  • Tidskriftsartikel (refereegranskat)abstract
    • Staphylococcus aureus can produce small colony variants (SCVs) during infections. These cause significant clinical problems because they are difficult to detect in standard microbiological screening and are associated with persistent infections. The major causes of the SCV phenotype are mutations that inhibit respiration by inactivation of genes of the menadione or hemin biosynthesis pathways. This reduces the production of ATP required to support fast growth. Importantly, it also decreases cross-membrane potential in SCVs, resulting in decreased uptake of cationic compounds, with reduced susceptibility to aminoglycoside antibiotics as a consequence. Because SCVs are slow-growing (mutations in men genes are associated with growth rates in rich medium similar to 30% of the wild-type growth rate) bacterial cultures are very susceptible to rapid takeover by faster-growing mutants (revertants or suppressors). In the case of reversion, the resulting fast growth is obviously associated with the loss of antibiotic resistance. However, direct reversion is relatively rare due to the very small genetic target size for such mutations. We explored the phenotypic consequences of SCVs evolving faster growth by routes other than direct reversion, and in particular whether any of those routes allowed for the maintenance of antibiotic resistance. In a recent paper (mBio 8: e00358-17) we demonstrated the existence of several different routes of SCV evolution to faster growth, one of which maintained the antibiotic resistance phenotype. This discovery suggests that SCVs might be more adaptable and problematic that previously thought. They are capable of surviving as a slow-growing persistent form, before evolving into a significantly faster-growing form without sacrificing their antibiotic resistance phenotype.
  •  
36.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella
  • 2019
  • Ingår i: Bio-protocol. - : Bio-Protocol LLC. - 2331-8325. ; 9:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous recombination between two locations with sequence similarity within the same bacterial chromosome. For this purpose, a selectable/counter-selectable genetic cassette is inserted into one of the locations and homologous recombination repair rates are measured as a function of recombinational removal of the inserted cassette. This recombinational repair process is called gene conversion, non-reciprocal recombination. We used this method to measure the recombination rates between genes within gene families and to study the stability of mobile genetic elements inserted into members of gene families.
  •  
37.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Mechanisms of fitness cost reduction for rifampicin-resistant strains with deletion or duplication mutations in rpoB.
  • 2018
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • Rifampicin resistance (Rif(R)) is caused by mutations in rpoB, encoding the beta-subunit of RNA polymerase. Rif(R )mutations generally incur a fitness cost and in resistant isolates are frequently accompanied by compensatory mutations in rpoA, rpoB or rpoC. Previous studies of fitness compensation focused on Rif(R) caused by amino acid substitutions within rpoB. Rif(R) is also caused by deletion and duplication mutations in rpoB but it is not known whether or how such mutants can ameliorate their fitness costs. Using experimental evolution of Salmonella carrying Rif(R) deletion or duplication mutations we identified compensatory amino acid substitution mutations within rpoA, rpoB or rpoC in 16 of 21 evolved lineages. Additionally, we found one lineage where a large deletion was compensated by duplication of adjacent amino acids (possibly to fill the gap within the protein structure), two lineages where mutations occurred outside of rpoABC, and two lineages where a duplication mutant reverted to the wild-type sequence. All but the two revertant mutants maintained the Rif(R) phenotype. These data suggest that amino acid substitution mutations are the major compensatory mechanism regardless of the nature of the primary Rif(R) mutation.
  •  
38.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Mutant RNA polymerase can reduce susceptibility to antibiotics via ppGpp-independent induction of a stringent-like response
  • 2021
  • Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 76:3, s. 606-615
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundMutations in RNA polymerase (RNAP) can reduce susceptibility to ciprofloxacin in Escherichia coli, but the mechanism of transcriptional reprogramming responsible is unknown. Strains carrying ciprofloxacin-resistant (CipR) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear.ObjectivesTo assess the potential for CipRrpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of CipRrpoB mutations. To understand how CipRrpoB mutations reprogramme RNAP.MethodsE. coli strains carrying five different CipRrpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MICCip. The effects of dksA mutations and a ppGpp0 background on growth and susceptibility phenotypes associated with CipRrpoB alleles were determined.ResultsThe number of distinct CipRrpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of CipRrpoB mutations. Deletion of dksA reduced the MICCip for strains carrying CipRrpoB alleles. A ppGpp0 phenotype had no effect on drug susceptibility.ConclusionsCipRrpoB mutations induce an ppGpp-independent stringent-like response. Approximately half of the reduction in ciprofloxacin susceptibility is caused by an increased affinity of RNAP to DksA while the other half is independent of DksA. Stringent-like response activating mutations might be the most diverse class of mutations reducing susceptibility to antibiotics.
  •  
39.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • Operon Concatenation Is an Ancient Feature That Restricts the Potential to Rearrange Bacterial Chromosomes
  • 2019
  • Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 36:9, s. 1990-2000
  • Tidskriftsartikel (refereegranskat)abstract
    • The last common ancestor of the Gammaproteobacteria carried an important 40-kb chromosome section encoding 51 proteins of the transcriptional and translational machinery. These genes were organized into eight contiguous operons (rrnB-tufB-secE-rpoBC-str-S10-spc-alpha). Over 2 Gy of evolution, in different lineages, some of the operons became separated by multigene insertions. Surprisingly, in many Enterobacteriaceae, much of the ancient organization is conserved, indicating a strong selective force on the operons to remain colinear. Here, we show for one operon pair, tufB-secE in Salmonella, that an interruption of contiguity significantly reduces growth rate. Our data show that the tufB-secE operons are concatenated by an interoperon terminator-promoter overlap that plays a significant role regulating gene expression. Interrupting operon contiguity interferes with this regulation, reducing cellular fitness. Six operons of the ancestral chromosome section remain contiguous in Salmonella (tufB-secE-rpoBC and S10-spc-alpha) and, strikingly, each of these operon pairs is also connected by an interoperon terminator-promoter overlap. Accordingly, we propose that operon concatenation is an ancient feature that restricts the potential to rearrange bacterial chromosomes and can select for the maintenance of a colinear operon organization over billions of years.
  •  
40.
  • Brandis, Gerrit, 1985-, et al. (författare)
  • The SNAP hypothesis : Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation
  • 2020
  • Ingår i: PLOS Genetics. - : PUBLIC LIBRARY SCIENCE. - 1553-7390 .- 1553-7404. ; 16:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Author summary All life on earth has evolved from a universal common ancestor with a specific order of genes on the chromosome. This order is not maintained in modern species and the standard hypothesis is that changes reflect a lack of strong selection on gene order. Here, we propose an alternative hypothesis, SNAP. The occupation of a novel environment by bacteria is generally a trade-off situation. For example, while the bacteria may not be adapted to grow well under the new conditions, they may benefit by not having to share available resources with other microorganisms. Bacterial populations frequently acquire duplications of chromosomal segments containing genes that can help them adapt to a new environment. Other genes that are also duplicated are not required in two copies so that over time a superfluous copy can be lost. Eventually, the process of duplication and gene loss can lead to the rearrangement of the gene order in the chromosomal segment. The major benefit of this model over the standard hypothesis is that the process is driven by positive selection and can reach fixation rapidly. The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.
  •  
41.
  • Cao, Sha, et al. (författare)
  • Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus
  • 2017
  • Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 8:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.
  •  
42.
  • Cao, Sha, et al. (författare)
  • Positive Selection during Niche Adaptation Results in Large-Scale and Irreversible Rearrangement of Chromosomal Gene Order in Bacteria
  • 2022
  • Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 39:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Analysis of bacterial genomes shows that, whereas diverse species share many genes in common, their linear order on the chromosome is often not conserved. Whereas rearrangements in gene order could occur by genetic drift, an alternative hypothesis is rearrangement driven by positive selection during niche adaptation (SNAP). Here, we provide the first experimental support for the SNAP hypothesis. We evolved Salmonella to adapt to growth on malate as the sole carbon source and followed the evolutionary trajectories. The initial adaptation to growth in the new environment involved the duplication of 1.66 Mb, corresponding to one-third of the Salmonella chromosome. This duplication is selected to increase the copy number of a single gene, dctA, involved in the uptake of malate. Continuing selection led to the rapid loss or mutation of duplicate genes from either copy of the duplicated region. After 2000 generations, only 31% of the originally duplicated genes remained intact and the gene order within the Salmonella chromosome has been significantly and irreversibly altered. These results experientially validate predictions made by the SNAP hypothesis and show that SNAP can be a strong driving force for rearrangements in chromosomal gene order.
  •  
43.
  • Cotman, Andrej Emanuel, et al. (författare)
  • Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold- Based DNA Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa
  • 2023
  • Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 66:2, s. 1380-1425
  • Tidskriftsartikel (refereegranskat)abstract
    • We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aerugi-nosa, which are both on the WHO priority list of antibiotic -resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase II alpha, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.
  •  
44.
  • Durcik, Martina, et al. (författare)
  • New Dual Inhibitors of Bacterial Topoisomerases with Broad-Spectrum Antibacterial Activity and In Vivo Efficacy against Vancomycin-Intermediate Staphylococcus aureus
  • 2023
  • Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 66:6, s. 3968-3994
  • Tidskriftsartikel (refereegranskat)abstract
    • A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125–0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1–4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.
  •  
45.
  • Ehrenberg, Måns, et al. (författare)
  • tRNA-ribosome interactions
  • 1995
  • Ingår i: Biochemistry and Cell Biology. - 0829-8211 .- 1208-6002. ; 73:11-12, s. 1049-1054
  • Forskningsöversikt (refereegranskat)abstract
    • Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.
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46.
  • Frimodt-Møller, Niels, et al. (författare)
  • Apramycin efficacy against carbapenem- and aminoglycoside-resistant Escherichia coli and Klebsiella pneumoniae in murine bloodstream infection models
  • 2024
  • Ingår i: International Journal of Antimicrobial Agents. - : Elsevier. - 0924-8579 .- 1872-7913. ; 64:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundThe aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, the potential of apramycin in the treatment of drug-resistant bloodstream infections (BSIs) has not yet been assessed.MethodsThe resistance gene annotations of 40 888 blood-culture isolates were analysed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for a total of nine Escherichia coli and Klebsiella pneumoniae isolates.ResultsGenotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, colistin resistance was shown in 46.4% and apramycin in 2.1%. Apramycin activity against methylated ribosomes was > 100-fold higher than that for other aminoglycosides. Frequencies of resistance were < 10-9 at 8 × minimum inhibitory concentration (MIC). Tentative epidemiological cut-offs (TECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log colony-forming unit (CFU) reduction in the blood and peritoneum. Two doses of 80 mg/kg resulted in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans and led to complete eradication of carbapenem- and aminoglycoside-resistant bacteraemia.ConclusionEncouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants the conduct of clinical studies to validate apramycin as a drug candidate for the prophylaxis and treatment of BSI.
  •  
47.
  • Garmendia, Eva, et al. (författare)
  • Chromosomal Location Determines the Rate of Intrachromosomal Homologous Recombination in Salmonella
  • 2021
  • Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 12:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Homologous recombination is an important mechanism directly involved in the repair, organization, and evolution of prokaryotic and eukaryotic chromosomes. We developed a system, based on two genetic cassettes, that allows the measurement of recombinational repair rates between different locations on the chromosome. Using this system, we analyzed 81 different positional combinations throughout the chromosome to answer the question of how the position and orientation of sequences affect intrachromosomal homologous recombination. Our results show that recombination was possible between any two locations tested in this study and that recombinational repair rates varied by just above an order of magnitude. The observed differences in rate do not correlate with distance between the recombination cassettes or with distance from the origin of replication but could be explained if each location contributes individually to the recombination event. The relative levels of accessibility for recombination vary 5-fold between the various cassette locations, and we found that the nucleoid structure of the chromosome may be the major factor influencing the recombinational accessibility of each chromosomal site. Furthermore, we found that the orientation of the recombination cassettes had a significant impact on recombination. Recombinational repair rates for the cassettes inserted as direct repeats are, on average, 2.2-fold higher than those for the same sets inserted as inverted repeats. These results suggest that the bacterial chromosome is not homogenous with regard to homologous recombination, with regions that are more or less accessible, and that the orientation of genes affects recombination rates. IMPORTANCE Bacterial chromosomes frequently carry multiple copies of genes at separate chromosomal locations. In Salmonella, these include the 7 rrn operons and the duplicate tuf genes. Genes within these families coevolve by homologous recombination, but it is not obvious whether their rates of recombination reflect general rates of intrachromosomal recombination or are an evolved property particularly associated with these conserved genes and locations. Using a novel experimental system, we show that recombination is possible between all tested pairs of locations at rates that vary by just above 1 order of magnitude. Differences in rate do not correlate with distance between the sites or distance to the origin of replication but may be explained if each location contributes individually to the recombination event. Our results suggest the existence of bacterial chromosomal domains that are differentially available for recombination and that gene orientation affects recombination rates.
  •  
48.
  • Garmendia, Eva, et al. (författare)
  • Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella
  • 2018
  • Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 9:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Highly expressed genes are commonly located close to the origin of replication of bacterial chromosomes (OriC). This location skew is thought to reflect selective advantages associated with gene dosage effects during the replication cycle. The expression of constitutively expressed genes can vary up to fivefold based on chromosomal location, but it is not clear what level of variation would occur in naturally regulated operons. We tested the magnitude of the chromosome location effect using EF-Tu (tufA, tufB), an abundant protein whose cellular level correlates with, and limits, the maximum growth rate. We translocated the Salmonella tufB operon to four locations across the chromosome. The distance from OriC had only a small effect on growth rate, consistent with this operon having the natural ability to upregulate expression and compensate for reduced gene dosage. In contrast, when the total EF-Tu concentration was limiting for the growth rate (tufA deleted), we observed a strong gene dosage effect when tufB was located further from OriC. However, only a short period of experimental evolution was required before the bacteria adapted to this EF-Tu starvation situation by acquiring genetic changes that increased expression levels from the translocated tufB gene, restoring growth rates. Our findings demonstrate that, at least for the tufB operon, gene dosage is probably not the dominant force selecting for a chromosomal location close to OriC. We suggest that the colocation of highly expressed genes close to OriC might instead be selected because it enhances their coregulation during various growth states, with gene dosage being a secondary benefit. IMPORTANCE A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.
  •  
49.
  • Garoff, Linnéa, et al. (författare)
  • Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli
  • 2018
  • Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 73:12, s. 3285-3292
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.
  •  
50.
  • Garoff, Linnéa, 1989- (författare)
  • Exploring the Ciprofloxacin Resistome
  • 2018
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • This thesis presents an exploration of the resistance evolution in Escherichia coli towards the antibiotic ciprofloxacin. High level ciprofloxacin resistance is typically acquired by an accumulation of mutations and plasmid borne genes reducing drug target binding, increasing drug efflux, and modifying the drug.Paper I describes the finding that novel mutations in tRNA synthetase gene leuS conferred resistance to ciprofloxacin. We also provided evidence for a mechanism, where the leuS mutations induced global changes in transcription that generated a net effect of increased drug efflux.In Paper II we observed that the evolutionary trajectory towards high level ciprofloxacin resistance in E. coli is repeatable and predictable in in vitro evolution experiments. However, the types and order of appearance of selected mutations was highly dependent on the bottleneck size used. In addition to the findings in Paper I, we found that mutations involved in transcription and translation were repeatedly selected upon subjection to high concentrations of ciprofloxacin.Paper III explored the resistance capacity of the plasmid-borne gene qnr, which reduces ciprofloxacin susceptibility by a target protection mechanism. We found that upon increased expression, the gene qnrS was able to bring E. coli to clinically resistant levels of ciprofloxacin without the addition of other resistance elements.  In Paper IV we aimed for a similar study as described above but with another plasmid-borne gene, the inner-membrane efflux pump qepA. However, we ran into the interesting finding of a potentially undescribed regulatory mechanism of qepA expression, which we are currently investigating.The work in this thesis presents a new addition of mutations causing ciprofloxacin resistance, and evidence that the dogma of accumulative mutations being a requirement to develop clinical resistance to ciprofloxacin in E. coli can be circumvented. This shows that there is still much to explore, even with a drug used for several decades with an already well documented resistome. We need to learn more about the evolutionary trajectories leading to antibiotic resistance, in order to slow down its development towards existing and future antibiotics to the furthest extent possible.
  •  
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