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Hughes, Diarmaid
Publications (10 of 111) Show all publications
Cao, S., Huseby, D. L., Brandis, G. & Hughes, D. (2017). Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus. mBio, 8(3), Article ID e00358-17.
Open this publication in new window or tab >>Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus
2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, e00358-17Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2017
Keyword
ATP, experimental evolution, growth compensation, hemin biosynthesis, menaquinone, SrrAB, transcriptional regulation, translational suppression
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-330055 (URN)10.1128/mBio.00358-17 (DOI)000404733300009 ()
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2017-11-16Bibliographically approved
Banin, E., Hughes, D. & Kuipers, O. P. (2017). Bacterial pathogens, antibiotics and antibiotic resistance: Editorial. FEMS Microbiology Reviews, 41(3), 450-452, Article ID fux016.
Open this publication in new window or tab >>Bacterial pathogens, antibiotics and antibiotic resistance: Editorial
2017 (English)In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, 450-452 p., fux016Article in journal, Editorial material (Other academic) Published
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-332902 (URN)10.1093/femsre/fux016 (DOI)000402064900012 ()28486583 (PubMedID)
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06Bibliographically approved
Pietsch, F., Bergman, J. M., Brandis, G., Marcusson, L. L., Zorzet, A., Huseby, D. L. & Hughes, D. (2017). Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects. Journal of Antimicrobial Chemotherapy, 72(1), 75-84.
Open this publication in new window or tab >>Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects
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2017 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 72, no 1, 75-84 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the beta-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Methods: Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Results: Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. Conclusions: These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2017
National Category
Infectious Medicine Microbiology in the medical area Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-319816 (URN)10.1093/jac/dkw364 (DOI)000394038700010 ()27621175 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2017-04-10 Created: 2017-04-10 Last updated: 2018-01-13Bibliographically approved
De Rosa, M., Lu, L., Zamaratski, E., Szałaj, N., Cao, S., Wadensten, H., . . . Karlen, A. (2017). Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB). Bioorganic & Medicinal Chemistry, 25(3), 897-911.
Open this publication in new window or tab >>Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)
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2017 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, 897-911 p.Article in journal (Refereed) Published
Abstract [en]

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

Keyword
Antibacterials, Escherichia coli, Oligopeptides, Solid-phase peptide synthesis, Type I signal peptidase
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-314110 (URN)10.1016/j.bmc.2016.12.003 (DOI)000394201900009 ()28038943 (PubMedID)
Funder
Swedish Research Council, 521-2014-6711 521-2013-2904 521-2013-3105 621-2014-6215Swedish Foundation for Strategic Research , RIF14-0078Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

Maria De Rosa and Lu Lu contributed equally to this work.

Available from: 2017-01-27 Created: 2017-01-27 Last updated: 2018-01-13Bibliographically approved
Hughes, D. & Andersson, D. I. (2017). Environmental and genetic modulation of the phenotypic expression of antibiotic resistance. FEMS Microbiology Reviews, 41(3), 374-391, Article ID fux004.
Open this publication in new window or tab >>Environmental and genetic modulation of the phenotypic expression of antibiotic resistance
2017 (English)In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, 374-391 p., fux004Article, review/survey (Refereed) Published
Abstract [en]

Antibiotic resistance can be acquired by mutation or horizontal transfer of a resistance gene, and generally an acquired mechanism results in a predictable increase in phenotypic resistance. However, recent findings suggest that the environment and/or the genetic context can modify the phenotypic expression of specific resistance genes/mutations. An important implication from these findings is that a given genotype does not always result in the expected phenotype. This dissociation of genotype and phenotype has important consequences for clinical bacteriology and for our ability to predict resistance phenotypes from genetics and DNA sequences. A related problem concerns the degree to which the genes/mutations currently identified in vitro can fully explain the in vivo resistance phenotype, or whether there is a significant additional amount of presently unknown mutations/genes (genetic 'dark matter') that could contribute to resistance in clinical isolates. Finally, a very important question is whether/how we can identify the genetic features that contribute to making a successful pathogen, and predict why some resistant clones are very successful and spread globally? In this review, we describe different environmental and genetic factors that influence phenotypic expression of antibiotic resistance genes/mutations and how this information is needed to understand why particular resistant clones spread worldwide and to what extent we can use DNA sequences to predict evolutionary success.

Keyword
persisters, pan genome, epistasis, successful clones, heteroresistance, virulence
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-332901 (URN)10.1093/femsre/fux004 (DOI)000402064900008 ()28333270 (PubMedID)
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2017-11-06Bibliographically approved
Kacar, B., Garmendia, E., Tuncbag, N., Andersson, D. I. & Hughes, D. (2017). Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio, 8(4), Article ID e01276-17.
Open this publication in new window or tab >>Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs
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2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 4, e01276-17Article in journal (Refereed) Published
Abstract [en]

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Keyword
EF-Tu, ancient genes, horizontal gene transfer, proteobacteria, tuf
National Category
Microbiology
Research subject
Biology with specialization in Microbiology; Biology with specialization in Molecular Biology; Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-330501 (URN)10.1128/mBio.01276-17 (DOI)000409384300045 ()28851849 (PubMedID)
Available from: 2017-10-02 Created: 2017-10-02 Last updated: 2017-12-19Bibliographically approved
Brandis, G., Cao, S., Huseby, D. L. & Hughes, D. (2017). Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance. MICROBIAL CELL, 4(8), 275-277.
Open this publication in new window or tab >>Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance
2017 (English)In: MICROBIAL CELL, ISSN 2311-2638, Vol. 4, no 8, 275-277 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
SCV (small colony variant), suppressors, SrrAB, tRNA mutations, intragenic suppressor, extragenic suppressor, ATP generation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-332676 (URN)10.15698/mic2017.08.587 (DOI)000407316900005 ()28845425 (PubMedID)
Funder
Swedish Research Council, 2013-02904, 2016-04449
Available from: 2017-10-31 Created: 2017-10-31 Last updated: 2018-01-13Bibliographically approved
Huseby, D. L., Pietsch, F., Brandis, G., Garoff, L., Tegehall, A. & Hughes, D. (2017). Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli. Molecular biology and evolution, 34(5), 1029-1039.
Open this publication in new window or tab >>Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli
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2017 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 5, 1029-1039 p.Article in journal (Refereed) Published
Abstract [en]

Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.

Keyword
ciprofloxacin, multistep evolution, population bottleneck, modeling evolution, clinical isolates
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-264874 (URN)10.1093/molbev/msx052 (DOI)28087782 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2015-10-20 Created: 2015-10-19 Last updated: 2017-05-11Bibliographically approved
Andersson, D. I. & Hughes, D. (2017). Selection and Transmission of Antibiotic-Resistant Bacteria. Microbiology Spectrum, 5(4), Article ID UNSP MTBP-0013-2016.
Open this publication in new window or tab >>Selection and Transmission of Antibiotic-Resistant Bacteria
2017 (English)In: Microbiology Spectrum, ISSN 2165-0497, Vol. 5, no 4, UNSP MTBP-0013-2016Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-336646 (URN)10.1128/microbiolspec.MTBP-0013-2016 (DOI)000412228600001 ()
Available from: 2017-12-15 Created: 2017-12-15 Last updated: 2017-12-15Bibliographically approved
Paulsson, J., El Karoui, M., Lindell, M. & Hughes, D. (2017). The processive kinetics of gene conversion in bacteria. Molecular Microbiology, 104(5), 752-760.
Open this publication in new window or tab >>The processive kinetics of gene conversion in bacteria
2017 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 104, no 5, 752-760 p.Article in journal (Refereed) Published
Abstract [en]

Gene conversion, non-reciprocal transfer from one homologous sequence to another, is a major force in evolutionary dynamics, promoting co-evolution in gene families and maintaining similarities between repeated genes. However, the properties of the transfer - where it initiates, how far it proceeds and how the resulting conversion tracts are affected by mismatch repair - are not well understood. Here, we use the duplicate tuf genes in Salmonella as a quantitatively tractable model system for gene conversion. We selected for conversion in multiple different positions of tuf, and examined the resulting distributions of conversion tracts in mismatch repair-deficient and mismatch repair-proficient strains. A simple stochastic model accounting for the essential steps of conversion showed excellent agreement with the data for all selection points using the same value of the conversion processivity, which is the only kinetic parameter of the model. The analysis suggests that gene conversion effectively initiates uniformly at any position within a tuf gene, and proceeds with an effectively uniform conversion processivity in either direction limited by the bounds of the gene.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-327241 (URN)10.1111/mmi.13661 (DOI)000402864900005 ()28256783 (PubMedID)
Available from: 2017-08-08 Created: 2017-08-08 Last updated: 2017-08-08Bibliographically approved
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