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Mechanisms of Adaptation to Deformylase Inhibitors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan Andersson)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 68
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 571
Keywords [en]
antibiotic resistance, compensatory evolution, compensatory mutations, protein synthesis, initiation factor 2, initiator tRNA, formylation, peptide deformylase inhibitors
National Category
Microbiology in the medical area
Research subject
Microbiology; Biochemistry; Geography
Identifiers
URN: urn:nbn:se:uu:diva-123242ISBN: 978-91-554-7828-5 (print)OAI: oai:DiVA.org:uu-123242DiVA, id: diva2:313609
Public defence
2010-06-11, Sal C8:301, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2010-05-21 Created: 2010-04-26 Last updated: 2018-01-12
List of papers
1. Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes
Open this publication in new window or tab >>Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 18, p. 6976-6981Article in journal (Refereed) Published
Abstract [en]

Deformylase inhibitors belong to a novel antibiotic class that targets peptide deformylase, a bacterial enzyme that removes the formyl group from N-terminal methionine in nascent polypeptides. Using the bacterium Salmonella enterica, we isolated mutants with resistance toward the peptide deformylase inhibitor actinonin. Resistance mutations were identified in two genes that are required for the formylation of methionyl (Met) initiator tRNA (tRNAi)(fMet): the fmt gene encoding the enzyme methionyl-tRNA formyltransferase and the folD gene encoding the bifunctional enzyme methylenetetrahydrofolate-dehydrogenase and -cyclohydrolase. In the absence of antibiotic, these resistance mutations conferred a fitness cost that was manifested as a reduced growth rate in laboratory medium and in mice. By serially passaging the low-fitness mutants in growth medium without antibiotic, the fitness costs could be partly ameliorated either by intragenic mutations in the fmt/folD genes or by extragenic compensatory mutations. Of the extragenically compensated fmt mutants, approximately one-third carried amplifications of the identical, tandemly repeated metZ and metW genes, encoding tRNAi. The increase in metZW gene copy number varied from 5- to 40-fold and was accompanied by a similar increase in tRNAi levels. The rise in tRNAi level compensated for the lack of methionyl-tRNA formyltransferase activity and allowed translation initiation to proceed with nonformylated methionyl tRNAi. Amplified units varied in size from 1.9 to 94 kbp. Suppression of deleterious mutations by gene amplification may be involved in the evolution of new gene functions.

Keywords
Amidohydrolases/antagonists & inhibitors/genetics/metabolism, Animals, Anti-Bacterial Agents/metabolism/pharmacology, Drug Resistance/*genetics, Evolution; Molecular, Gene Expression Regulation; Bacterial, Hydroxamic Acids/metabolism/pharmacology, Mice, Mutation, RNA; Transfer; Met/*genetics/metabolism, Salmonella typhimurium/drug effects/enzymology/genetics
National Category
Biological Sciences Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-80312 (URN)10.1073/pnas.0602171103 (DOI)16636273 (PubMedID)
Available from: 2007-01-24 Created: 2007-01-24 Last updated: 2017-12-14Bibliographically approved
2. Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance
Open this publication in new window or tab >>Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance
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2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, ISSN 20132454, Vol. 75, no 5, p. 1299-1313Article in journal (Refereed) Published
Abstract [en]

Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNA(i)) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNA(i) IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNA(i) the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNA(i) conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNA(i). IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2010
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-121733 (URN)10.1111/j.1365-2958.2010.07057.x (DOI)000274808800020 ()
Available from: 2010-04-26 Created: 2010-03-29 Last updated: 2018-01-12Bibliographically approved
3. Initiation factor 2 mutants promoting fast joining of ribosomal subunits in the absence of initiator tRNA or GTP
Open this publication in new window or tab >>Initiation factor 2 mutants promoting fast joining of ribosomal subunits in the absence of initiator tRNA or GTP
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have previously identified several mutations in initiation factor 2 (IF2) located outside the tRNA binding domain IV of IF2 that compensate for lack of initiator tRNA (Met-tRNAi) formylation in vivo. We have also shown that these IF2 mutants promote fast joining of ribosomal subunits even when non-formylated Met-tRNAi or deacylated tRNAi was present in the 30S pre-initiation complex (30S PIC) instead of formylated fMet-tRNAi. We demonstrate here in vitro that these IF2 mutants do not require any tRNA present on the 30S subunit to promote fast subunit joining provided that GTP is present. Moreover, in the presence of fMet-tRNAi the mutants promote fast subunit joining in the presence of only GDP. Thus, A-type IF2 mutants require either GTP or fMet-tRNAi for fast subunit joining. In contrast, fast subunit joining with wild type IF2 requires the presence of both GTP and fMet-tRNAi in the 30S PIC. These results imply that the presence of tRNA on the 30S subunit is per se not required for fast subunit joining but rather for switching the 30S:IF2 complex containing wild type IF2 into its 50S docking conformation. We show also that the rate of subunit joining with A-type IF2 is much less sensitive to the energy level in the reaction mixture than the rate of subunit joining with wild type IF2. We speculate that this insensitivity of initiation to energy levels may result in deteriorated adaptation of formylation-proficient strains harbouring A-type mutations in IF2 to growth under energy-limited conditions.

Keywords
protein synthesis, initiation factors, initiator tRNA, ribosome, formylation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Microbiology in the medical area
Research subject
Biochemistry; Microbiology
Identifiers
urn:nbn:se:uu:diva-123147 (URN)
Available from: 2010-04-25 Created: 2010-04-25 Last updated: 2018-01-12
4. Compensatory evolution restores fitness to actinonin-resistant Staphylococcus aureus
Open this publication in new window or tab >>Compensatory evolution restores fitness to actinonin-resistant Staphylococcus aureus
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have studied the emergence of actinonin-resistant mutants in S. aureus. In accordance with earlier studies we identified resistance mutations in the fmt gene that apart from conferring high-level resistance also reduced the growth rate. To study how fitness could be restored we performed compensatory evolution by serial passage of cells in liquid culture. Compensated mutants arose quickly, within 40 generations and sometimes in less than 10 generations. The mutants were fully resistant to actinonin and showed increased growth rates on plates. However, exponential growth rates in liquid media were not higher than for the parental resistant mutants. We sequenced the whole genomes of one slow-growing strain and three compensated and found alterations in three genes. These genes were SAOUHSC_01699 coding for shikimate 5-dehydrogenase, SAOUHSC_00945 coding for a magnesium transporter and SAOUHSC_02264, coding for accessory gene regulator protein C (AgrC). None of these mutants were obvious candidates for compensating lack of formylation. When sequencing these three loci in the remainder of the compensated strains, mutations in agrC were found in 11 of 16 strains. Further studies will show if these mutations are due to general environmental adaption, specific adaptation for slow growth or specific compensation for loss of formylation.

 

Keywords
formylation, compensatory evolution, peptide deformylase
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:uu:diva-123149 (URN)
Available from: 2010-04-25 Created: 2010-04-25 Last updated: 2018-01-12

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