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Sökning: WFRF:(Andersson Dan Professor) > (2015-2019)

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1.
  • Malik, Sohaib Zafar (författare)
  • Interaction of cyclotides and bacteria : A study of the cyclotide action and the bacterial reaction
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). Thesepotent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults.    Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics.     Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.
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2.
  • Knopp, Michael, 1984- (författare)
  • Mechanisms of Antibiotic Resistance Evolution
  • 2018
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The continuing emergence and spread of antibiotic resistant bacteria are a threat to various applications in modern medicine and impose a strong economic burden on health systems. The development of new antibiotics is slow and cannot counterbalance the dissemination of resistant bacteria. Thus, we need to find ways to reduce the rate of antibiotic resistance development. For this, we need to acquire a deeper understanding of the mechanisms underlying the evolution of antibiotic resistance.Here, we investigate the factors that govern how antibiotic resistance mechanisms affect bacterial fitness and the overall level of resistance. Using porin-deficient mutants of Escherichia coli, we show that upregulation of alternative porins provides compensatory mechanisms that can ameliorate the fitness costs associated with resistance. Furthermore, we demonstrate that the phenotypic effects of antibiotic resistance mutations are largely predictable, both in combination with each other as well as in different bacterial strains. However, outliers from this trend exemplify the limitations of solely relying on laboratory strains for the characterization of antibiotic resistance mechanisms. In contrast, strong epistatic interactions were observed in mutants evolved at sub-lethal concentrations of streptomycin. Despite these low concentrations and weak selective pressure, strains of Salmonella Typhimurium evolved high-level resistance, which followed completely different mutational pathways compared to high-level selection. Finally, we show that aminoglycoside resistance genes can be selected de novo from the expression of completely randomized nucleotide sequences. This demonstrates that new genes can arise from pools of non-coding sequences and that this process is relatively common.The studies presented in this thesis provide insights into the mechanistic basis of resistance evolution, including the mutational spectrum causing antibiotic resistance, compensatory pathways for growth-restoration and the influence of epistatic interactions on the phenotypic expression of resistance mutations. Understanding these factors in detail will enable us to better predict and prevent the emergence of antibiotic resistance development, through improvements in surveillance, treatment regimens and drug development.
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3.
  • Knöppel, Anna, 1984- (författare)
  • Experimental Evolution : and Fitness Effects of Mutations
  • 2016
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations.In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions.Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations.In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components.Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%.
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4.
  • Kremel, Anna (författare)
  • Heterogeneity in the level and handling of the Liability of Newness : Female and immigrant entrepreneurs’ need for and use of business advisory service
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • In the start-up entrepreneurs face the Liability of Newness when problems and challenges often threaten business survival. Business advisory service, provided by public and private supplier contacts, can offer important knowledge and information, accompanied by various forms of assistance, and thereby decrease the entrepreneurs’ risk of failure and reduce their Liability of Newness. However, it is difficult to match the entrepreneurs’ need for such advice with the available advice. The support must meet the need. Most nations in the European Union have programs and projects that provide such support for entrepreneurs and SMEs. Special programs often support female entrepreneurs and/or immigrant entrepreneurs.This thesis examines the level and handling of the Liability of Newness with special focus on female entrepreneurs and immigrant entrepreneurs in Sweden. The four papers of this thesis take the perspective of these entrepreneurs. The research is based on a sample of 2 832 entrepreneurs who were interviewed (in a telephone survey) on their impressions and recollections on their need for and use of business advisory service in the start-up processes of their companies. Fulfilment of need is achieved when the need for business advisory service is matched with the right use of business advisory service.Heterogeneities as far as the level and handling of the Liability of Newness exist related to female entrepreneurs (vs. male entrepreneurs) and immigrant entrepreneurs (vs. non-immigrant entrepreneurs). Female entrepreneurs have a higher need for business advisory service than male entrepreneurs and also use more business advisory service than male entrepreneurs. As a result, female entrepreneurs can decrease the hazard rate for their companies and also reduce the Liability of Newness as their companies move toward the standard risk in their industry. Immigrant entrepreneurs also have a higher need for business advisory service than non-immigrant entrepreneurs. However, because immigrant entrepreneurs use business advisory service to the same extent that non-immigrant entrepreneurs do, immigrant entrepreneurs are unable to decrease the hazard rate for their companies or to reduce the Liability of Newness.The thesis makes both theoretical, methodological and practical contributions.The thesis may be of interest to government policy makers with its attention to the need and use of business advisory service by female entrepreneurs and immigrant entrepreneurs.
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5.
  • Linkevičius, Marius, 1985- (författare)
  • Evolution and Mechanisms of Tigecycline Resistance in Escherichia coli
  • 2015
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections.In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates.Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines.Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.
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6.
  • Lofton Tomenius, Hava, 1976- (författare)
  • Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides
  • 2016
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development.In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4.The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways.Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host.In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.
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7.
  • Thulin, Elisabeth, 1984- (författare)
  • Mechanisms and Dynamics of Mecillinam Resistance in Escherichia coli
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The introduction of antibiotics in healthcare is one of the most important medical achievements with regard to reducing human morbidity and mortality. However, bacterial pathogens have acquired antibiotic resistance at an increasing rate, and due to a high prevalence of resistance to some antibiotics they can no longer be used therapeutically. The antibiotic mecillinam, which inhibits the penicillin-binding protein PBP2, however, is an exception since mecillinam resistance (MecR) prevalence has remained low. This is particularly interesting since laboratory experiments have shown that bacteria can rapidly acquire MecR mutations by a multitude of different types of mutations.In this thesis, I examined mechanisms and dynamics of mecillinam resistance in clinical and laboratory isolates of Escherichia coli. Only one type of MecR mutations (cysB) was found in the clinical strains, even though laboratory experiments demonstrate that more than 100 genes can confer resistance Fitness assays showed that cysB mutants have higher fitness than most other MecR mutants, which is likely to contribute to their dominance in clinical settings.To determine if the mecillinam resistant strains could compensate for their fitness cost, six different MecR mutants (cysB, mrdA, spoT, ppa, aspS and ubiE) were evolved for 200-400 generations. All evolved mutants showed increased fitness, but the compensation was associated with loss of resistance in the majority of cases. This will also contribute to the rarity of clinical MecR isolates with chromosomal resistance mutations.How MecR is mediated by cysB mutations was previously unclear, but in this thesis I propose and test a model for the mechanism of resistance. Thus, inactivation of CysB results in cellular depletion of cysteine that triggers an oxidative stress response. The response alters the intracellular levels of 450 proteins, and MecR is achieved by the increase of two of these, the LpoB and PBP1B proteins, which rescue the cells with a mecillinam-inhibited PBP2.Mecillinam is used for UTI treatments and to investigate mecillinam resistance in a more host-like milieu, MecR strains were grown in urine and resistance was examined. Interestingly, this study showed that neither laboratory, nor clinical cysB mutants are resistant in urine, most likely because the cysteine present in the urine phenotypically reverts the bacteria to susceptibility. These findings suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests, and that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant results.In summary, the work described in this thesis has increased ourgeneral knowledge of mecillinam resistance and its evolution. Hopefully this knowledge can be put to good use in clinical settings to reduce the negative impact of antibiotic resistance.
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8.
  • Lundin, Erik (författare)
  • Evolution of New Genes and Functions
  • 2018
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • To answer major evolutionary questions, we need a better understanding of the effects of mutations on specific functions and organism fitness. The aim of this thesis was to elucidate how new functions evolve and potential trade-offs with the original function.Paper I identified a bimodal distribution of fitness effects of mutations in Salmonella enterica HisA protein. Most mutations negatively affected protein function but the effect was masked at high gene expression. Expression levels and the extent to which the studied protein limited growth were important in determining the fitness effects of mutations. No fitness prediction tool was satisfactorily alone but in combination predictions were improved.In Paper II, S. enterica HisA was evolved to acquire TrpF activity. Numerous pathways towards improved TrpF activity were examined and several improvement mechanisms were identified. Improved TrpF activity extensively reduced the original activity, generalist enzymes were rare and restoring original activity after an initial loss was difficult. Furthermore, expression levels had a major impact on the shape of the trade-off curve.In Paper III, adaptation during serial passage of Escherichia coli and S. enterica under laboratory conditions were examined. Adaptive mutations were identified in four different laboratory media and their fitness effects were determined. Little overlap in mutation spectra was found in the different media and species suggesting that adaptation was media-specific. Furthermore, media adaptation mutations reduced the accuracy of fitness assays and the use of pre-adapted strains improved the sensitivity of fitness assays 10-fold.Paper IV examined evolution of novel metabolic capabilities in S. enterica by analyzing growth on 124 non-native carbon sources. Growth was observed on 25 compounds and for five of these, the causative mutation was identified. Increased gene expression of cryptic genes was a major mechanism for acquiring the novel phenotypes.In conclusion, my results show that in most cases many types of mutations can improve a function and allow adaptive evolution but this often is associated with a trade-off and loss in other abilities. Increased gene expression was a major mechanism by which bacteria could compensate for loss of an activity as well as acquire new metabolic capabilities.
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