| 1. |
- Andrésen, Cecilia, et al.
(author)
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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
- 2010
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In: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
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Journal article (peer-reviewed)abstract
- The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
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| 2. |
- Islam, Sohidul
(author)
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Chromosomal antibiotic resistance mechanisms in Pseudomonas aeruginosa and Neisseria gonorrhoeae
- 2008
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Doctoral thesis (other academic/artistic)abstract
- The progressive increase in fluoroquinolone resistant N. gonorrhoeae and emergence of multiple antibiotic resistant P. aeruginosa are growing concerns among physicians and health policy makers. In N. gonorrhoeae chromosomal gene mutations encoding different subunits of DNA gyrase and topoisomerase IV have been considered the main mechanism of fluoroquinolone resistance even though these changes do not explain the varying MICs of resistant strains. Whereas in P. aeruginosa the MexXY efflux pump is described as the predominant manifestation of aminoglycoside resistance in isolates from cystic fibrosis lungs, the aminoglycoside modifying enzymes (AME) contribute to resistance in P. aeruginosa strains isolated from other infections. Modification of target (16s rRNA) also accounted for several cases of aminoglycoside resistance but has not been investigated in clinical isolates. The main mechanism of carbapenem (meropenem and imipenem) resistance in P. aeruginosa is down regulation of porin protein OprD and/or increased efflux by MexAB-OprM. Alterations of penicillin binding proteins have also been accounted for increased resistance to carbapenems in clinical isolates of P. aeruginosa. The major focus of this thesis is to study the chromosomally mediated antibiotic resistance mechanisms of fluoroquinolones in Neisseria gonorrhoeae and aminoglycosides and carbapenems in Pseudomonas aeruginosa. To assess fluoroquinolone resistance mechanisms in N. gonorrhoeae we transformed bacterial DNA from clinically resistant strains to sensitive strain and studied the involvement of chromosomal genes gyrA, parE, porB1b and lysR by PCR and sequencing. A total of 40 cystic fibrosis isolates of P. aeruginosa were included in this study to understand the aminoglycoside and in particular the amikacin resistance mechanism in CF environment. An array of chromosomal determinants, which might have role in aminoglycoside resistance including mexY, mexB, oprM, oprD, mexZ, aph (3´)-IIps, PA5471, galU, rplY and genes involved in electron transport chains were analyzed by sequencing or real time PCR. We examined 16S rRNA A-site by pyrosequencing of some clinically relevant strains where other mechanisms failed to explain aminoglycoside resistance properly. To assess the carbapenem resistance mechanism, conjugation experiments have been performed between resistant clinical strains and a laboratory strain of P. aeruginosa. The transconjugants with low susceptibility to carbapenems were further studied for the expression and sequence of OprD by realtime PCR and presence of mutations in different hotspots of penicillin binding protein genes by sequencing. We have concluded that alteration in GyrA subunit of DNA gyrase is the main determinant of fluoroquinolone resistance in N. gonorrhoeae. Our study suggests that introduction of additional mutations in gyrA and/or parE as well as alterations of porB1b contribute to ciprofloxacin resistance. In P. aeruginosa, clinical strains and transconjugants, we found that downregulation of OprD porin protein is the main mechanism for carbapenem resistance. Many cystic fibrosis patients are infected with P. aeruginosa in their early age and generally the same strains persist and colonize the CF lungs over the period of their lifetime. In most cases progenies with different phenotype of the same strains perpetuate in CF lung. We focused on changes in chromosomal determinants of aminoglycoside resistance in CF P. aeruginosa isolates. The major chromosomal changes in our study are in the regulatory genes for the efflux pump MexXY followed by the overexpression of pump protein MexY as the dominating mechanism of aminoglycoside resistance in CF P. aeruginosa isolates.
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| 3. |
- Lindbäck, Emma, et al.
(author)
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Transformation of ciprofloxacin-resistant Neisseria gonorrhoeae gyrA, parE and porB1b genes
- 2006
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 28:3, s. 206-211
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Journal article (peer-reviewed)abstract
- In several transformation experiments, we have shown that introduction of an alteration in GyrA at position 95 of a ciprofloxacin-susceptible Neisseria gonorrhoeae strain (minimum inhibitory concentration (MIC) 0.008 mg/L) increases the MIC to 0.064 mg/L. Two alterations (positions 91 and 95) increase the MIC to 0.125-0.25 mg/L. Transformants with ciprofloxacin MICs of 0.5-16 mg/L were obtained from a moderately ciprofloxacin-resistant strain (MIC 0.25 mg/L). These transformants had alterations in the gene for PorB1b and probably other genes. In one transformant, an alteration in ParE was also introduced, which probably contributed to ciprofloxacin resistance. The ciprofloxacin-resistant transformants had the donor porB1b sequence, and most of them also had altered serovars. We conclude that alterations in N. gonorrhoeae PorB1b could be involved in ciprofloxacin resistance.
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