Effect of E. coli topa mutation on SOS and Antibiotic Response

Transcription

1 Effect of E. coli topa mutation on SOS and Antibiotic Response Presentation by Jenny Yang Board of Education Meeting February 27, 2012 NY Medical College Dr. Yuk Ching Tse-Dinh

2 Introduction Prevalent use has led to the emergence of drug resistant bacterial strains (File, 1999) Increased mortality rates and health-care costs (US Congress, 1995; Archibald et al, 1997) 1

3 Introduction Continued Antibiotics can only be temporary measures (Medeiros, 1997) Innate resistance and horizontal and vertical evolution Mutagenesis allows microbes to baffle bactericides Importance of suppression of mutagenesis 2

4 Review of Literature: SOS Response SOS response is a cell damage repair response that transcribes over twenty genes known as the Din genes (Walker, 1987) 3

5 Review of Literature: SOS Response Continued Induce rampant mutagenesis and reactionary antimicrobial resistance (Hastings, 2004) Thus, inhibition would lessen mutagenesis and suppress antimicrobial resistance 4

6 Review of Literature: Antibiotic Quinolones The exacerbated damage ultimately leads to recovery SOS response and antibiotic resistance (Newmark, 2005) The objective of this research was to genetically alter and impair topoisomerase, a key element in the SOS response 5

7 Review of Literature: Topoisomerase Continued Enzyme that relieves topological problems that may cause bactericide At least one type I and type II topoisomerase is present in all organisms and are essential to life (Bergerat et al, 1997, Forterre, 2001) Topoisomerase I as encoded by the topa gene relieves torsional stress: major part of SOS response (Rui and Tse-Dinh 2007) 6

8 Process Quinolone Cell damage Cell Damage SOS response SOS response Mutations Mutations Drug resistance Drugs targeting topa gene Decreased antiobiotic resistance Lower health care costs and mortality rates 7

9 Research Objective Investigate the effect of a topa66 gene mutation on the vulnerability, the SOS response levels, and the mutation rate of an altered E. coli strain 8

10 Hypothesis H1: A mutation in the topa gene of E. coli leads to increased antibiotic sensitivity H2: TopA mutant E. coli will have a lower SOS response compared to that of wild type E. coli H3: Decreased SOS response for E. coli topa mutant results in lower inclination to develop drug resistance after quinolone treatment 9

11 Materials Table 1: Bacterial Strains and Plasmid Strain Genotype Source or Reference DPB635 F-, λ -, zci-2250::minikan, rph-1 Yale E. coli Genetic Stock Center DPB636 DPB635, topa66 Yale E. coli Genetic Stock Center pdinlux SOS reporter plasmid with dind1 ::luxcadbe fusion Reference 8 Luria broth (LB) used as growth media Mueller Hinton Broth (MHB) Antibiotics 10

12 Method 1: Microdilutions Four 1:10 serial dilutions for both strains were made and spotted in grid position Mueller Hinton agar (MHA) plates were used as controls To compare sensitivity, the dilutions were spotted onto MHA plates with different antibiotics Plates were incubated at 37 C, and pictures were taken the following day 11

13 Results: Microdilutions FIG. 1 Wild type and topa mutant E. coli were plated on Mueller Hinton Agar to observe the uninhibited growth of each strain. The wild type strain was placed on the first row, whereas the mutant is located on the second row. On 80 ng/ml trimethropin plate, it can be H1 observed is shown that both the to wild be type and true: the mutant a were mutation more sensitive to in the antibiotics the topa66 than the wild gene type is. leads to increased sensitivity sensitive to the antibiotic. It is evident that the mutant is Plated on 4 ng/ml ciprofloxacin, the growth of the wild type and the growth of the topa mutant evince the increased sensitivity of the mutant E. coli compared to that of the wild type. Like on the 4 ng/ml ciprofloxacin plate, the topa mutant growth on the 25 ng/ml norfloxacin plate is much less compared to that of the wild type, though the difference in the amount of growth is more than that of what is observed on the ciprofloxacin plate. 12

14 Discussion: Microdilutions Unmanaged supercoils may lead to cell damage topa gene mutation results in supercoils which may lead to cell damage (Drolet, 1995; Cheng et al, 2003) Mutation in the topa gene leads to increased sensitivity to antibiotics due to the inhibited interaction between top I and RNA polymerase 13

15 Discussion: Microdilutions Continued Since SOS induction is used as a reparation for cell damage, the decreased cell viability of the mutant may be due to requirement of topa Thus, a luciferase assay was conducted to measure SOS response 14

16 Method 2: Luciferase Assay The Perkin Elmer 7000 Bio Assay was used to measure luminescence at 37 C for 35 cycles Each cycle was ten minutes long with 60 seconds of shaking during and 300 seconds of shaking between each cycle (Sutherland and Tse-Dinh, 2010) Luciferase response ratio was obtained as the ratio of luciferase reading of treated v. untreated culture 15

17 Results: Luciferase Assay FIG. 2. (A) Effects of topa mutation on SOS response can be viewed after treatment with 25 ng/ml norfloxacin from measurement of induction of luciferase from dind1::luxcadbe fusion. Mutant DPB636 response ratio is stagnant throughout the time course; whereas the response ratio of DPB635 increases. At 100 minutes, the response ratio of DPB635 is over three fold greater than that of the mutant. (B) In 10 ng/ml ciprofloxacin, response ratio of DPB636 is also less compared to that the response ratio of DPB635. The response ratio of DPB635 is nearly two times that of DPB

18 Discussion: Luciferase Assay Without the induction of SOS, mutant topa66 cells would be hypersensitive to quinolones topa gene is a necessary component for SOS induction 17

19 Discussion: Luciferase Assay Continued Exposure to quinolones Wild type Mutant SOS response NO SOS response Mutagenesis NO mutagenesis Antibiotic Resistance NO antibiotic resistance 18

20 Method 3: Mutation Rate Experiment Both strains of overnight cultures were diluted into LB with norfloxacin (50 ng/ml) as treatment Seven independent treated and control cultures were diluted and spread onto ten 100 μg/ml riframpicin plates and LB plates Plates were incubated for 48 hours and counted for number of rifampicin resistant mutants/ml Mutation rate was calculated by dividing the mutants/ml by viable CFU/mL (Kohanski et al, 2010) Fold increase was calculated as mutation rate of norfloxacin treated culture v. control culture 19

21 Results: Mutation Rate Experiment FIG. 3. The increase in mutation rate (mutation rate of treated culture versus untreated culture) of DPB635 from norfloxacin treatment is nearly two folds that of DPB636. The mean and standard mean error of the results from three experiments are shown here. 20

22 Discussion: Mutation Rate Experiment Results support previous studies: decreased SOS response will lead to lower inclinations of multidrug resistance No topoisomerase I Inhibition of topoisomerase I activity may limit development of resistance to antibiotics No SOS response No mutagenesis and drug resistance 21

23 Limitations and Future Research Continuance of seeing whether topoisomerase I would be a useful target for cotherapy with antibiotics to limit drug resistance 22

24 References Bergerat, A. et al. An atypical topoisomerase II from Archaeawith implications from meiotic recombination. Nature.386. (1997): Drlica K, Malik M. Fluoroquinolones: action and resistance. Curr. Top. Med. Chem. 3.(2003): Drolet, M. et al.overexpressionof RNase H partially complements the growth defect of an Escherichia coli ΔtopA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc. NatlAcad. Sci. USA 92. (1995): Cheng, B., I. Liu, Tse-Dinh, Y.C., Compounds with antibibacterialactivity that enhance DNA cleavage by bacterial DNA topoisomerasei.j. Antimicrob. Chemother. 59. (2007): Cheng, B., Zhu, C.Z., Ji, C., Ahumada, A., Tse-Dinh, Y.C., Direct Interaction between Escherichia coli RNA polymerase and the zinc ribbon domains of DNA topoisomerasei. J. Bio. Chem (2003): File, T.M. Jr. Overview of Resistance in the 1990s. Chest. 115(3 Suppl). (1999):3S-8S Hastings, P.J., Rosenberg, S.M., Slack, A. Antibiotic-induce lateral transfer of antibiotic resistance. TRENDS in Microbiology (2004): Kohanski, M.A., DePristo, M.A., Collins, J. J. SublethalAntibiotic Treatment Leads to Multidrug Resistance via Radical-Induced Mutagenesis. Molecular Cell. 37. (2010): Qi H, Menzel R, Tse-DinhYC. Effect of the deletion of the sigma 32-dependent promoter (P1) of the escherichiacoli topoisomerase I gene on. MolMicrobiol. 21. (1996): Qi H, Menzel R, Tse-DinhYC. Increased thermosensitivity associated with topoisomerase I deletion and promoter mutations in escherichia coli. FEMS Microbiol. Lett.178. (1997): Rui S, Tse-DinhY.C. Topoisomerase function during bacterial responses to environmental challenge. Front. Biosci.8. (2003):D256 D263. Shlaes DM, Rice LB. Emerging mechanisms of B-lactam resistance: an update. Infect Dis in Clin Prac. 15. (1995): Sutherland JH, Cheng B, Liu IF, Tse-DinhYC. SOS induction by stabilized topoisomerase IA cleavage complex occurs via the RecBCDpathway.JBacteriol.190. (2008): Sutherland J.H., Tse-DunhY.C., Analysis of RuvABC and RecG Involvement in the Escherichia coli Response to the Covalent Topoisomerase-DNA Complex. Journal of Bacteriology. (2010); Tabary X, Moreau N, DureuilC, Le Goffic F. Effect of DNA gyrase inhibitors pefloxacin, other quinolones, novobiocin, and clorobiocinon escherichiacoli topoisomerase I.Antimicrob. Agents Chemother. 31. (1987): Thornsberry C. Trends in antimicrobial resistance among today s bacterial pathoegns. Pharmacotherapy.15(1 pt 2) (1995):3S-8S. Tse-DinhYC. Increased sensitivity to oxidative challenges associated with topa deletion in escherichiacoli. J. Bacteriol.182. (2000): Tse-Dinh, Y.C. Bacterial topoisomerase I as a target for discovery of antibacterial compounds. Nucleic Acids Research. (2008): 1-7. US Congress, Office of Technology and Assessment. Impacts of antibiotic-resistance bacteria [abstract]. Washington, DC: Office of Technology and Assessment. (1995): OTA -H-629. Wang, J.C. Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3. (2002):

25 Conclusion and Major Finding A drug utilizing this mutagenesis-inhibiting mechanism would prolong the potency of bactericidal compounds, and ideally ought to be used in antibiotic synergy BhdUg/s1600/Pills2.jpg 24

26 Acknowledgements I d like to thank Mr. Inglis, my mentor, Dr. Yuk Ching Tse-Dinh, my class, and my family and friends for their help and support in my research.