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

Presentation by Jenny Yang

Board of Education Meeting

February 27, 2012

NY Medical CollegeDr. Yuk Ching Tse-Dinh

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)

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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

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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)

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Review of Literature: SOS Response Continued

Induce rampant mutagenesis and reactionary antimicrobial resistance (Hastings, 2004)

Thus, inhibition would lessen mutagenesis and suppress antimicrobial resistance

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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

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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)

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Process

Cell damage

QuinoloneSOS

responseCell

Damage MutationsSOS response

Drug resistance

Mutations

Drugs targeting topA gene Decreased antiobiotic

resistanceLower health care costs

and mortality rates

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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

Research Objective

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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

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Materials

Table 1: Bacterial Strains and Plasmid

Strain Genotype Source or Reference

DPB635 F-, λ-, zci-2250::mini-

kan, 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

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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

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Results: Microdilutions

H1 is shown to be true: a mutation in the topA66 gene leads to increased sensitivity

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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 observed that both the wild type and the mutant were sensitive to the antibiotic. It is evident that the mutant is more sensitive to the antibiotics than the wild type 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.

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

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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

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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 luciferasereading of treated v. untreated culture

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Results: Luciferase Assay

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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 DPB636.

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

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Discussion: Luciferase Assay Continued

Exposure to quinolones

Wild type

SOS response

Mutagenesis

Antibiotic Resistance

Mutant

NO SOS response

NO mutagenesis

NO antibiotic resistance

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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

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Results: Mutation Rate Experiment

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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.

Discussion: Mutation Rate Experiment

Results support previous studies: decreased SOS response will lead to lower inclinations of multidrug resistance

Inhibition of topoisomerase I activity may limit development of resistance to antibiotics

• No topoisomerase I

• No SOS response

• No mutagenesis and drug resistance

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Limitations and Future Research

Continuance of seeing whether topoisomerase I would be a useful target for cotherapy with antibiotics to limit drug resistance

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Drolet, M. et al.Overexpressionof RNase H partially complements the growth defect of an Escherichia coli Δ topA mutant: R-loop formation is a major problem in the absence of DNA

topoisomerase I. Proc. NatlAcad. Sci. USA 92. (1995): 3526-3530.

Cheng, B., I. Liu, Tse-Dinh, Y.C., Compounds with antibibacterialactivity that enhance DNA cleavage by bacterial DNA topoisomeraseI.J. Antimicrob. Chemother. 59. (2007): 640-645.

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Hastings, P.J., Rosenberg, S.M., Slack, A. Antibiotic-induce lateral transfer of antibiotic resistance. TRENDS in Microbiology.12.9. (2004): 401-404.

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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

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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.