Kevin Wyllie Week 11

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Milestones This Week

  1. Understand experimental design.
  2. Establish sample-data relationship
    • raw.zip and .sdrf.txt files
    • Construct sample-data flowchart/diagram
  3. If possible develop compiled raw data file

10 Unfamiliar Terms

  1. anaplerotic pathway - "Anaplerotic reactions replenish TCA cycle intermediates and allow respiration to continue" *1
  2. aminoglycosides - "Group of antibiotics active against many aerobic Gram-negative and some Gram-positive bacteria. Composed of two or more amino sugars attached by a glycosidic linkage to a hexose nucleus" *1
  3. catalase - "An enzyme that catalyzes the reaction of H2O2 → H2O + ½ O2. Catalase is especially abundant in the liver, where it is contained in peroxisomes." *2
  4. plasmid transformation - "Any alteratoin in the properties of a cell that is stably inherited by its progeny." *1
  5. triparental mating - "[Describes the process of] three different phages simultaneously infect[ing] the host cell. The observation of triparental recombinants demonstrates that repeated recombinational events occur in the infected cell between replicating nucleic acid molecules derived from all parent viruses." *2
  6. sonication - "Subject[ing] a biological sample to ultrasonic vibration so as to fragment the cells, macromolecules, and membranes.*2
  7. RT-PCR - "Reverse transcriptase polymerase chain reaction; PCR in which the starting template is RNA, implying the need for an initial reverse transcriptase step to make a DNA template." *1
  8. flow cytometry - "Slightly imprecise but common term for the use of the fluorescence activated cell sorter. Cells are labelled with fluorescent dye and then passed, in suspending medium, through a narrow dropping nozzle so that each cell is in a small droplet. A laser-based detector system is used to excite fluorescence, and droplets with positively fluorescent cells are given an electric charge. Charged and uncharged droplets are separated as they fall between charged plates and so collect in different tubes." *1
  9. exconjugants - "Ciliates that were partners in conjugation and therefore have exchanged genetic material." *2
  10. catalase - "An enzyme that catalyzes the reaction of H2O2 → H2O + ½ O2. Catalase is especially abundant in the liver, where it is contained in peroxisomes." *2
  11. rhamnose - "6-deoxy L-mannose, a sugar found in plant glycosides." *1
  12. catechol - "A type of biogenic amine derived from tyramine, characterized as alkylamino derivatives of o-dihydroxybenzene." *1


Sources:

  • 1. Lackie, John M.. Dictionary of Cell and Molecular Biology. Kidlington, GBR: Academic Press, 2007. ProQuest ebrary. Web. 16 November 2015.

Copyright © 2007. Academic Press. All rights reserved.

  • 2. King, R C.. Dictionary of Genetics. Oxford University Press, 2013. Web. 17 November 2015. Copyright © 2014. doi: 10.1093/acref/9780199766444.001.0001

Article Outline

Significance of this Study

  • Burkholderia cenocepacia is a a member of the Burkholderia cepacia complex (Bcc), which is comprised of 17 similar species.
  • B. cencocepacia is an opportunistic pathogen, which causes severe lung infection in cystic fibrosis patients, as well as those who are generally immunocompromised.
  • Infections with Bcc organisms are difficult to eliminate for three reasons:
    • Widespread antibiotic resistance.
    • Ability to form biofilms (forming a physical barrier from antibiotics).
    • "Persister cells," which are a phenotypic variant exhibiting even greater antibiotic resistance. Though they comprise a very small fraction of a population, persister cells can linger during/after antibiotic treatment, and can then repopulate when the antibiotic is removed.
  • One of the proposed mechanisms by which antimicrobials work is overstimulation of NADH oxidation, hyperactivating the electron transport chain. This releases large amounts of reactive oxygen species (ROS), which damage proteins containing iron-sulfur clusters, releasing ferrous iron. Finally, this ferrous iron damages DNA, lipids and proteins, leading to eventual cell death.
  • The glyoxylate cycle can be induced in many microorganisms to lessen the generation of ROS, as it skips two of the steps in the TCA cycle which would normally product NADH. This may be a primary mechanism of antibiotic resistance in Bcc (also mentioned are the ubiquitous drug efflux pumps and beta-lactamases).
    • A prior study (Van Schaik et al.) found that inhibition of isocitrate lyase - the enzyme which initiates the shunting of TCA into the glyoxylate cycle - forced Burkholderia pseudomallei back into a state of antibiotic susceptibility, supporting this hypothesis.
  • This study investigates:
    • Levels of persister cells in Bcc biofilms.
    • The molecular root of antibiotic resistance.
    • Effective methods for eradicating Bcc biofilms.

Methods

Minimal Inhibitory Concentration

  • Minimal Inhibitory Concentrations (MIC) were determined, using the EUCAST broth microdilution protocol, for the following antimicrobials:
    • Tobramycin
    • Ciproflaxin
    • Itaconate with 3-nitropropionate
    • 2-thenoyltrifluoroacetone
  • This was done in with duplicates, in 96-well microtiter plates, treated and incubated for 24 hours.
  • MIC's were defined as the lowest concentration at which no significant difference in OD590 was observed, between blank and inoculated wells.

Quantification of Persister Cells in Biofilms and Planktonic Cultures

  • Biofilm and planktonic cultures were exposed to tobramycin and ciproflaxin and the numbers of surviving cells were determined.
  • Cultures were grown for 24 hours, and then exposed to either antibiotic (or a physiological saline control) for another 24 hours.
    • For biofilm cultures:
      • Cultures were grown in 96-well microtiter plates. Twelve wells were used for each condition, but it appears that not all were quantified (see below).
      • Both incubation periods were at 37°C.
      • Cells were harvested and plated on LB media for quantification. All experiments had two or more replicates.
    • For planktonic cultures:
      • Cultures were grown overnight in a shaking warm water bath. After 24 hours, cell suspensions with OD590's of 1 were centrifuged in falcon tubes, and then resuspended in either antibiotic or the control.
      • Cells were resuspended in physiological saline and quantified by plating on LB media.

RNA Extraction and Microarray Analysis

  • Biofilms of B. cenocepacia J2315 were grown in 96-well microtiter plates for 24 hours, and then incubated in the presence of either tobramycin (4x MIC) or a saline control solution for another 24 hours.
    • Cells were collected by vortexing and sonication.
    • RNA was:
      • Extracted with the Ambion RiboPure Bacteria Kit.
      • Treated with DNase I for 1 hour.
      • Concentrated with Microcon YM-50 filters.
      • Linearly amplified with the Ambion MessageAmp II-Bacteria Kit.
  • cDNA was synthesized and hybridized to custom made 4x44K Agilent arrays.
  • GeneSpring GX 7.3 was used for array analysis.
  • Data was normalized using Agilent's recommended procedure for two-color micorarrays, and a T-test was performed.

Note: Although neither the methods nor results sections mention the amount of replicates used, the .sdrf file indicates that five replicates were used for untreated samples, and three replicates were used for the tobramycin treated samples. The author has also stated that the reference sample for each array was the organism's genomic DNA.

Quantitative RT-PCR

  • 11 genes were selected to be quantified, as a means of validating the microarray results.
  • Treated and untreated biofilms were grown as described in the microarray analysis methods.
    • RNA was extracted, and cDNA was was synthesized with BioRad's iScript cDNA Synthesis Kit.
  • Forward and reverse primers were designed using tools on NCBI's website, and BLAST was used (with B. cenocepacia J2315's genome) to verify the specificity of these primers.
  • Samples were spotted in duplicate, along with a control, containing no cDNA.
  • BioRad's CFX96 Real-Time System C1000 Thermal Cycler was used.
  • A melting curve was generated at the end of each run.
  • Three "reference genes" were used to normalize the data. An algorithm called GeNorm was used to confirm a stable expression for these three genes.

Flow Cytometry

  • After being resuspended in physiological saline, both treated and untreated B. cenocepacia J2315 biofilms (again, both prepared as described in the microarray methods) were stained with an ROS-specific dye, and the passed through a Cyan ADP flow cytometer for quantification.
    • Biofilms were dyed by incubating for 30 minutes in the dark, in the presence of the dye.
  • Data for at least 50,000 cells was collected in each sample.

Inhibition of SOD, ICL and SDH

  • To evaluate the importance of superoxide dismutase (SOD), isocitrate lyase (ICL) and succinate dehydrogenase (SDH), B. cenocepacia J2315 biofilm persister cells (presumably prepared as described above, though not explicitly stated in the article) were quantified after exposure to 4x MIC tobramycin in conjunction with the following inhibitory agents:
    • Diethyldithiocarbamate, inhibiting SOD.
    • Itaconate or 3-NP, both inhibiting ICL.
    • 2-thenoyltrifluoracetone, inhibiting SDH.
  • The exact method of quantification is not stated, however quantification was presumably done by plating on LB media, as described in the "quantification of persister cells" section.

Construction of ICL Mutant

  • Both B. cenocepacia J2315 ICL-encoding genes were knocked out via introduction of a mutant plasmid.
    • Several methods were used to verify successful conjugation and mutagenesis, including:
      • Spraying colonies with catechol, resulting in a yellow color due to xylE, encoded by the plasmid.
      • Testing for tetracycline resistance, also encoded by the plasmid.
      • PCR, followed by sequencing, to ensure successful gene deletion.

Construction of SDH Antisense Overexpression Mutants

  • Two of the four subunits composing SDH were amplified by PCR.
  • PCR product was purified and ligated into a plasmid, which was transferred to B. cenocepacia J2315 cells via triparental mating.
  • Successful transformation was screened for by plating cells on LB media in the presence of "Tp" (resistance to which was encoded by the plasmid).

Figures and Tables

  • Figure 1 shows the percentage of cells which survived treatment of tobramycin and ciproflaxin, at different concentrations. Though significantly fewer planktonic cells survived in both treatments, their curves exhibited a similar shape, suggesting that persister cells do exist in planktonic cultures as well.
  • Figure 2 shows the fluorescent signal for tobramycin-treated and untreated biofilms, explicitly indicating that 21% of the cells in the untreated biofilms had a "high" fluorescent signal, while the respective value for treated biofilms was 72%.
  • Figure 3 shows percentage of surviving B. cenocepacia C5424 cells, as well as two catalase mutants, in biofilms treated with tobramycin. dkatA saw a slightly reduced persister fraction, while dkatB saw approximately fourty times smaller persister fraction.
  • Figure 4 shows the persister fraction for SOD-inhibited B. cenocepacia J2315 biofilms, treated with tobramycin (versus the non SOD-inhibited control). The persister fraction was approximately 100 times lower in the SOD-inhibited biofilms.
  • Figure 5 shows the persister fraction for ICL-inhibited B. cenocepacia J2315 biofilms, treated with both tobramycin and ciproflaxin (versus non-inhibited controls). Inhibition with itaconate and 3-NP, followed by treatment with tobramycin resulted in a 10-fold lower persister fraction, while no significant effect was seen in the case of ciproflaxin.
  • Figure 6 shows the same information as in Figure 5 (for tobramycin only) for several different species in the Bcc group. The results for some of these species - including B. pyrrocinia and B. ambifaria - are similar to that of B. cenocepacia J2315.
  • Figure 7A shows the persister fraction for B. cenocepacia J2315 wild-types double ICL mutant (D2) biofilms, treated with either tobramycin or tobramycin plus 3-NP. No significant decrease in persister fraction was seen for the D2 mutant compared the wild-type. However, there was a difference between D2 and wild-type when 3-NP was used.
  • Figure 7B shows persister fraction for D2 biofilms treated with either tobramycin or tobramycin plus the succinate dehydrogenase inhibitor, 2-thenoyltrifluoroacetone. Addition of the inhibitor brought about a significant decrease in persister fraction.
  • Table 5 shows the fold changes of several pertinent genes (involved in the glyoxylate shunt, TCA cycle, oxidative phosphorylation, NAD(P)H production, and response to oxidative stress) from the microarray analysis, along with the RT-PCR results for the 11 genes tested. The results suggest that pathways responsible for ROS production were downregulated in response to tobramycin exposure.

Microarray Methods Flowchart

KWVPMethoddiagram.jpg

Previous Studies

The primary difference between this article and prior literature is its emphasis on targeting the cell's mechanisms to deal with ROS, versus "reawakening" persister cells to counteract the phenotypic changes which allow for antimicrobial resistance, as previous studies have attempted. Prior literature focuses on undoing dormancy, while this study focuses on circumventing dormancy.

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