BIOL368/F14:Isabel Gonzaga Week 11

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Contents

Finding a Journal Club Article/Microarray Dataset

Potential Articles

  1. Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program - Voskuil et al. (2003)
  2. The Stringent Response of Staphylococcus aureus and Its Impact on Survival after Phagocytosis through the Induction of Intracellular PSMs Expression - Geiger et al. (2012)
  3. Role of the Accessory Gene Regulator agr in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis - Cheut et al. (2004)

Chosen Article

Voskuil, M.I., Schappinger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M., Sherman, D.R., and Schoolnik, G.K. (2003). Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198(5), 705-713. doi:10.1084/jem.20030205.

Preparation for Journal Club 3

Vocabulary

  1. Anhydrobiosis: (n.) A state of apparent suspended animation entered by certain invertebrate animals in order to survive desiccation or other extreme stresses. Also known as Cryptobiosis. Source: Oxford Dictionary of Biology, 6ed.
  2. Granulomas (n.) A localized collection of cells, usually produced in response to an infectious process, that is characterized by the presence of aggregates of epithelioid histiocytes; giant cells, monocytes, or lymphocytes may also be present. The types of cells comprising a granuloma (of which there may be many or few) and their arrangement can assist in diagnosing the cause of the response. Source: Oxford Concise Medical Dictionary, 8ed.
  3. Hexamer: (n.) any oligomer consisting of six monomers. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  4. Hypoxic: (n.) A deficiency of oxygen in body tissues, which can result from living in an oxygen-deficient environment, inadequate inspiration, or deficiency of red blood cells or haemoglobin (required for oxygen transport). Source: Oxford Dictionary of Biology, 6ed.
  5. Immunocompetent cell: (n.) A cell capable of carrying out its immune function when given the proper stimulus. Source: Oxford Dictionary of Genetics, 7ed.
  6. Latency: (n.) The state or quality of being latent. Latent: existing in a potential, dormant, or suppressed form but usually capable of being expressed or evoked. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  7. Murine (adj.) Of, belonging to, characteristic of, affecting, transmitted by, or being a member of the Muridae, a family of small rodents that includes mice and rats; of or relating to the mouse genus, Mus. Source: Oxford Dictionary of Biochemistry and Molecular Biology, 2ed.
  8. Pleiotropic: (adj.) Describing an allele that has more than one effect in an organism. Source: A Dictionary of Biology, 6ed.
  9. Quiescent: (adj.) Describing a disease that is in an inactive or undetectable phase. Source: Oxford Concise Dictionary of Medicine, 8ed.
  10. Regulon: (n.) A group of operons that are under the control of the same regulatory protein. Source: Oxford Dictionary of Genetics, 7ed.

Article Outline

Introduction

  • Tuberculosis models state a three-stage development of disease
    1. Aerosal infection causes Mycobacterium tuberculosis to aggregate and divide in alveolar macrophages
    2. Immune system causes bacteriostasis; bacteria persists in granulomatous lesions. Host shows no signs of symptoms.
    3. ~10% of infected hosts immune system declines; bacteria wins and continues replication, host shows disease
  • Approximately 1/3 of the world population is latently infected with the disease; little known about the disease
    • Using murine lung example studies have found:
      • Isocytrate lyase necessary for long term survival
      • Nutrient deprivation can cause persistant or dormant state
      • O2 depletion effects most widely studied, able to use this to link mechanism for pathway
  • O2 Depletion Model of latency
    • Gradual depletion leads to nonreplicating, persistant state
    • Further lack of O2 lead to quiescent state
      • Sensitive to metornidazole, resistant to antimocrials
    • Provision of O2 resucitated dormant bacteria
  • Present Study
    • Roles of Nitric Oxide
      1. Immune factor to reduce mycobacterial activity
      2. reversible inhibitor of aerobic respiration for mitochondria and bacteria
      3. Signaling agent
    • Hypothesis: NO controls M. tuberculosis genetic program to adapt the organism for survival during extended periods of in vitro dormancy

Methods and Results

RNA Isolation and Culture
  • Clinical isolate 1254, 7H9 medium (with BSA, NaCl, glucose and glycerol), 350ml vented tissue culture flasks at 90rpm shaking with culture density of OD 0.5 used.
    • Same day RNA samples isolated from OD 0.15 cultures of M. tuberfulosis strain; cells collected from 4 min centrifuge and frozen on dry ice
    • Pellets suspended in 1ml TRIzol reagent and transfered to 0.5mL 0.1mm diameter zirconia/silica beads.
    • Cells disrupted, supernatant transferred to chloroform, inverted, incubated and centrifuged before adding 270μL isopropanol, and 270μL of a mixture of 0.8M sodium citrate and 1.2M NaCl
    • Samples incubated an centrifuged
    • RNA pellets isolated, washed with EtOH, centrifuged and air dried
    • RNA Pellets resuspended in 90μL H2O, 10μL DNaes I 10X buffer and 6 U DNase I (Ambion)
    • Samples incubate 30mins
    • RNA purification with RNeasy column
  • Murine Infection RNA
    • 15 6-8 week old C57BL.6 mice were infected with 87 cfu aerosol wild-type H37Rv strain of M.tuberculosis. Mice killed 3 weeks later and tissue was retained
    • Viable plate counts conducted using serial dilutions of the left lung in PBS 0.5% NB-40 on 7H10 agar, incubated at 37°C
    • Cultures were monitored at 6, 12 and 21 week timepoints and indicated steady-state infection
    • RNA was prepared by grinding mouse lungs with tissue homogenizer with phenol, chloroform and guanidium thiocyanate. RNA was isolated.
cDNA and Microarray Hybridization
  • cDNA prepared from 2μG RNA combined with oligonucleotide hexamers, RTase buffer, nucleotides, Cy3 or Cy5, Stratascript RTase and incubated.
    • cDNA purified using microcon-10 filtration
  • DNA Microarray Hybridization
    • Hybridization solution (containing labeled cDNA, tRNA, SSC and SDS) were sealed and hybridized overnight
  • Oligonucletoide microarray (using tuberculosis oligonucleotide set: QIAGEN)
    • Prehybridized in SSC, BSA and SDS for one hour before washing with H2O and isopropanol
    • Then, hybridized with Hybridization solution (containing labeled cDNA, tRNA, SSC, formamide and SDS) overnight
Data Analysis
  • Microarrays scanned using GenePix4000A (Axon Instruments, Inc.)
  • Dye intensities quantified using ScanAlyze (M. Eisen, Lawrence Berkeley National Lab, Berkeley, cA)
  • Cy3 and Cy5 intensities normalized to exclude the top 5% and bottom 5% induction ratios for all gene specific spots
  • Noise value calculation per channel: average intensity for 20% lowest intensity spots; spots below this average was raised to the osier value
  • Microarray determined ratios calculated from 3 replicates, 2 microarrays per replicate (with exception of Figure 2, which is 2 replicates and 4 microarrays)
Low Concentrations of NO induce 48 genes
  • Microarrays used to test transcription responses to high and low NO concentrations
    • Time course studies show that induction was rapid with diethylenetriamine/nitric oxide adduct (DETA/NO); removal of NO caused transcription levels to decline to basal levels
    • Coincubation of GSNO and carboxyl-PTIO showed that the NO induced gene set was upregulated
    • Low concentrations of NO are not part of a general stress response
      • Response not desensitized after repeated experiments
      • Previous literature shows that other factors in the intraphagosomal environment of the macrophage do not regulate the same genes
    • High concentrations pleiotriopic and cause oxidative stress response, inducing many genes different tot hose controlled by the low NO concentration
Dormancy Gene Regulation by NO and Hypoxia=
  • Expression profiles taken after 4 days of lowO2 state exposure, produced by sealing the culture tube and bacteria consuming the O2
  • The same 48 genes induced by low NO concentrations were induced by the low O2 concentraten
    • Authors suggest that these genes are located in clusters in 9 modules in the chromosome, with 7 contiguous genes, which allows supra-operonic organization and a fast and coordinated transcriptional response to environmental factors


Figure 1 shows the dormancy regulon, which is the gene set of the up-stream promoter region that controls the genes induced in the dormant state.

In Vivo Gene Expression of the NO/Dormancy/Hypoxia-induced Response
  • Researchers sought to see if the 48 gene set was induced in living infected tissue
  • C57BL/6 mice killed 21 days after aerosol infection of M. tuberculosis. Lung tissue total RNA isolated and quantified with qRT-PCR
    • M. tuberculosis must encounter NO and/or hypoxia during course of infection (at least in regards to murine lung)
    • All 5 sentinel groups of the 48 gene region were expressed
    • Supported by Schnappinger et al. - five dormancy genes remain expressed in high levels after infection for many weeks
  • Researchers believe this may be applicable to infection in human lungs because:
    • Human Tb granulomas show hihgl evils of inducible and endothelial NO syntheses
    • 85% pf Tb patients have antibodies encoded by acr, which is induced by NO/dormancy/hypoxia


  • Figure 2 shows dormancy regulon induction at various levels of DETA/NO. Increased levels were measured through qRT-PCR, in vitro and in vivo, and showed similar results.
48 gene NO/Dormancy/Hypoxia Response Defines the Dormancy Regulon
  • Dos R (2 component response regular and dormancy survival regulator) is encoded by Rv3133c, required for acr induction in hypoxia
    • Part of 3 gene operon: Rv3134c, dosR and R/Rv3132c
      • Inactivating Rv3134c fully or particually decreased induction amounts
        • Reversible with complementing mutant
      • Hypoxia does not induce genes if dosR disrupted; thus the effect of Rv3134c is possibly due to the decreased levels of the DosR expressed
      • Thus, dormancy regulon designated
  • Microarray expression profiling was used to compare wild type bacteria and a Rv3134c mutant.
    • Both strains grew to the same level in initial adaptive phase (After O2 limitation), but 40-50 days later, wildtyper bacteria grew 200fold more
    • Dormancy regulon increases overall M. tuberculosis fitness in this in vitro model


Figure 3 shows the comparison of growth between the dormancy regulon mutant and the wild type. After 40-50 days, the mutant gene had significantly reduced fitness compared to the wild type.

Inhibition of Respiration and Growth by NO
  • O2 consumption and bacterial growth rate was measured during NO exposure, to see if NO inhibits respiration and slows bacterial growth
  • NO inhibits respiration and causes growth arrests, depending on the dosage


Figure 4 shows the three effects (dormancy regulon induction, inhibition of respiration and growth rate arrest) occur at the same time and at the same NO concentrations

  • A: NO inhibits respiration and causes growth arrest depending on dose
  • B: DETA/NO concentration decomposed below threshold
  • C: NO exposure in limited quantities does not affect true viability of the bacterial strain, thus explaining why effects are reversible at these levels. However high levels show a slight effect on growth
  • D: NO growth inhibition with dormancy regulon induction showed resumption of growth and disappearance of NO
Characterization of NO and O2 Sensor
  • Researchers investigated if NO presence and O2 absence ould be sensed by the same molecular sensor
  • Found O2 could competitively inhibit NO mediated induction of the dormancy rgulon
    • Used miroarray profiles after exposing M. tuberculosis in high or low aerated cultures and at different concentrations of NO
    • Aeration controlled by culture stirring speed
    • Low aeration: only 1-5μL DETA/NO needed to initiate induction of dormancy regulon
    • High aeration: at least 5x more NO necessary
    • Thus, O2 able to inhibit NO mediated region induction - thus same molecular sensor can monitor O2 and NO levels
  • Researchers tested possibility of the sensor being a heme containing protein bending NO and O2
    • Microarray profiling used to see if low levels of CN- (heme inhibitor) could prevent dormancy regulon induction by NO and hypoxia


    • Figure 5 shows that CN- blocked dormancy regulon expression, and did not affect overall transcription or induction - this supports the proposed heme-containing protein in NO/low O2 signal transduction



Based on the methods section of the paper:

  • What samples did they collect and use for the microarray experiment?
    • Mtb 1254 exposed to 50μM DETA/NO for 40 minutes
    • Mtb 1254 control condition
    • Mtb H37Rv exposed to 0.2% O2 for 2 hours (hypoxia)
    • Mtb H37Rv at 4 days gradual adaptation and low O2 (dormancy)
    • Mtb exposed to 5μM DETA/NO for 40 minutes
    • Mtb exposed to 50μM DETA/NO for 40 minutes
    • Mtb exposed to 500μM DETA/NO for 40 minutes
    • Mtb exposed to 1000μM DETA/NO for 40 minutes
    • Mtb exposed to 5000μM DETA/NO for 40 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes
    • Mtb exposed to 500μM DETA/NO for 10 minutes
    • Mtb exposed to 500μM DETA/NO for 20 minutes
    • Mtb exposed to 500μM DETA/NO for 40 minutes
    • Mtb exposed to 500μM DETA/NO for 60 minutes
    • Mtb exposed to 500μM DETA/NO for 2 hours
    • Mtb exposed to 500μM DETA/NO for 8 hours
    • Mtb exposed to 500μM DETA/NO for 16 hours
    • Mtb exposed to 500μM DETA/NO for 24 hours + 40 minutes with additional NO pulse
    • Mtb exposed to 50μM DETA/NO for 40 minutes
    • Mtb exposed to 50μM KCN for 40 minutes
    • Mtb exposed to 50μM KCN + DETA/NO for 40 minutes
    • Mtb exposed to 2 hour hypoxia
    • Mtb exposed to 500μM KCN + 2 hour hypoxia
    • Mtb exposed to 0μM DETA/NO at low aeration
    • Mtb exposed to 1μM DETA/NO at low aeration
    • Mtb exposed to 5μM DETA/NO at low aeration
    • Mtb exposed to 10μM DETA/NO at low aeration
    • Mtb exposed to 50 μM DETA/NO at low aeration
    • Mtb exposed to 0μM DETA/NO at high aeration
    • Mtb exposed to 1μM DETA/NO at high aeration
    • Mtb exposed to 5μM DETA/NO at high aeration
    • Mtb exposed to 10μM DETA/NO at high aeration
    • Mtb exposed to 50 μM DETA/NO at high aeration
  • How many microarray chips did they hybridize in the experiment? 131 chips
  • Which samples were paired to hybridize on the chip?
    • Mtb 1254 control / Mtb 1254 exposed to 50μM DETA/NO for 40 minutes
    • Mtb H37Rv control / Mtb H37Rv exposed to 0.2% O2 for 2 hours (hypoxia)
    • Mtb H37Rv control / Mtb H37Rv at 4 days gradual adaptation and low O2 (dormancy)
    • Mtb 1254 control / Mtb 1254 exposed to 5μM DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb 1254 exposed to 50μM DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb 1254 exposed to 500μM DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb 1254 exposed to 1000μM DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb 1254 exposed to 5000μM DETA/NO for 40 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 10 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 20 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 40 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 60 minutes
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 2 hours
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 8 hours
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 16 hours
    • Mtb exposed to 500μM DETA/NO for 0 minutes / Mtb exposed to 500μM DETA/NO for 24 hours + 40 minutes with additional NO pulse
    • Mtb 1254 control / Mtb exposed to 50μM DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb exposed to 50μM KCN for 40 minutes
    • Mtb 1254 control / Mtb exposed to 50μM KCN + DETA/NO for 40 minutes
    • Mtb 1254 control / Mtb exposed to 2 hour hypoxia
    • Mtb 1254 control / Mtb exposed to 500μM KCN + 2 hour hypoxia
    • Mtb exposed to 0μM DETA/NO at low aeration / Mtb exposed to 1μM DETA/NO at low aeration
    • Mtb exposed to 0μM DETA/NO at low aeration / Mtb exposed to 5μM DETA/NO at low aeration
    • Mtb exposed to 0μM DETA/NO at low aeration / Mtb exposed to 10μM DETA/NO at low aeration
    • Mtb exposed to 0μM DETA/NO at low aeration / Mtb exposed to 50 μM DETA/NO at low aeration
    • Mtb exposed to 0μM DETA/NO at high aeration / Mtb exposed to 1μM DETA/NO at high aeration
    • Mtb exposed to 0μM DETA/NO at high aeration / Mtb exposed to 5μM DETA/NO at high aeration
    • Mtb exposed to 0μM DETA/NO at high aeration / Mtb exposed to 10μM DETA/NO at high aeration
    • Mtb exposed to 0μM DETA/NO at high aeration / Mtb exposed to 50 μM DETA/NO at high aeration
  • Which was labeled red (Cy5)? Which was labeled green (Cy3)?
    • The control samples were labeled green with Cy3 with experimental samples labeled red with Cy5
  • How many replicates did they perform of each type?
    • For all experiments, they performed 3 biological replicates with 2 technical replicates for a total of 6 replicates per type
    • For the experiments in Figure 2, they performed 2 biological replicates with 4 technical replicates


Discussion

  • Low NO concentrations induce 48 gene regulon using the DosR regulator. This inhibits aerobic respiration and slows replication
  • Regulon is used to increase fitness, during periods if dormancy
    • Predicted roles of genes within the dormancy regulon are supported by previous research of the physiological properties in the doormant state
      • See: Crowe et al (1992), Yuan et al. (1996), Garbe et al. (1995) and Narberhaus (2002)
    • Literature has yet to prove in vivo functioning's of M. tuberculosis in humans
  • NO is usually viewed as a 'bad' molecule that causes cell damage (explaining why it's bad in high doses), but it is also important in intercellular signaling
    • Positive effects of NO for M. tuberculosis respiration, growth, and dormancy regulon induction shows how it may stay in inactive forms in hosts for the hosts' lifetime
      • This effect is seen in the parasite T. gondii which exists in an inactive bradyzoite stage and goes through a stage conversion (initiated by NO) into its active form
  • Hypothesized NO signaling functions are different than those reported in literature (on physiology and gene regulation)
    • Other literature shows NO to modulate gene regulation to reproduce the effect of reactive O2 species
      • Only seen in M. tuberculosis at high concentrations, to initiate oxidative stress response
      • Previous literature does not look at low concentrations of NO; this paper shows low concentrations to mediate an opposing effect on gene regulation (NO induces and O2 represses regulon expression)
      • Thus, the mechanism employed for the dormancy regulon expression must be different than that described in previous literature (using OxyR, SoxR or FNR)
  • Cytochrome C Oxidase is proposed for the NO/low O2 sensor
    • CcO is shown to be reversibly inhibited by low concentrations of NO
    • This proposal must be supported by further functional studies comparing purified wild type and CcO mutant


Figure 6 shows the model proposed by the present article. NO competes with O2 to inhibit respiration, slow growth and induce the dormancy regulon. This is based on the idea that M. tuberculosis is an obligate aerobe and granuloma formation and NO production functions to limit the aerobic respiration.

  • Decreasing respiration initiates transcriptional response, and the pathogen is transformed to stabilize the virus. This lets the pathogen endure longer latency periods
  • NO thus serves as an environmental signal for activation of the bacteria by the immune system

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