Msaeedi23 Week 11

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Process for GenMapp analysis

Microarray paper: <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243> Genome Paper: <http://www.nature.com/ng/journal/v35/n1/full/ng1227.html>

Journal Club Presentation

File:Microarray Journal Club Presentation.pdf

Week 11

  • For the microarray paper (GenMAPP Users only), include the following:
  • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
  • Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
  • Construct a flow chart that illustrates the above.

Unknown Term Definitions

  • Phosphorelay: A multi-stage process, involving the movement of phophoryl groups by histidine kinases in bacterial signal transduction.

< https://en.wiktionary.org/wiki/phosphorelay>

  • Putative: Believed or deemed to be the case; accepted by supposition rather than from proof.

< https://en.wiktionary.org/wiki/putative>

  • Supernatant: Floating on the surface or over something; the chemistry of a liquid-lying above a sediment or settled precipitate.

< http://dictionary.reference.com/browse/supernatant?s=t>

  • Chemiluminescent: The emission of light as the result of a chemical reaction.

< https://en.wikipedia.org/wiki/Chemiluminescence>

  • Endogenous: Growing or developing from within; originating within.

< http://dictionary.reference.com/browse/endogenous>

  • Non-Permeabilized: Made not permeable, unable to infiltrate a surface.

< https://en.wiktionary.org/wiki/nonpermeabilized>

  • Albeit: Although, even if.

< http://dictionary.reference.com/browse/albeit?s=ts>

  • Attenuated: To render less virulent, as a strain of pathogenic virus or bacterium.

< http://dictionary.reference.com/browse/attenuated?s=t>

  • Modulating: To regulate or adjust to a certain measure or proportion; soften or tone down.

< http://dictionary.reference.com/browse/modulating?s=t>

  • Homodimerization: Any reaction which leads to the formation of a homodimer, a molecule composed of paired identical proteins.

< http://dictionary.reference.com/browse/modulating?s=t>

Article Outline

Write an outline of the article. The length should be a minimum of the equivalent of 2 pages of standard 8 1/2 by 11 inch paper (you can use the "Print Preview" option in your browser to see the length). Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article. What is the importance or significance of this work (i.e., your species)?

Introduction/Significance

(Used in accordance with GenMAPP user lolufson)

    • The Gram-negative bacterium Bordetella pertussis is the causative agent of pertussis or whooping cough.
      • Pertussis is responsible for 300,000-400,000 deaths each year as it is one of the top ten most infectious diseases worldwide.
    • B. pertussis creates a lot of different virulence factors that include adhesions, toxins, and multiple others that are regulated by the BvgA/S two-component system when a change in the environment is detected.
      • B. pertussis produces an intact PS microcapsule on the surface of its bacteria.
      • The B. pertussis capsule does not play a role in the capsule-mediated defense mechanisms, some of which include adherence to mammalian host cell, complement-mediated killing and antimicrobial attack.
    • This study characterizes the role of the capsule locus and the expression in pertussis pathogenesis.
    • This study showed that a protein, KpsT, located on the membrane is involved via a structural role in the transportation of the PS capsule across the cell envelope, and this protein is necessary for the greatest BvgA/S-mediated signal transduction.

What were the methods used in the study? (Used in accordance with GenMAPP user lolufson)

Methods

    • Bacterial Strains and growth conditions
      • All B. pertussis strains were grown at 37°C on Bordet-Gengou (BG) agar (Difco) supplemented with 10% defibrinated sheep blood with 1% glycerol or in modified Stainer-Scholte (SS) medium containing 2,6-O-dimethyl-β-cyclodextrin (Sigma Chemical) at 1 g/liter supplemented with either 10 μg/ml gentamicin, 100 μg/ml streptomycin or 30 μg/ml chloramphenicol.
      • E. coli strains were grown at 37°C overnight in fresh Luria-Bertani broth or on LB agar (Difco) plates, and suitably 100 µg/ml ampicillin, 50 µg/ml kanamycin, 10 µg/ml gentamicin, 30 µg/ml chloramphenicol was added to select for antibiotic-resistant strains.
    • Construction of B. pertussis capsule-deficient mutant strains
      • Non-polar single gene deletion was performed for kpsT, kpsE, and vipC ORFs by using the double homologous recombination method.
      • The final products of the vector insertions yielded pJQT1-2, pJQE1-2, and pJQV1-2, and the recombinant pJQ constructs were used for allelic exchange in wild-type BPSM to yield the mutant strains.
    • Construction of recombinant B. pertussis BPSH strain
      • The B. pertussis BPSH strain that expressed a histidine tag at the N-terminal end of BvgS was created by PCR amplification.
      • The final products of the vector insertions yielded pJQ-His-BvgS, and this was used to further develop mutant and wild-type BPSH strains.
    • Southern blot analysis
      • Chromosomal DNA was extracted and purified from BPSM and ΔkpsT and ΔkpsE bacteria and the B. pertussis strains were digested using restriction enzymes.
      • Detection of hybridization was done using alkaline phosphatase-conjugates anti-DIG antibody, and the membrane was developed using NBT/BCIP AP substrate (Chemicon).
    • Whole cell extract and supernatant concentration preparation
      • Whole cell extract was harvested from bacteria previously grown and then washed to prepare it for incubation with centrifugation proceeding it.
      • The bacteria pellet created was solubilized and the cell lysate produced from this was centrifuged to pellet unsolubilized cell and debris.
    • Purification of His-BvgS affinity chromatography
      • Protein lysate was mixed with Qiagen to allow overnight binding to occur, and then the cellular lysate underwent chromatography column for gravity flow purification.
    • SDS-Page and Western blot analysis
      • Ten times concentrated supernatant of B. pertussis and whole cell extract were run on an SDS-PAGE and then the transferred membranes were incubated with mouse anti-FHA monoclonal antibody, mouse anti-PT monoclonal antibody, rabbit anti-BrkA polyclonal antibodies.
      • The purified His-BvgS were run on 10% SDS-PAGE and were then either stained or transferred onto PVDF membranes and incubated with other antibodies.
    • RNA extraction from in vitro B. pertussis culture
      • B. pertussis bacteria in virulent phase were harvested and then incubated with an RNA bacteria reagent designed to stabilize the RNA.
      • After the pelleted bacteria were resuspended, RNA extraction was performed and the purified RNA were treated to remove contaminant DNA and finally reverse transcription was performed on 10 ng of bacterial RNA.
    • RNA extraction from B. pertussis infected mice lungs.
      • B. pertussis-infected mice were euthanized so their lungs could be removed to be drowned in RNAprotect Bacteria Reagent for 1 hour.
      • The lung homogenates from the individual mice lungs were filtered and then centrifuged to pellet the remaining cell debris.
      • Bacterial RNA was extracted using lysozyme and then in-solution DNA digestion was performed, finally ending with the elution of total RNA in DPEC water.
    • Real-time polymerase chain reaction
      • PCR was performed in a 96 well-plate with each well containing 2 µl of cDNA mix, 0.5 µl of forward and reverse primers (0.5 µM final), and 25 µl SYBR-Green Supermix with ROX (Bio-Rad) to a final volume of 50 µl.
      • Samples were run in triplicate.
      • Expression of each target gene was based on relative quantification using the comparative critical threshold value method.
      • Relative quantification of a specific gene was measure in each reaction by normalization to the Ct value obtained for the endogenous control gene, recA.
      • Control reactions without cDNA were used as negative controls.
    • Microarray production and analysis
      • Oligonucleotides were synthesized and spotted on slides in a buffer.
      • For each of the samples done, 15 µg of total RNA was reverse transcribed in the presence of either 100 µM Cy3-dCTP or Cy5-dCTP (GE).
      • The newly labeled cDNA was treated with NaOH to degrade RNA and then purified to allow hybridization to occur.
      • Hybridization was then performed and the slides were washed and then scanned using an Innoscan microarray scanner to be analyzed.
      • The LIMMA package was used for the differential expression and normalization, while moderated t-statistic was used to identify statistically significant regulation.
      • Due to multiple testing, obtained P-values were corrected using Benjamini & Hochberg method to control the false discovery rate.
      • Microarray data were deposited in the GEO online database.
    • Reverse Transcribed (RT)-PCR
      • RNA was purified from exponentially grown B. pertussis bacteria in virulent phase, and the cDNA was obtained as described previously.
      • PCR was performed using recA primers (endogenous control) or using primers mapping to the respective downstream region of the deleted ORFs.
    • FACS
      • In order to make an accurate estimation of the surface polysaccharide capsule expression, fluorescence-activated cell sorting (FACS) analysis was done on the B. pertussis strains grown in a medium containing MgSO4 by using the anti-Vi polyclonal mouse immune serum.
      • Each assay was performed 3 times independently.
    • Intranasal infection
      • Female adult mice were selected for their specific pathogen free qualities and then infected intranasally with B. pertussis strains for lung colonization, as well as BPSM for bacterial RNA extraction.
      • Using an interval, four mice were killed using CO2 in order to allow their lungs to be removed and homogenized.
      • The serial dilutions of the individual lung homogenates were plated and then counted for CFU quantities after 4 days of incubation.
      • Each experiment was performed at least twice independently.

Briefly state the result shown in each of the figures and tables

  • Table 1: Lists all of the B. Pertussis strains used in the study along with their genotypes/relevant features, and the source where the strain was obtained.
  • Table 2: A complete list of all of the forward and reverse primers used in cloning are provided with their sequences, 5'-3', and their indicated mode of amplification (forward or reverse).
  • Table 3: Lists specific reverse and forward primers used in the real-time PCR analysis.
  • Figure 1: Two biological replicates were performed and the expression of the capsule locus was monitored using real-time PCR and observed to be significantly up-regulated in the mouse lungs compared to its in vitro Bvg+ expression level, with a peak of expression at day 3 p.i. A similar trend of expression was seen for another bvg-repressed gene, vrg6. Concurrently, the expression of vags including bvgA, fhaB and ptx were all up regulated at day 3 and day 7 p.i. compared to Day 0 and to the in vitro Bvg+ expression level.
  • Figure 2: Outlines the construction of the kpsT, kpsE, vipC, and BPSM B. pertussis mutants. Goes on to portray the Southern blot analysis and the transcription efficacy of the downstream gene. Finally it looked at the growth kinetic profiles of each mutant. "The ΔkpsT, ΔkpsE and ΔvipC mutant strains displayed hemolytic and domed colony morphology on blood agar plates (data not shown) and in vitro growth kinetics comparable to the parental BPSM strain (Fig. 2 D), implying that these single gene deletions within the capsule locus did not affect the in vitro fitness of the bacteria." <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243#pone-0115243-g002>
  • Figure 3: Depicts phenotypic characterization of the kpsT, kpsE, and vipC mutants. It was observed that ΔkpsT, ΔkpsE and ΔvipC cells consistently displayed levels of fluorescence shift comparable to that measured with KOcaps, corresponding to nonspecific background shift. "The FACS data supports that in-frame deletion of the single ORF kpsT, kpsE or vipC within the capsule locus is sufficient to prevent the presence of the PS capsule at the bacterial surface."
  • Figure 4:Compared to BPSM, the band signal intensity for FHA in ΔkpsT 10x concentrated culture supernatant was about 30% down-regulated (Fig. 4 A). In contrast, signal intensities for FHA in the whole cell lysates appeared comparable between mutant strains (Fig. 4A), indicating that secretion into the culture supernatant may be compromised in ΔkpsT. Similarly, secretion but not production of PT appeared to be impaired in ΔkpsT (Fig. 4C). "In contrast, both production and secretion of BrkA in ΔkpsT were visibly reduced (Fig. 4 B). Production/secretion levels of FHA, PT and BrkA were partially restored to parental level in ΔkpsTcom strain (Fig. 4 A, B and C)."
  • Figure 5: Consistent with the Real-time PCR analysis, expression of the bvgAS locus and fhaB was not found to be down-regulated in the ΔkpsT mutant (S1 Table).In addition to further support this analysis, the microarray data revealed that the absence of KpsT affects negatively the expression of a large number of bvg-regulated genes. "Such overall down-regulation is likely to be responsible for the attenuated phenotype observed with the ΔkpsT mutant in mice."
  • Figure 6: "Higher amounts of BrkA, PT and to a lesser extent FHA were detected with the BvgS-VFT2- ΔkpsT double mutant compared to ΔkpsT single mutant, with band signal intensities comparable to those observed for wild type BPSM and BvgS-VFT2 strain (Fig. 6A). Also, real-time PCR analysis showed that down-regulation of the brkA, ptx and sphB1 genes observed with the ΔkpsT single mutant was not seen with the BvgS-VFT2- ΔkpsT double mutant (Fig. 6B). These data thus indicated that deletion of kpsT in a Bvg+-phase locked mutant did not affect the virulence genes expression therefore suggesting that VFT2 mutation in the BvgS sensor is dominant over kpsT deletion. Consistently, a parental in vivo virulent phenotype was observed with BvgS-VFT2- ΔkpsT double mutant in mice (Fig. 6C). Altogether, these results indicate that deletion of kpsT ORF in a constitutive bvgS background had no effect on the expression of bvg-regulated genes."
  • Figure 7: "Western blot analysis using anti-His and anti-BvgS antibodies confirmed the identity of the 140 kDa eluted protein as His-BvgS monomers in E2 and E3 from BPSH but not from BPSM extracts (Fig. 7B). A band at 50 kDa MW was also observed in E3 and E4 from both BPSM and BPSH, suggesting that this unknown protein bound to the Ni-NTA beads likely due to high His content (Fig. 7A). A stronger signal intensity of the 140 kDa band was observed under reducing/heat treatment conditions (Fig. 7C). Altogether these data supported that KpsT is necessary for BvgS oligomerization although no direct physical interaction seems to occur between both proteins."
  • Figure 8: The results portrayed in this figure support that neither KpsT alone nor the KpsMT complex is enough to restore fully a parental colonization profile in KOcaps. "Therefore, instead of KpsT alone, the entire capsule transport machinery may play a role in the BvgS-mediated signal transduction and indirectly in the overall bacterial virulence."

How do the results of this study compare to the results of previous studies?

  • Previous research showed that B. pertussis PS capsule is not involved in the classical capsule-defense mechanisms including phagocytosis, complement-mediated killing and antimicrobial peptides attack. This new study provides evidence that the PS capsule transport proteins, in particular the membrane associated KpsT protein, are necessary for B. pertussis virulence in an indirect mode-of-action. The data suggest a structural role for the entire PS capsule transport machinery in the cell envelope and a consequential impact on the BvgA/S-mediated signal transduction and virulence gene expression. The work presented in this paper demonstrates for the first time that a vrg locus (the PS capsule locus) plays a role in pertussis pathogenesis. Therefore, it is demonstrated for the first time that a vrg locus (specifically the capsule locus) i) is expressed and modulated during in vivo infection, and ii) plays an indirect role in B. pertussis in vivo virulence. This finding apparently contradicts a previous study which claimed that the Bvg- phase is dispensable for bacterial in vivo virulence based on a Bvg+ phase locked mutant. This new observation thus suggests that in vivo, dynamic gene regulation occurs thereby implying that over time the micro-environmental conditions encountered by the bacteria vary. The PS capsule locus in B. pertussis is physically distinct from the bps locus which encodes proteins involved in biofilm formation. The data indicates that absence of the membrane-associated protein KpsT (and to a lesser extent KpsE) affects significantly pertussis pathogenesis. "In conclusion, this research portrays that the B. pertussis PS capsule transporter-export machinery and in particular KpsT are necessary for optimal expression of virulence genes and therefore play an important role in pertussis pathogenesis."

<http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243#pone-0115243-g006>

For the microarray paper (GenMAPP Users only)

    • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.

This paper identified polysaccharide (PS) capsules as important virulence determinants for bacterial pathogens and and KpsT as a membrane protein involved in the transport of PS polymers across the cellular envelope in Bordetella pertussis. To determine the impact of PS capsules on the virulence of B. pertussis, a microarray experiment was run testing a ∆KpsT mutant against the wild-type.

  • Treatment Group: ∆KpsT Mutant
  • Control Group: Wild-type (BPSM)

Biological replicates: 3 replicates of both the control and treatment groups; Technical replicates: 2 per biological replicate (dye-swapped)

  • Replicate Listings (taken from NCBI GEO):
    • GSM1519507 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 1
    • GSM1519509 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 1 by dye-swap color
    • GSM1519511 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 2
    • GSM1519514 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 2 by dye-swap color
    • GSM1519516 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 3
    • GSM1519519 Strain of Bordetella pertussis deleted of KpsT versus wild-type B. pertussis replicate 3 by dye-swap color
  • Construct a flow chart that illustrates the above.

Media: Flow_chart_db_project.docx

DOCUMENT ALL STEPS TAKEN, MANIPULATIONS OF FILES AND ALL.

1. Experimental design-what is the treatment and what is the control

  • Biological vs technical replicants- for vibrio control was the untreated strain, technical replicates = splitting a sample, different from biological replicates. Biological replicates are derived from the original strain. *note how many replicates of each. # of chips(microarrays) (for vibrio it was 9, total number of samples)
      • Dye swaps, cy3 green dye and cy5 red. note how many swaps. Swaps done to avoid dye playing an effect in replication.

2. Sample + data relationship. In raw data there should be one file to each chip. Match file name to sample. raw.zip on arrayexpress has one file for each chip. Sdrf.txt open in excel to show whats in the samples-should tell you all of the samples, their matches, and dye swaps.

3. Compiled raw data file. Include columns for: ID, Sample log2FC, sample log2FC. Compiling raw data files into one spreadsheet to prepare for statistical analysis.

4. Normalization + statistical analysis. Customized for each dataset due to different treatments. Either t-test or one way anova. Also due sanity check, which will be a result in the paper. Compare it to what was written in the paper, corresponding to specific genes. Comparing log-fold changes of specific genes between our data and the paper's.

5. GenMAPP + mappfinder. Run data through database created by groupmates.

6. Deliverables-describing methods, production of tables including the sanity check, etc.

  • In discussion compare what we found vs to what they found.

Msaeedi23 (talk) 23:42, 16 November 2015 (PST)

Mahrad Saeedi

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