BIOL368/F14:Chloe Jones Week 11

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Contents

Journal Club Article/Microarray Dataset Possibilities

  1. An Outbreak of Severe Infections with Community-Acquired MRSA Carrying the Panton-Valentine Leukocidin Following Vaccination
    • Link to paper
    • This first article is not suitable because it is not measuring gene expression (mRNA). Kam D. Dahlquist 17:30, 5 November 2014 (EST)
  2. Two Distinct Clones of Methicillin-Resistant Staphylococcus aureus (MRSA) with the Same USA300 Pulsed-Field Gel Electrophoresis Profile: a Potential Pitfall for Identification of USA300 Community-Associated MRSA
    • Link to paper
    • This second article is not suitable because it is not measuring gene expression (mRNA). Kam D. Dahlquist 17:33, 5 November 2014 (EST)
  3. Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs
    • Link to paper
    • This third article is not suitable because they only did duplicates instead of at least three replicates. Kam D. Dahlquist 17:38, 5 November 2014 (EST)
  4. Cyclic di-GMP mediates Mycobacterium tuberculosis dormancy and pathogenecity
    • Link to paper
    • This fourth article is not suitable because they only did duplicates for one condition and only one replicate for the other condition, instead of at least three replicates. Kam D. Dahlquist 17:41, 5 November 2014 (EST)
  5. Global Network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus

Selected Article

Overton et al., 2011 I.M. Overton, S. Graham, K.A. Gould, J. Hinds, C.H. Botting, S. Shirran, G.J. Barton, P.J. Coote Global Network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus

Vocabulary

  1. Anabolism: (N.) The synthesis of complex molecules, such as proteins and fats, from simpler ones by living things Source:Oxford Concise Medical Dictionary 8ed.
  2. Antimicrobial: (N.) Describing a drug, antibiotic agent, physical process, radiation, etc. that is inimical to microbes. Source: Oxford Dictionary of Biochemistry and Molecular Biology 2ed.
  3. Attenuator: (N.) Sequence in DNA that is located between the operator and the gene for the first protein in an operon. Source: Oxford Dictionary of Biochemistry and Molecular Biology 2ed.
  4. Autolysis: (N.) The process of self-destruction of a cell, cell organelle, or tissue. It occurs by the action of enzymes within or released by lysosomes Source: Oxford Dictionary of Biology 6ed.
  5. Glycosyltransferase: (N.) abbr.: GT; any enzyme transferring a glycosyl group. Source: Oxford Dictionary of Biochemistry and Molecular Biology 2ed.
  6. Transcriptome: (N.) The entire set of mRNAs transcribed from a cell's genome. Source: Oxford Dictionary of Biochemistry and Molecular Biology 2ed.
  7. Topology: (N.) The study of the properties of geometrical figures that are subjected to deformations such as bending or twisting. Source: A Dictionary of Genetics 7ed.
  8. Tropoelastin: (N.) The soluble polypeptide precursor of elastin. Secreted tropoelastin molecules are aligned on a scaffold of fibrillin-rich microfibrils and crosslinked by the formation of desmosine to form elastin fibres.Source: Oxford A Dictionary of Biomedicine
  9. Up-regulation: (N.) The process of reducing or suppressing the response of a cell to a stimulus. Source: Oxford Dictionary of Biochemistry and Molecular Biology 2ed.
  10. Vancomycin: (N.) A glycopeptide antibiotic used as a last resort for treatment of infections by Gram-positive bacteria, although resistance has emerged. Source: Oxford A Dictionary of Biomedicine

Global network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus

A. Methicillin Resistant Staphylococcus aureus (MRSA): human pathogen with strains resistant to existing treatments
  1. MRSA infection have significantly risen
  2. Strains resistant to existing treatments continue to emerge, therefore prevention and treatment strategies are imperative
a.) Antimicrobial peptides (AMPs) possible method of unique antibiotics
i.)Part of the innate immune response (host defense peptides)
b.) Ranelexin (20a.a. peptide, isolated from bullfrog)
i.) Activity against Gram-positive bacteria, in vitro
ii.) Therapeutic potential against MRSA
B. Transcriptome and proteome profiling
  1. Understanding the mechanism, able to developed new therapeutic strategies
a.) mRNAn (Transcriptome) and protein profiles (proteome) generated in response to antimicrobial stress reflect the change in particular cell functions, provide marker for the type of stress
b.) Expression profiling, analysis of drug mode of action, examples drug interactions or gene regulation
C. Global function association network, Linking disease associations with regulatory information
  1. high confidence functional association network that shows pathway relationships for 95% of S. aureus MRSA-252 genes
a.) MRSA-252isolate of most prevelant epidemic MRSA clones, EMRSA-16
b.) Approach showed 22 unique MRSA virulence factors (molecules expressed or secreted by pathogens) , and corresponding killing mechanisms for ranalexin
  1. VrsRS, two-component system in cationic peptide
a.) Stimulus-response coupling mechanism to allow organisms to sense and respond to changes
  1. FtsH, proposed as a candidate drug target
  2. PhoU mediated persistor, aids in drug tolerance
a.) persistence gene, phoU, inactivation leads to a generalized higher susceptibility than that of the parent strain to a diverse range of antibiotics.
D. Ranalexin elicits significant change in transcript and protein levels
  1. sublethal ranalexin concentration (20ug/ml) determined, impaired but did not abolish growth of MRSA- 252
  2. From transcriptome an proteome profiling, Microarrays identified (93 upregulated and 105 downregulated genes) between control and ranalexin exposed
a.) No inconsistencies observed betweentranscriptome and proteome profiles
b.) Overlap in Gene Ontology (GO), (gene functional similarity) was observed, only few observed that directly overlapped.
i.) Identified 290 significant enriched terms (identify shared associations between proteins), showing effects that ranalexin has
E. Global gene functional association network
  1. . Help to show model for global gene function of MRSA and analysis of ranalexin responses
  2. MRSA-252 genes were network nodes and cell signaling and metabolism were the connections (edges) between genes (nodes)
  3. Final network contained 2494 nodes(genes) and 19076 edges (connections).
a.) network edges (connections) relate 94.5% of MRSA-252 genes with high confidence-the global gene very similar to the actual bacteria.
  1. gene functional association network topology closer to that of metabolic pathways, rather than protein interaction networks which are less modular.
  2. 11 modules showed that there was a significant altered expression in MRSA-252 cultures exposed to ranalexin
a.) 5 were upregulated, 6 downregulated
b.) 58 nodes outside modlues classified as intermodular hubs that are important regulators of system behavior.
F. Impact on virulence and inference of novel determinants
  1. genes that downregulated with introduction of ranalexin (RanaDown) included all six of MRSA-252 ESAT-6 secretion system componenets , which are essential to S. aureus pathogenesis.
  2. Highly significant module included for ESAT-6 components, six gene not assigned to module, but RanaDown
  3. RanaDown gene in module ”hypothetical”
a.) SAR0287:predicted 6 transmembrane proteins and SAR0288: secred or cell wall anchored
i.) were able to be matched , agree with virulence factors
b.) other 5 hypothetical genes matched to conserved domains , consistent with roles in virulence
  1. 2 significant RanaDown modules, because associated with high affinity metal ion transport, crucial for establishment of infection
a.) SAR0787 and SAR0790 representing the sst iron-uptake operon
b.) 5th gen in module SAR101, substrate binding protein for iron complex transport
  1. further modules 12 genes implicated in colonization and immune-modulation putative ::# ::#Remaining 4 putative genes: SAR1489, SAR2421, SAR1223, and SAR1802.
a.) Autolysis for SAR2421 (lyrA) and SAR1223 (lytN) and SAR1802 hypothetical, SAR1489 (EBPS) binds tropoelastin and not associated with adhension
  1. All 16 genes were predicted to encode cell wall anchored, transmembrane or secreted proteins
  2. TMHMM2 indicated one transmembrane and two secreted/cell wall proteins
  3. most significant enriched GO term was ranalexin downregulated gene set was ‘pathogenesis’, mapping GO annotation based on pathogenesis
a.) SAR2122, SAR1022, and SAR2648 strongly downregulated
  1. . Ranalexin dependent repression of virulence gene corresponds with the upregualtion of sarA attenuator SARR1374, which inhibits sarA-dependent virulence gene expression
  2. RanaDwon genes (SAR0115 and SAR2474) are transcriptional regualtors (sarS, sarZ) from the sarA family, that activate virulence factor transcription.
  3. . Ranalexin inhibits S. aureus MRSA-252 pathogenicity, including ESAT-6 system, and 22 new virulence factors
G. Ranalexin induces cell wall stress
  1. strong upregulation of several peptiglycolyan synthesis genes, in response to ranalexin
a.) hint at ranalexin acting on cell wall
  1. VraR (SAR1974) regulator protein of vancomyscin resistance associated with 2 component system (VraSR) was RanaUp
a.) VraSR controls expression of genes induced by vancomycin, participate in cell wall biosynthesis
i.) induced by exposure to cell wall antibiotics and glycolpeptides and beta lactams
  1. FtsH, RanaUp, intermodular hub, regulating system behavior
a.) connect to different biological modules, interacting with different partners in different moments and/or different cellular compartments
b.) AAA ( ATPase Associated with diverse cellular Activities) family, chaperon required for incorporation of penicillin binding proteins (pbp2) into cell membrane.
i.) pbp2 RanaUp
  1. Ftsh proposed as drug target, key role in MRSA response to antimicrobials (i.e ranalexin)
  2. vraX overexpressed in isolates of vancomycin-intermedidate s. aureus, >200 fold increase in in expression in vancomycin sensitive s. aureus treated with vancomycin
a.) Vancomycin and ranalexin cause similar cell wall stress
  1. Ranalexin and gylcopeptide intermediate-resistant S. aureus (GISA) strains induce a similar cell wall stress response
H. Further investigation of ranalexin cell wall effects
  1. Protein expression chang3es were studied by producing vraR mutants and tcaA disruption mutants, examine for peptide sensitivity
a.) prior to mutants, ranalexin induced enhanced expression was verified for vraR and tcaA
b.) vraR mutant identical to parent strain when ranalexin not present , inhibited when ranalexin added.
c.) no significant differences in rate of loss of viability between the tcaA mutant and the parent strain exposed to ranalexin were detected.
  1. measure osmotic fragility of MRSA exposed to sublethal concentration of antimicrobials
a.) osmotic fragility symbol for cell wall disruption-tells if more likely to break
b.) insignificant loss to hypo osmotic stress in in control cells
c.) cells treated pre-treated with antimicrobials (vancomycin & ranalexin) were sensitive
  1. Ranalexins inhibitory action involves effects at staphylococcal cell wall and not only due to membrane disruption.
I. MRSA persister formation and drug tolerance
  1. When ranalexin present produce strong upregulation of proteins determined by pstSCAB-phoU operon
  2. MRSA gets antimicrobial resistance by adopting a PhoU-mediated persister phenotype
a.) Pi(inorganic phosphate) transport not a major component for pstSCAB-phoU operon
  1. . Persister bacteria show a thicker wall and loss of virulence factors
J. Multiple actions in MRSA killing
  1. Ranalexin makes cell wall permeable, cation antiport upregulated
a.) permeabilisation leads to cation(+) influx and dissipation of transmembrane electrochemical gradient
  1. membrane and cell wall complementary kill MRSA -252
  2. Upregulation of dlt operon increased positive charge at cell surface and reduced peptide binding
  3. Ranalexin exposure: upregulation of glycolysis, LDH1(L-lactate dehydrogenase 1) RanaDown, and upregulation of Val/Leu/Ile synthesis
a.) repurposing pyruvate into production of hydrophobic proteins for peptide sequestration
K. Conclusion
  1. Functional network analysis allowed us to see the mode of action for cationic AMPs and MRSA-252 resistance mechanisms
  2. Ranalexin had effects on cell wall as well as the membrane, act in coordination
  3. FtsH membrane (degrades both soluble and integral membrane proteins in E.coli) (intermodular hub) chaperone was upregulated in response to ranalexin, drug target
  4. VraR, suggested to be involved n mediating tolerance to AMPs, maybe 2-compnenet response regulator involved in cationic peptide resistane with apsRS
  5. 22 novel virulence factors inferred from the reduction of MRSA-252 due to exposure of ranalexin exposure
  6. PhoU-mediated persistor, mechanism of drug tolerance in MRSA, therapeutic target
  7. Multiple inhibitory actions for ranalexin, cell wall effects, cation influx, and disappearing transmembrane electrochemical gradient

Figures and Tables

  • Figure 1. MRSA-252 exposed to sublethal ranalexin concentration ( 20μgml-1), showed that in the presence of ranalexin had a brief reduction in growth rate .
  • Table 1. Ranalexin response modules (11). 5 are upregulated denoted by “+”, and 6 were downregulated denoted y “-.“ Shows the module description, ranalexin altered gene set , and additional genes in the module (unaltered).
  • Figure 2. Displays node pair degree connectivity between pairs., with the axes representing the degree of interacting nodes (genes) in MRSA network. Bottom left can be considered low degree values (less interaction), top right higher degree values (more interaction). Z-axis is the ratio.
  • Table 2. Modules that are significant in virulence. (10). Marked with ‘Pathogenesis ‘.Shows the module description, genes marked with ‘Pathogenesis ‘, and additional genes in the module (unaltered). If underlined downregulated. If bolded candidate novel virulence factors
  • Figure 3. ESAT-6 system plays important role in pathogenesis, when exposed to ranalexin, RanaDown (in pink). Showing novel/hypothetical genes ESAT-6
  • Figure 4. Showign the upregulation of vraR and tcaA expression at different exposure times when exposed to ranaflexin. Peaked at 30 minutes (upregulation) and then declined after an hour. (VraR: involved in the control of the cell wall peptidoglycan biosynthesis)( tcaA teicoplanin resistance)
  • Figure 5. Hyposomotic stress in response to ranalexin and/or vancomycin at different concentrations. The contrl experienced the least amount of stress , and a combination of vancomycin and ranalaexin experienced the most as well as ranalexin on its own.

Microarray hybridization and analysis/Methods

  • For the microarray experiment they collected and used samples form MRSA-252.
  • They hybridized 6 microarray chips in this experiment
  • The paired samples on the chip were: Control 1 and Ranaflexin 1, Control 2 and Ranaflexin 2, Control 3 and Ranaflexin 3
  • Cy5 and Cy3 were both used for each individual technical replicate.
  • In this experiment they performed three biological replicates, with each having two technical replicates for a total of six.
  • The paper used ImaGene to quantify the florescence signal in each spot, calculate the ratio of red/green, and to transform the ratio to log (base2).
  • The paper used GeneSpring v7 to normalize the log ratios as well as perform statistical analysis.
  • To compare against known data, used mapping GO annotations, only reference to known data that was proven by them (discovery-driven experiment)
  • The UniProt GO mapping for Staphylococcus aureus was used to assign GO terms to the reference datasets. Fisher exact test was performed for every GO term associated with each of the test datasets(TU, TD, PU, PD)
  • Because this was discovery-driven experiment they only were able to map the microarray data based on its own pathway

Journal Club 3 Powerpoint

Journal Club 3 Powerpoint

Electronic Lab Notebook

Weekly Assignments

Class Journals


Chloe Jones 03:46, 15 October 2014 (EDT)Chloe Jones

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