Robert W Arnold Week 11

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===PowerPoint===
===PowerPoint===
*here is mine and Alex's portion of the [[Media:JournalClub-3bobbyandalex.pptx | powerpoint]].
*here is mine and Alex's portion of the [[Media:JournalClub-3bobbyandalex.pptx | powerpoint]].
 +
*Final powerpoint can be found [[Media:JournalClubFinal.pptx |here]].
==Links==
==Links==

Current revision

Contents

Electronic Lab Notebook 11

Robert W Arnold

Week 11 Assignment

Week 12 Journal Club

Word Definitions

  1. morbidity - A diseased condition or state, the incidence of a disease or of all diseases in a population
  2. Autolysis - Breakdown of whole cell or tissue by self produced enzymes
  3. virulence - The degree or ability of a pathogenic organism to cause disease
  4. pathogenicity - The ability of a parasite to inflict damage on the host
  5. peptidoglycan - A glycan (a polysaccharide) attached to short cross-linked oligopeptides in the cell wall of eubacteria
  6. Putative Substrate - Commonly thought or deemed; supposed; reputed
  7. stimulon - A set of proteins whose synthesis responds to a single stimulus
  8. Gene Ontology - That department of the science of metaphysics which investigates and explains the nature and essential properties and relations of all beings, as such, or the principles and causes of being
  9. Antimicrobial - A drug for killing microorganisms or suppressing their multiplication or growth
  10. Peptide - A compound of two or more amino acids where the alpha carboxyl group of one is bound to the alpha amino group of another

Journal Club Outline

Abstract

  • Staphylococcus Aureus - human pathogen with strains that can resist treatment
  • New treatments being developed, antimicrobial peptides used for antibiotics to treat MRSA (Methicillin-Resistant Staphylococcus Aureus)
  • Ranalexin is an antimicrobial peptide which should potential against gram-positive bacteria, specifically S. aureus
  • Important to understand microbial physiology and antibiotic mechanism to develop drugs to kill pathogens
  • Ranalexin shows multiple killing mechanisms with cell wall activity
  • Findings show that newly developed strategies in dealing with S. aureus and MRSA will need to be found using many different aspects including microbial activity, peptide reaction and drug resistance

Background

  • MRSA can kill and the rates of MRSA infection have increased in recent years
  • Strains continue to pop up that are resistant to current treatment
  • Important to develop new treatments
  • Antimicrobial peptides (AMPs) can be used to treat MRSA
  • AMPs are made by all living creatures, over 880 types
  • Ranalexin is from the American bull frog, Rana catesbeiana, and is a cationic 20 amino acid peptide
  • Ranalexin shows strong activity against gram-positive bacteria, specifically Staphylococcus aureus, good to use against MRSA
  • Profiling transcriptome and proteome allows for insight into antimicrobial inhibitory actions
  • Used to predict how antibiotics will work
  • Big picture approached used to integrate transcriptome and proteome profiling with MRSA-252 gene and Ranalexin
  • MRSA-252 is one of the most prevalent epidemic causing strains
  • 22 virulence factors found from MRSA-252 along with killing mechanisms by the AMP Ranalexin

Results and Discussion

  • Found a "sublethal ranalexin concentrationon 20μg/ml" which hindered but did not stop growth of MRSA-252
  • Microarrays found 93 upregulated and 105 downregulated genes
  • iTRAQ found 56 upregulated and 15 downregulated proteins
  • Network was made to create a model of gene function in MRSA-252 and its responses to ranalexin
  • Network had a total of 2494 nodes, which are genes, and 19076 edges, connections between genes
  • Gene network comparable to a metabolic network
  • MRSA-252 gene network will have analysis based on module decomposition
  • 11 modules were found to have genes that showed altered expression do to ranalexin expression
  • 5 of the 11 were upregulated and 6 were downregulated
  • Genes downregulated known as "RanaDown" and they included all 6 MRSA-252 ESAT-6 system
  • Ranalexin restricts S. aureus MRSA-252 "pathogenicity" including the ESAT-6 system and 22 virulence factors
  • Ranalexin promotes a response that includes upregulation of peptidoglycan synth genes
  • Shows that ranalexin may induce cell wall stress
  • RanaUp genes include VraR which is a part of a system VraSR that controls vancomycin induced genes, many of which are used in cell wall creation
  • FtsH is another RanaUp protein and it is an intermodular hub that regulates behavior
  • FtsH looks after penicillin binding proteins (PBPs) and is also involved with osmotic stress and mutant reaction
  • FtsH is very important in MRSA's response to ranalexin and other AMPs
  • Possible targeting of FtsH by AMPs could support Ranalexin action
  • RanaUp genes also include transcription regulation proteins that are in cell well antibiotics
  • Ranalexin exposure can make cell walls stressed like antibiotics can in resistant S. aureus strains
  • Ranalexin induced cell wall gene and protein changes were examined for peptide sensitivity
  • Ranalexin expression detected after 15 minutes, peaked at 30 minutes and declined at 60 minutes
  • Osmotic fragility also tested for in MRSA cells
  • Sublethal concentrations of AMPs used and cultures were incubated and harvested
  • Control cells showed negligible loss while those treated with sublethal AMPs had induced stress at the staphylococcal cell wall
  • Ranalexin is able to permeate cell membranes by the upregulation of genes allowing for cation influx to help ranalexin exposure
  • Upregulation also effects osmolarity which should increase cell fragility and affect integrity of the cell
  • Ranalexin therefore can be effective in killing S. aureus MRSA-252
  • There is evidence to show ranalexin's multiple actions that can degrade the intergrity of a MRSA cell
  • MRSA attempts to deal with ranalexin stress but inhibiting actions of ranalexin can kill it

Conclusions

  • Evidence showed ranalexin effect on bacterial cell wall and action at the cell membrane
  • Cationic AMps exert inhibatory actions
  • FtsH membrane chaperone -->upregulated in response to ranalexin, potential drug target
  • VraRS --may be two component staphylococcal response regulator that is involved with cationic peptide resistance --> 22 virulence factors were inferred
  • Evidence for PhoU-mediated persister switching as a mech. of drug tolerance in MRSA
    • Further investigation needed
  • This work is important and informative to the development of stratagies against S. aureus and shows approach to study of drug resistance and mode of action

Figures and Tables

  • Figure 1 - sublethal effects of ranalexin on MRSA-252 growth
  • Figure 2 - network degree pairs probability, shows genes interacting on MRSA network
  • Table 1 - shows responses of ranalexin and the genes altered in relation to what occurs
  • Figure 3 - downregulated genes when exposed to ranalexin in ESAT-6 module
  • Table 2 - shows genes associated with pathogenesis
  • Figure 4 - shows upregulation of vraR and tcaA when exposed to ranalexin
  • Figure 5 - hypoosmotic stress shown in relationship to ranalexin exposure

PowerPoint

  • here is mine and Alex's portion of the powerpoint.
  • Final powerpoint can be found here.

Links

Biol 368 Homepage

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