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Abstract

  • Background
    • Staphylococcus aureus - human pathogen. Treatments are needed. Antimicrobial peptides potential antibiotic to MRSA.
      • Such as Renalexin - fights against gram positive bacteria, S. aureus in particular.
    • Study of drug resistance and mode of action is important.
  • Results
    • Bayesian logistic regression - allows identification of renalexin response modules from MRSA-252 preteome and transcriptome profiling.
    • Relaxin displayed killing mechanisms & cell wall activity.
      • Gene disruption and osmotic fragility experiments supported cell wall effects.
    • 22 Virulence factors inferred --> VraRS two-component system & PhoU-mediated persister formation --> MRSA tolerance to cationic antimicrobial peptides.
  • Conclusions
    • Integrative approach to study drug resistance and their mode of action. Finds lead to development of trategies against Staphylococcus aureus (MRSA in particular)

Background

  • MRSA - major cause of mortality and mobility. Resistant strains to treatments continue to emerge. Global problem --> community-associated MRSA.
    • Prevention and treatment is needed!
  • Antimicrobial peptides (AMPs) - novel antibiotic --> could be developed to combat bacteria that is resistant (such as MRSA).
    • Produces by all living creatures for defenses -- 880 have been described.
  • Renalexin
    • 20 a.a. peptide (cationic)
    • Possesses single intramolecular dispulphide bond --> forming heptapeptide ring at carboxyl terminus.
    • Displays strong activity against Staphylococcus aureus (Gram-positive bacteria).
    • Offers potential against staphylococcal infections (including MRSA).
  • Need to understand antimicrobial action mechanisms to develop strategies.
  • Antimicrobial inhibitory action can by studied by transcriptome and proteome profiling.
  • Generation of protein and mRNA in response to antimicrobial stress are shown in certain cell functions and provide stress type imposed.
  • Modeled pathway relationships for 95% of S. aureus MRSA-252 genes by integrating proteome and transcriptome profiling of bacteria that was drug-exposed w/ high-confidence functional association network.
    • Approach showed 22 virulence factors and killing mechs. for renalexin along with cell wall effects.
    • Evidence of VraRS two-component system in cationic peptide resistance.
  • Drug target candidate - FtsH. Role of PhoU-mediated persister formation in S. aureus drug tolerance.

Results and Discussion

Renalexin elicits significant changes in transcript and protein levels

  • Sublethal ranalexin impaired but didn't abolish growth of MRSA-252 at concentration of 20μg/ml.
  • Proteome and transcriptome profiling applied between control and renalexin exposed MRSA-252 cultures. Microarrays showed:
    • 93 upregulated genes.
    • 105 downregulated genes.
      • No inconsistencies shown
  • Gene Ontology (GO) --> only few cases of overlap and not uncommon. Identified 290 enriched terms --> highlighting effects of renalexin on MRSA.

Global gene functional association network

  • Developed to give probabilistic model of global gene fn. and lay framework for renalexin response profiles.
  • MRSA-252 genes were network nodes and cell signalling and metabolism were the connections (edges) between genes (nodes).
  • 2494 nodes (genes) and 19076 edges (connections) were contained in final network. Network edges rate was 94.5% of MRSA-252 genes.
  • Hierarchical structure w/ embedded modularity are denoted from node pair degree connectivity along with network degree clustering coefficient distributions.
  • When compared to protein interaction networks, gene functional association is closer to metabolic networks.
    • Seems reasonable because gene interactions are shared amongst all members of a functional group.
  • Network was clustered into 597 putative functional modules --> transcriptome and proteome were mapped into these --> 11 modules had rich genes that showed altered expression in MRSA-252 cultures that were exposed to renalexin.
    • Of theses 11 --> 5 upregulated and 6 downregulated.
    • 58 nodes classified as intermodular hubs that are important regulators of system behavior.

Impact on virulence and inference of novel determinants

  • Upon renalexin exposure(RanaDown), genes significantly downregulated that included all 6 MRSA-353 ESAT-6 secretion system components --> central to Staphylococcus aureus pathogenesis.
  • Significant module included five ESAT-6 components; sixth gene (esaA, SAR0280) not assigned to module. 2 RanaDown genes (SAR0287, SAR0288) - uncharacterized hypothetical proteins.
  • Six transmembrane regions predicted from SAR0280 gene --> 'membrane ABC permease' match found.
  • Predicted that SAR0287 was cell wall anchored or secreted --> also matched conserved domains of protein families and virulence-associated families.
  • Other five genes on module matched conserved domains of unknown fn.
  • TMHMM2 showed one transmembrane protein and two secreted/cell wall.
  • Module showed good correspondence w/ operon structure --> 7 hypothetical genes may be co-regulated with the ESAT-6 system.
  • Two RanaDown modules (4/5 RanaDown; 4/9 RanaDown) had high-affinity metal ion transport --> crucial for establishment of infection.
    • 4/5 RanaDown genes included SAR0787-SAR0790 --> displaying the sst iron-uptake. 5 gene was binding protein for iron complex transport.
  • 12 genes associated with virulence function found in another module (4/16 nodes RanaDown).
    • 12 implicated Colonization and immuno-modulation.
    • Remaining 4 genes: SAR1489, SAR2421, SAR1223, and SAR1802.
    • All 16 genes encode transmembrane/secreted proteins that are anchored on the cell wall.
  • 'Pathogenesis' was significant for this module.



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