Alex A. Cardenas Week 11

Final Presentation

• Working with both Zeb and Bobby for the final project.
• We are looking at and research Staphylococcus aureus.
  • The microarray data we will be using is on this article. [Found here].
  • Microarray data article can be found [here].

Article Outline

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. Virulence-coupled autolysis for SAR2421, SAR1223, and SAR1802.
• Transmembrane protein that binds tropoelastin --> SAR1489
• Two secreted/cell wall proteins and one transmembrane --> TMHMM2.
• GO annotations showed strongly downregulated gene sets.
• Showed that ranalexin results in repression of virulence factor expression --> potential clinical application benefits.
• Methods predicted additional S. aureus virulence factors and a total of 10 modules were significant.
  • ESAT-6 & colonization-associated modules included.
• S. aureus MRSA-252 pathogenicity (along with ESAT-6 system and 22 virulence factors) are restricted by ranalexin.

Ranalexin induces cell wall stress

• VraSR -- controls expression of genes mainly for cell wall biosynthesis. Genes reulated by this were found to be RanaUp --> SAR1461, SAR1964, SAR1030 and SAR 2442.
• FtsH - RanaUp protein that had highest betweeness centrality in the network. --> key role in regulation of cell wall behavior.
• FtsH - key player in MRSA response to antimicrobials (ranalexin) --> potential drug target in combination of cell wall agents.
• Largest ranalexin dependent induction of transcription -- SAR0584.
• Transcriptional regulatory proteins that were RanaUp genes inlcluded SAR1689 and SAR0625. --> induced in the presence of cell wall active antibiotics.
• Aspartate family amino acid biosynthesis --> supports upregulation of cell wall anabolism .
• Cell wall stress response was induced by exposure to ranalexin.
  • Associated with cell wall active antibiotics or GISA strains.

Further investigation of ranalexin cell wall effects

• vraR and tcaA disruption mutants and examination of peptide sensitivity was investigated.
• Enhanced production of craR and tcaA was observed in ranalexin exposure.
• Dependent induction of expression was observed after 15 min exposure that peaked after 30 mind and declined after 60 min.
• Genes from MRSA-252 were identified in RN4220 and disrupted.
• Dose responses showed viability loss from vraR mutant and increasing concentrations and duration of exposure when compared to parent stains.
• Results show that ranalexin induces cell wall stress.
• Cell wall effects of ranalexin were examined by measuring osmotic fragility for MRSA cells exposed to concs. of antimicrobials.
• Pre-treated cells with a sub-lethal conc. of vancomycin were sensitieve and sub-lethal conc. of ranalexin showed induced sensitivity to hypo-osmotic stress. And when treated with both, they showed a similar degree of osmotic fragility.
• Ranalexins inhibitory action involves effects at staphylococcal cell wall and not only due to membrane disruption.

MRSA persister formation and drug tolerance

• Strong upregulation of proteins encoded by pstSCAB-phoU operon PstS, PstC, PstA, PhoU, and PstB when exposed to ranalexin.
• Impacts on processes beyond Pi transport --> PhoU.
• MRSA adopts a PhoU-mediated persister phenotype to gain antimicrobial tolerance.
• Cell division proteins that were RanaDown - SAR0017, SAR1047, SAR2000, SAR1180, SAR1040 --> candidates for negative regulation by PhoU in MRSA-252.

Multiple actions in MRSA killing

• Cation antiport --> significantly upregulated.
• Major effect of ranalexin exposure --> membrane permeabillisation leading to cation influx and dissipation of transmembrane electrochemical gradient.
• opuC was contained in significant module and osmoprotectant transporters --> strongly depended on oB induction.
  • Upregulation of these transporters is probably a product of oB induction by influx of cations.
• Actions of ranalexin --> may show effects in killing S. aureus MRSA-252.
• Response included upregulation of dlt operton --> leads to increased + charge at cell wall surface and decreased peptide binding.
• A module showed cahperone proteins and stress response genes.
• Cell wall effects, increased influx of cations and possible transmembrtane electrochemical gradient dissipation --> multiple inhibitory actions of ranalexin.
• When exposed to ranalexin, MRSA tries to make survival strategies --> but it was found that there were inhibitory actions in MRSA killing.

Conclusions

• Evidence showed effects of ranalexin 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 - shows the sublethal effects that ranalexin has of samples of MRSA-252 growth.
• Figure 2- shows the network degree pairs probability. It shows different genes interacting on the MRSA network. Low degree values are shown in the bottom left and high degree values are shown in the top right.
• Table 1 - this table just shows the different responses to ranalexin and the genes that are altered in relation to the description of what is occuring.
• Figure 3 - shows genes that are downregulated when exposed to ranalexin in the ESAT-6 module. Pink are genes that are significantly downregulated and yellow are others.
• Table 2 - this table shows genes that are associated with pathogenesis and the summary of the different mechanisms that are occurring or what part of the cell function is being disrupted.
• Figure 4 - this figure shows the upregulation of vraR and tcaA when exposed to ranalexin. It shows the time since exposure of ranalexin and a control is in place for reference.
• Figure 5 - hypo osmotic stress is shown in the figure in relationship to ranalexin exposure. Controls were used and there were different solutions being experimented on. There was 2 different concentrations of ranlexin, vancomycin, and ranalexin plus vancomycin were observed.

10 Biological Terms

1. Virulence - the degree or ability of a pathogenic organism to cause disease. [Biology Online] 09 November 2011
2. Putative Substrate - Commonly thought or deemed; supposed; reputed. [Biology Online] 09 November 2011
3. Cationic - referring to positively charged ions and their properties. [Biology Online] 09 November 2011
4. 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. [Biology Online] 09 November 2011
5. Autolysis - breakdown of whole cell or tissue by self produced enzymes. [Biology Online] 09 November 2011
6. Staphylococcus aureus - genus of nonmotile gram-positive bacteria that are found in clusters and that produce important exotoxins. Staphylococcus aureus (Staphylococcus pyogenes) is pyogenic, an opportunistic pathogen and responsible for a range of infections including severe sepsis, pneumonia, endocarditis and soft tissue infections. It has protein A on the surface of the cell wall. Coagulase production correlates with virulence: hyaluronidase, lipase and staphylokinase are released in addition to the toxins. [Biology Online] 09 November 2011
7. Antimicrobia - a drug for killing microorganisms or suppressing their multiplication or growth. An agent (as heat or radiation or a chemical) that destroys microorganisms that might carry disease.An agent (as heat or radiation or a chemical) that destroys microorganisms that might carry disease.[Biology Online] 09 November 2011
8. Chaperones - cytoplasmic proteins of both prokaryotes and eukaryotes that bind to nascent or unfolded polypeptides and ensure correct folding or transport. Chaperone proteins do not covalently bind to their targets and do not form part of the finished product. Heat-shock proteins are an important sub set of chaperones. [Biology Online] 09 November 2011
9. Pathogen - An agent causing disease or illness to its host, such as an organism or infectious particle capable of producing a disease in another organism. [Biology Online] 15 November 2011
10. Vancomycin- an antibiotic drug used to fight resistant bacterial infection. [Cancer.Gov] 09 November 2009

Powerpoint Presentation

• First half of powerpoint can be found here.
• Bobby and Alex portion of powerpoint.

Links

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