BACKGROUND
methicillin resistant staphylococcus aureus  is a major cause of morbidity and mortality  <cit> .
indeed, rates of mrsa infections have risen significantly in recent years  <cit> .
strains that are resistant to existing treatments continue to emerge and community-associated mrsa is a major global problem  <cit> .
therefore, the development of novel prevention and treatment strategies is a pressing concern.
antimicrobial peptides  are a potential source of novel antibiotics that may be developed to combat resistant bacteria such as mrsa  <cit> .
amps are produced by virtually all living creatures as part of their innate defences and more than  <dig> have been described  <cit> .
ranalexin is a cationic  <dig> amino acid peptide, first isolated from the american bullfrog, rana catesbeiana, and has a single intramolecular disulphide bond to form a heptapeptide ring at the carboxyl terminus  <cit> .
ranalexin has potent activity against gram-positive bacteria in vitro, particularly staphylococcus aureus  <cit> .
therefore, ranalexin offers therapeutic potential against staphylococcal infections, including mrsa.
understanding the molecular mechanisms of antimicrobial action is an important facet of developing new therapeutic strategies, particularly where drug resistance is a problem  <cit> .
transcriptome and proteome profiling offers a powerful approach for studying antimicrobial inhibitory action  <cit> .
in principle, the mrna and protein profiles generated in response to the imposition of antimicrobial stress reflect modulation of particular cellular functions, and provide a signature of the type of stress imposed.
for example, expression profiling has been applied to predict mode of action  <cit>  and to identify molecular targets of uncharacterised antibiotics  <cit> .
a network biology approach in combination with expression profiling enables systems analysis of drug mode of action, for example with networks of drug interactions  <cit>  or gene regulation  <cit> ; for a recent review see  <cit> .
we took a systems-wide approach to integrate transcriptome and proteome profiling of drug-exposed bacteria with a high-confidence functional association network  <cit>  that modelled pathway relationships for 95% of s. aureus mrsa- <dig> genes.
mrsa- <dig> is an isolate of one of the most prevalent epidemic mrsa clones, emrsa- <dig>  <cit> .
this approach enabled inference of twenty-two novel mrsa virulence factors and novel complementary killing mechanisms for the antimicrobial peptide ranalexin, including effects at the cell wall.
we also found evidence supporting involvement of the vrars two-component system in cationic peptide resistance.
furthermore, ftsh was proposed as a candidate drug target, and a role was inferred for phou-mediated persister formation in s. aureus drug tolerance.
these results demonstrate the power of this global functional association network approach to study drug resistance and mode of action.
RESULTS
ranalexin elicits significant changes in transcript and protein levels
we determined a sublethal ranalexin concentration  that impaired, but did not abolish growth of mrsa- <dig> .
transcriptome and proteome profiling were applied to identify changes in the ranalexin exposed mrsa- <dig> cultures compared to controls .
microarrays identified  <dig> upregulated and  <dig> downregulated genes , while itraq lc-ms/ms coupled with proquant  analysis identified  <dig> upregulated and  <dig> downregulated proteins by stringent criteria .
no inconsistencies were observed between the transcriptome and proteome profiles.
overlap in gene ontology   <cit>  annotation was observed for these datasets although there were only a few cases of direct overlap at the gene level.
this is not uncommon for the integration of proteome and transcriptome data  <cit> .
go-based functional profiling of these transcripts and proteins identified  <dig> significantly enriched terms , underlining the multi-faceted effects of ranalexin on mrsa.
global gene functional association network
a functional association network  <cit>  was developed in order to give a probabilistic model of global gene function in mrsa- <dig>  and to provide a framework for systems-wide analysis of the ranalexin response profiles.
the uniprot  <cit>  mrsa- <dig> genes were network nodes, while connections  between genes  represented relationships in cell signalling and metabolism.
details of network construction and evaluation are given in methods.
briefly, edges were generated using a bayesian logistic regression approach, which integrated information from gene ontology  <cit>  coannotations, string scores  <cit>  and kegg  <cit>  pathways .
the final network contained  <dig> nodes  and  <dig> edges , with a false positive rate  no greater than 3% over several independent test datasets .
interestingly, these fpr values are similar to the estimated value of the functional association prior , which suggests that a significant fraction of the 'false positives' may be genuine functional relationships that were not annotated in the test data.
overall, the network edges relate  <dig> % of mrsa- <dig> genes with high confidence.
the node pair degree connectivity , as well as the network degree and clustering coefficient distributions , denote hierarchical structure with embedded modularity that has been previously observed for metabolic networks  <cit> .
indeed, the gene functional association network topology is closer to that of metabolic networks, rather than protein interaction networks which are less modular  <cit> .
this result seems intuitively reasonable because gene  interactions in functional association networks are expected to be shared amongst all the members of a functional grouping, such as a signalling pathway, which would contribute to modular topology.
therefore, the s. aureus mrsa- <dig> gene association network structure fits well with further analysis based on module decomposition.
the network was clustered into  <dig> putative functional modules using the mcl algorithm  <cit> .
the transcriptome and proteome profiles were mapped into these modules, and significance was assessed against background distributions generated by network re-sampling .
a total of eleven modules were found to be enriched with genes that displayed significantly altered expression in mrsa- <dig> cultures exposed to ranalexin.
of the eleven modules, five were upregulated and six downregulated; these results are summarised in table  <dig>  a total of  <dig> nodes outside these modules were classified as intermodular hubs , which link to multiple highly interconnected subnetworks and are putatively important regulators of system behaviour  <cit> .
a prefix '+' in the first column indicates modules with upregulated genes, a prefix '-' downregulated genes.
*no instances of these module coverage levels were observed in the appropriate random sampling.
therefore the background frequency was set to 1/ <dig> for estimation of p-values.
impact on virulence and inference of novel determinants
the genes significantly downregulated upon ranalexin exposure  included all six of the mrsa- <dig> esat- <dig> secretion system components , which are central to staphylococcus aureus pathogenesis  <cit> .
a highly significant module  included five esat- <dig> components; the sixth mrsa- <dig> esat- <dig> gene  was not assigned to a module but was ranadown and shared edges with 10/ <dig> genes in the module.
the two further ranadown genes in this module  were relatively uncharacterised 'hypothetical' proteins  <cit> .
analysis of sar <dig> predicted six transmembrane regions  <cit>  and found a match to the 'membrane abc permease' domain pd <dig>  <cit> ; sar <dig> was predicted to be secreted or cell wall anchored  <cit> , and matched to several protein families including conserved domains of unknown function , plus virulence-associated families   <cit> .
these results agree with the network-inferred virulence function for sar <dig> and sar <dig>  the remaining five genes in the module were also hypothetical, while all five matched to conserved domains of unknown function  and virulence-associated families   <cit> .
consistent with roles in virulence for these remaining five genes, tmhmm <dig>  <cit>  indicated two secreted/cell wall and one transmembrane protein, while predictions were not clear in two cases.
the module had good correspondence with predicted operon structure  <cit> , implying that the seven hypothetical genes may be co-regulated with the esat- <dig> system.
the go term 'pathogenesis'  was significant for this module .
therefore, seven novel s. aureus  virulence factors were inferred.
modules with false discovery rate corrected fisher p-values ≤  <dig>  and size > <dig> are given.
all modules were manually inspected with reference to literature and relevant databases .
underlined locus identifiers were downregulated in response to ranalexin, none of the below loci were upregulated.
locus identifiers in bold are candidate novel virulence factors.
many genes that were not annotated with 'pathogenesis' in the gene ontology nevertheless had some association with virulence in the literature, and therefore are not indicated as candidate novel virulence factors in the table.
modules listed first 'colonization, immunomodulation' and fifth 'esat- <dig> system' were also significant for the ranalexin downregulated gene set .
two significant ranadown modules  were associated with high-affinity metal ion transport  <cit> , which is crucial for establishment of infection  <cit> .
the module with 4/ <dig> ranadown genes included sar0787-sar <dig>  representing the sst iron-uptake operon  <cit> .
the fifth gene in this module  was a putative substrate binding protein for iron complex transport  <cit> , which agrees with the functional relationship to the sst operon inferred from the network.
a further module  contained twelve genes with annotated virulence functions.
these twelve genes were largely implicated in colonization  and immuno-modulation   <cit> .
the remaining four genes in this module encoded ebps , a putative lyra protease , a putative lytn cell wall hydrolase , and a conserved hypothetical protein  matching to the pfam family 'hydrolase'  <cit> .
indeed, coordination of peptidoglycan hydrolase and virulence determinant expression has been observed in s. aureus  <cit> .
all sixteen genes were known or predicted to encode cell wall anchored, transmembrane or secreted proteins  <cit> .
the go term 'pathogenesis'  was significant for the module .
these data imply functions in virulence-coupled autolysis for sar <dig> , sar <dig>  and sar <dig> .
ebps  is a transmembrane protein that binds  tropoelastin, and not associated with adhesion  <cit> .
ebps function is not well defined, however involvement in virulence is suggested by the network structure.
a fully connected ranadown module  was composed of relatively uncharacterised, 'hypothetical' genes .
interestingly, the only edge from this module into the rest of the network connected with the esat- <dig> virulence module described above .
consistent with the network-based inference of virulence functions for these three genes, tmhmm <dig> indicated one transmembrane and two secreted/cell wall proteins, while database matches included conserved domains of unknown function  and rtx toxin family   <cit> .
additionally, the most significantly enriched go term in the ranalexin downregulated gene set was 'pathogenesis'  .
many genes annotated with this go term were strongly downregulated, for example: sar <dig> ; sar <dig> ; sar <dig> .
indeed, several agr locus genes additional to hld were ranadown .
ranalexin dependent repression of virulence genes agrees with the upregulation of sara attenuator sar <dig> , which inhibits sara-dependent virulence gene expression  <cit> .
furthermore, two ranadown genes  encoded putative hth-type transcriptional regulators  from the sara family, that activate virulence factor transcription  <cit> .
there is evidence linking traditional antibiotics to reduction of s. aureus virulence , however this had not previously been reported for a cationic amp.
the observation that ranalexin results in repression of virulence factor expression has clear beneficial implications for potential clinical applications .
prediction of additional s. aureus virulence factors was enabled by mapping go annotations for 'pathogenesis'  into the network .
a total of ten modules were significant , and were manually inspected .
these ten included the esat- <dig> and colonization-associated modules discussed above.
interestingly a significant module  had  <dig> known two-component sensors or regulators, and a further four poorly characterised genes which may form novel virulence-associated two-component systems.
manual inspection of these modules, with reference to literature and electronic resources , indicated eight genes not previously annotated as virulence factors .
in summary, the above analysis indicated that ranalexin restricts s. aureus mrsa- <dig> pathogenicity, including the esat- <dig> system, and inferred twenty-two novel virulence factors.
ranalexin induces cell wall stress
the response to ranalexin included strong upregulation of several peptidoglycan synthesis genes, which suggests that ranalexin may act on the cell wall .
indeed, the gene ontology  term 'peptidoglycan-based cell wall'  was significantly enriched in genes upregulated by ranalexin  .
the vrar  regulator protein of the vancomycin-resistance associated two-component system  was ranaup .
vrasr controls the expression of genes that are induced by vancomycin, many of which function in cell wall biosynthesis  <cit> .
several genes regulated by vrasr were found to be ranaup, including sar <dig> , sar <dig> , sar <dig>  and sar <dig> .
the above genes include members of a general cell wall stimulon induced in response to cell wall active agents  <cit> .
moreover, vrasr is upregulated in vancomycin-intermediate strains of s. aureus and induced by exposure to cell wall active antibiotics such as glycopeptides and β-lactams  <cit> .
a further ranaup protein, ftsh , had the highest betweenness centrality in the network, degree of  <dig> , and did not fall into a module.
therefore ftsh is an intermodular hub, which implies a key role in regulating system behaviour  <cit> .
indeed, ftsh is within the aaa  family and acts as a chaperone required for incorporation of penicillin binding proteins  into the cell membrane  <cit> .
as noted above, pbp <dig> was ranaup and is part of a cell wall stimulon, while pbps are upregulated in vancomycin-intermediate s. aureus   <cit> .
ftsh is also involved in response to osmotic stress and mutants are non-viable or have significantly attenuated virulence  <cit> .
considering the above, ftsh is proposed to be a key player in the mrsa response to antimicrobials such as ranalexin and a potential drug target.
indeed, these results suggest that a drug targeting ftsh would be particularly effective in combination with cell wall active agents.
the upregulation of ftsh in concert with the cell wall stimulon further supports action of ranalexin at the cell wall.
the largest ranalexin dependent induction of transcription was observed for sar <dig> , which is not well characterised.
however, vrax was over-expressed in isolates of vancomycin-intermediate s. aureus  and showed >200-fold increased expression in vancomycin-sensitive s. aureus  treated with vancomycin  <cit> .
overall, these data indicate that ranalexin caused cell wall stress similar to that produced by vancomycin - a cell wall active antibiotic.
ranaup genes also included transcriptional regulatory proteins encoded by sar <dig>  and sar <dig>  that are known to be induced in the presence of cell wall active antibiotics  <cit> .
a transcriptional attenuator of sara, sar <dig> , was induced by ranalexin.
significantly, msrr is induced upon exposure to cell wall active antibiotics, while its deletion results in sensitivity to methicillin and teicoplanin  <cit> .
an autolysis-deficient, teicoplanin-resistant strain of mrsa was found to upregulate fmta, sara, tcaa, msrr, vrar plus a large number of proteases  <cit> .
as noted above, these genes were found to be ranaup.
indeed, inactivation of mrsa methicillin resistance genes has been reported to increase susceptibility to the β-defensin and ll <dig> amps  <cit> .
evidence supporting upregulation of cell wall anabolism also included enrichment of the go term 'aspartate family amino acid biosynthesis' in the ranaup set .
aspartate metabolism produces essential components for pepdidoglycan biosynthesis, such as diaminopimelic acid, and components of the aspartate pathway represent possible drug targets  <cit> .
in summary, the above results suggest that ranalexin exposure induced a cell wall stress response, similar to that associated with cell wall active antibiotics or glycopeptide intermediate-resistant s. aureus  strains.
follow-up laboratory experiments were performed to further investigate these findings .
further investigation of ranalexin cell wall effects
the physiological relevance of ranalexin induced cell wall related gene and protein expression changes was investigated by producing vrar and tcaa disruption mutants and subsequent examination for peptide sensitivity.
prior to making mutants, ranalexin induced enhanced expression was verified by quantitative pcr for vrar  and tcaa  .
in both cases, we again detected ranalexin dependent induction of expression after  <dig> minute  exposure that peaked after  <dig> min and subsequently declined after  <dig> min.
the sigma targetron system was applied to generate disruption mutants, for which the s. aureus laboratory strain rn <dig> was recommended by the manufacturer.
thus, the corresponding genes from mrsa- <dig> were identified in rn <dig> and disrupted.
vrar disruption induced sensitivity to both ranalexin  and vancomycin , compared to the parent strain.
complementation experiments were attempted, but were inconclusive due to phenotypic effects induced by the empty cloning vector.
growth of the vrar mutant was identical to the parent strain in the absence of ranalexin, but was completely inhibited when ranalexin was added .
similarly, dose response experiments revealed dramatic viability loss for the vrar mutant both to increasing concentrations  and to increased duration of exposure  - contrasting with the parent strain which retained viability .
considering that the vrars two-component system regulates a response to cell wall damage  <cit> , these results support the view that ranalexin induces cell wall stress.
tcaa disruption resulted in minor sensitivity to ranalexin .
no significant differences in rate of loss of viability between the tcaa mutant and the parent strain exposed to ranalexin were detected .
tcaa is a member of the cell wall stress stimulon known to be induced by exposure to antibiotics in a vrars dependent fashion  <cit> .
therefore, these results suggest that vrars may be a staphylococcal two-component response regulator involved in cationic peptide resistance.
in s. aureus, vrars regulates the transcriptional induction of genes involved in cell wall peptidoglycan biosynthesis and is crucial for resistance to cell wall active antibiotics such as the glycopeptides vancomycin and teicoplanin  <cit> .
therefore, these results support the view that ranalexin's mode action includes disruption of the staphylococcal cell wall.
to further examine ranalexin cell wall effects, osmotic fragility was measured for mrsa cells exposed to sub-lethal concentrations of antimicrobials .
osmotic fragility is a hallmark of cell wall disruption  <cit> .
cultures were incubated in tsb, in the presence and absence of vancomycin or ranalexin, harvested and washed twice in sterile distilled water, followed by incubation in water for  <dig> min.
viability was determined throughout the experiment.
control cells  and vrar-disrupted cells showed negligible loss of viability or sensitivity to hypo-osmotic stress.
as expected, cells pre-treated with a sub-lethal concentration of vancomycin were sensitive.
notably, pre-treatment with a sub-lethal concentration of ranalexin also induced sensitivity to hypo-osmotic stress .
furthermore, exposure to both ranalexin and vancomycin induced a similar degree of osmotic fragility compared to treatment with ranalexin; suggesting commonalities in modes of action.
coupled with the induction of increased cell wall related gene/protein expression and hypersensitivity produced by vrar disruption, these results imply that the inhibitory action of ranalexin is not solely due to membrane disruption  <cit> , but also involves significant effects at the staphylococcal cell wall.
mrsa persister formation and drug tolerance
exposure to ranalexin was found to produce strong upregulation of proteins encoded by the pstscab-phou operon psts , pstc , psta , phou  and pstb  .
four of these  formed a significant network module .
sar <dig> did not fall into a module but had connections to all the above genes.
phou acts as a persister switch in e. coli multidrug tolerance and impacts on widespread processes beyond inorganic phosphate  transport  <cit> .
genetic disruption of the pstc transmembrane component of the pi transporter with the sigma targetron system did not have any effect on ranalexin sensitivity .
combined with the proteome profiling and network analysis this data suggests that mrsa adopts a phou-mediated persister phenotype to acquire antimicrobial tolerance, and that upregulation of the pi transporter is not a major component of this response.
indeed, the pstscab-phou operon was induced by streptococcus pneumoniae exposure to penicillin  <cit> , and targeted disruption correlated with increased sensitivity to ciprofloxacin in mycobacterium smegmatis  <cit> .
a pyrimidine metabolism module was ranadown  reflecting growth inhibition that is a hallmark of persister formation  <cit> .
additionally, several other cell division proteins were ranadown, including sar <dig> , sar <dig> , sar <dig> , sar <dig> , and the transcript sar <dig>  was ranadown.
these genes  are therefore tentative candidates for negative regulation by phou in mrsa- <dig>  the results presented in earlier sections, finding ranalexin exposure upregulated cell wall anabolism and reduced virulence, also align with persister formation.
indeed, persister bacteria exhibit thickening of the cell wall and loss of virulence factors  <cit> .
these findings underline the importance of persister formation in mrsa drug tolerance.
multiple actions in mrsa killing
ranalexin is canonically associated with cell membrane permeabilisation  <cit> .
in agreement with this, a cation antiport module was significant upregulated .
the ranaup genes in this module were subunits a-f  of the na+/h+ antiporter encoded by the mnh <dig> operon, sar <dig>  and subunit g  of the na+/h+ antiporter encoded by the mnh operon  <cit> .
the module also included subunits a-f from the mnh operon, and three hypothetical proteins .
the upregulation of these 'electrogenic monovalent cation proton antiporter-3' operons  <cit>  implies that membrane permeabilisation leading to cation influx and possible dissipation of transmembane electrochemical gradient is a major effect of ranalexin exposure.
the mnh and mnh <dig> operons are important for na+-dependent ph homeostasis in b. subtilis and are induced by alternative sigma factor b  in s. aureus  <cit> .
further, the go term 'ion transport'  was significantly enriched with upregulated genes.
additional to the above genes, these included sar <dig> , encoding a probable cadmium-effluxing p-type atpase that confers resistance to cd2+ toxicity  <cit> ; sar <dig>  encoding a putative na+/pi-cotransporter; sar <dig> , encoding a putative tetracycline, k+ or na+/h+ antiporter  <cit> ; and sar <dig> , encoding a regulatory transcription factor induced in response to zn2+ stress  <cit> .
a significant module  contained the opuc osmoprotectant transport operon ; osmoprotectant transporters are thought to be strongly dependent on σb induction  <cit> .
upregulation of osmoprotectant transport is likely a product of σb induction by cation influx, especially given the observed upregulation of the mnh and mnh <dig> operons.
interestingly, increased osmolarity would be expected to act in concert with the ranalexin cell wall effects presented above in producing hypo-osmotic fragility and disruption of cell integrity.
therefore the membrane and cell wall actions of ranalexin may exert complementary effects in killing s. aureus mrsa- <dig>
the response signature included upregulation of the dlt operon d-alanine and d-alanyl lipoteichoic acid synthesis, which leads to increased positive charge at the cell surface and reduced peptide binding  <cit> .
consistent with the expected ranalexin induced stress, a significant module  contained chaperones and stress response genes.
also, the chaperone proteins groel  and ftsh  were ranaup and intermodular hubs  <cit> .
another significant module  represented genes for val/leu/ile synthesis , which utilizes pyruvate  <cit> , and therefore may allow for greater glycolytic flux in powering ranalexin stressed cells.
indeed, several glucose catabolism and uptake proteins were upregulated, including eiia-glc , pgk  an intermodular hub, and pgi  substantiating upregulation of glycolysis upon ranalexin exposure.
interestingly, l-ldh <dig>  was ranadown; in conjunction with upregulation of val/leu/ile synthesis and glycolysis this suggests repurposing of pyruvate into production of hydrophobic proteins for peptide sequestration  <cit> .
these results, and those presented in above sections, detail several lines of defence employed by mrsa to escape the effects of cationic amps.
in summary, evidence supports multiple inhibitory actions for ranalexin including cell wall effects, increased cation influx and possible dissipation of transmembrane electrochemical gradient.
mrsa attempts several survival strategies when faced with antimicrobial peptide  stress, and evidence was found for complementary inhibitory actions in killing mrsa.
CONCLUSIONS
a global gene functional association network that covers 95% of s. aureus mrsa- <dig> genes was presented.
careful benchmarking against blind test datasets found a false positive rate no greater than 3% in the final network.
functional network analysis of drug response signatures enabled novel insights into the mode of action of a cationic antimicrobial peptide , ranalexin, as well as insights into mrsa- <dig> antimicrobial resistance mechanisms.
evidence was presented for novel ranalexin effects on the bacterial cell wall, in addition to the previously characterised action at the cell membrane  <cit> , and these actions were proposed to act in concert.
these findings agree with previous observations that cationic amps exert complex inhibitory actions  <cit> .
the ftsh membrane chaperone was an intermodular hub  <cit> , upregulated in response to ranalexin and suggested as a promising candidate drug target, particularly for combination therapy with cell wall active agents.
indeed, combination therapy has clear advantages for treatment of resistant bacteria  <cit> .
additionally, the two-component response regulator vrar was suggested to be important for mediating tolerance to antimicrobial peptides.
therefore, vrars may be a staphylococcal two-component response regulator involved in cationic peptide resistance, in addition to apsrs  <cit> .
ranalexin exposure produced mrsa- <dig> virulence reduction, and twenty-two novel virulence factors were inferred.
these novel roles in virulence are supported by several additional lines of evidence, including predicted cellular localisation  <cit> , domain matching  <cit>  and operon structure  <cit> .
finally, we present evidence to support phou-mediated persister switching as a mechanism of drug tolerance in mrsa; which merits further investigation as a route towards novel therapeutic targets.
this work is informative to the development of therapeutic strategies against s. aureus, and demonstrates an elegant approach to study drug resistance and mode of action.

 methicillin resistant staphylococcus aureus is a major cause of morbidity and mortality. indeed, rates of mrsa infections have risen significantly in recent years. strains that are resistant to existing treatments continue to emerge and community-associated mrsa is a major global problem. therefore, the development of novel prevention and treatment strategies is a pressing concern. antimicrobial peptides are a potential source of novel antibiotics that may be developed to combat resistant bacteria such as mrsa. amps are produced by virtually all living creatures as part of their innate defences and more than have been described. ranalexin is a cationic amino acid peptide, first isolated from the american bullfrog, rana catesbeiana, and has a single intramolecular disulphide bond to form a heptapeptide ring at the carboxyl terminus. ranalexin has potent activity against gram-positive bacteria in vitro, particularly staphylococcus aureus. therefore, ranalexin offers therapeutic potential against staphylococcal infections, including mrsa. understanding the molecular mechanisms of antimicrobial action is an important facet of developing new therapeutic strategies, particularly where drug resistance is a problem. transcriptome and proteome profiling offers a powerful approach for studying antimicrobial inhibitory action. in principle, the mrna and protein profiles generated in response to the imposition of antimicrobial stress reflect modulation of particular cellular functions, and provide a signature of the type of stress imposed. for example, expression profiling has been applied to predict mode of action and to identify molecular targets of uncharacterised antibiotics. a network biology approach in combination with expression profiling enables systems analysis of drug mode of action, for example with networks of drug interactions or gene regulation; for a recent review see. we took a systems-wide approach to integrate transcriptome and proteome profiling of drug-exposed bacteria with a high-confidence functional association network that modelled pathway relationships for 95% of s. aureus mrsa- genes. mrsa- is an isolate of one of the most prevalent epidemic mrsa clones, emrsa-. this approach enabled inference of twenty-two novel mrsa virulence factors and novel complementary killing mechanisms for the antimicrobial peptide ranalexin, including effects at the cell wall. we also found evidence supporting involvement of the vrars two-component system in cationic peptide resistance. furthermore, ftsh was proposed as a candidate drug target, and a role was inferred for phou-mediated persister formation in s. aureus drug tolerance. these results demonstrate the power of this global functional association network approach to study drug resistance and mode of action.  ranalexin elicits significant changes in transcript and protein levels we determined a sublethal ranalexin concentration that impaired, but did not abolish growth of mrsa-. transcriptome and proteome profiling were applied to identify changes in the ranalexin exposed mrsa- cultures compared to controls. microarrays identified upregulated and downregulated genes, while itraq lc-ms/ms coupled with proquant analysis identified upregulated and downregulated proteins by stringent criteria. no inconsistencies were observed between the transcriptome and proteome profiles. overlap in gene ontology annotation was observed for these datasets although there were only a few cases of direct overlap at the gene level. this is not uncommon for the integration of proteome and transcriptome data. go-based functional profiling of these transcripts and proteins identified significantly enriched terms, underlining the multi-faceted effects of ranalexin on mrsa. global gene functional association network a functional association network was developed in order to give a probabilistic model of global gene function in mrsa- and to provide a framework for systems-wide analysis of the ranalexin response profiles. the uniprot mrsa- genes were network nodes, while connections between genes represented relationships in cell signalling and metabolism. details of network construction and evaluation are given in methods. briefly, edges were generated using a bayesian logistic regression approach, which integrated information from gene ontology coannotations, string scores and kegg pathways. the final network contained nodes and edges, with a false positive rate no greater than 3% over several independent test datasets. interestingly, these fpr values are similar to the estimated value of the functional association prior, which suggests that a significant fraction of the'false positives' may be genuine functional relationships that were not annotated in the test data. overall, the network edges relate % of mrsa- genes with high confidence. the node pair degree connectivity, as well as the network degree and clustering coefficient distributions, denote hierarchical structure with embedded modularity that has been previously observed for metabolic networks. indeed, the gene functional association network topology is closer to that of metabolic networks, rather than protein interaction networks which are less modular. this result seems intuitively reasonable because gene interactions in functional association networks are expected to be shared amongst all the members of a functional grouping, such as a signalling pathway, which would contribute to modular topology. therefore, the s. aureus mrsa- gene association network structure fits well with further analysis based on module decomposition. the network was clustered into putative functional modules using the mcl algorithm. the transcriptome and proteome profiles were mapped into these modules, and significance was assessed against background distributions generated by network re-sampling. a total of eleven modules were found to be enriched with genes that displayed significantly altered expression in mrsa- cultures exposed to ranalexin. of the eleven modules, five were upregulated and six downregulated; these results are summarised in table a total of nodes outside these modules were classified as intermodular hubs, which link to multiple highly interconnected subnetworks and are putatively important regulators of system behaviour. a prefix'+' in the first column indicates modules with upregulated genes, a prefix'-' downregulated genes. *no instances of these module coverage levels were observed in the appropriate random sampling. therefore the background frequency was set to 1/ for estimation of p-values. impact on virulence and inference of novel determinants the genes significantly downregulated upon ranalexin exposure included all six of the mrsa- esat- secretion system components, which are central to staphylococcus aureus pathogenesis. a highly significant module included five esat- components; the sixth mrsa- esat- gene was not assigned to a module but was ranadown and shared edges with 10/ genes in the module. the two further ranadown genes in this module were relatively uncharacterised'hypothetical' proteins. analysis of sar predicted six transmembrane regions and found a match to the'membrane abc permease' domain pd; sar was predicted to be secreted or cell wall anchored, and matched to several protein families including conserved domains of unknown function, plus virulence-associated families. these results agree with the network-inferred virulence function for sar and sar the remaining five genes in the module were also hypothetical, while all five matched to conserved domains of unknown function and virulence-associated families. consistent with roles in virulence for these remaining five genes, tmhmm indicated two secreted/cell wall and one transmembrane protein, while predictions were not clear in two cases. the module had good correspondence with predicted operon structure, implying that the seven hypothetical genes may be co-regulated with the esat- system. the go term'pathogenesis' was significant for this module. therefore, seven novel s. aureus virulence factors were inferred. modules with false discovery rate corrected fisher p-values ≤ and size > are given. all modules were manually inspected with reference to literature and relevant databases. underlined locus identifiers were downregulated in response to ranalexin, none of the below loci were upregulated. locus identifiers in bold are candidate novel virulence factors. many genes that were not annotated with'pathogenesis' in the gene ontology nevertheless had some association with virulence in the literature, and therefore are not indicated as candidate novel virulence factors in the table. modules listed first'colonization, immunomodulation' and fifth'esat- system' were also significant for the ranalexin downregulated gene set. two significant ranadown modules were associated with high-affinity metal ion transport, which is crucial for establishment of infection. the module with 4/ ranadown genes included sar0787-sar representing the sst iron-uptake operon. the fifth gene in this module was a putative substrate binding protein for iron complex transport, which agrees with the functional relationship to the sst operon inferred from the network. a further module contained twelve genes with annotated virulence functions. these twelve genes were largely implicated in colonization and immuno-modulation. the remaining four genes in this module encoded ebps, a putative lyra protease, a putative lytn cell wall hydrolase, and a conserved hypothetical protein matching to the pfam family'hydrolase'. indeed, coordination of peptidoglycan hydrolase and virulence determinant expression has been observed in s. aureus. all sixteen genes were known or predicted to encode cell wall anchored, transmembrane or secreted proteins. the go term'pathogenesis' was significant for the module. these data imply functions in virulence-coupled autolysis for sar, sar and sar. ebps is a transmembrane protein that binds tropoelastin, and not associated with adhesion. ebps function is not well defined, however involvement in virulence is suggested by the network structure. a fully connected ranadown module was composed of relatively uncharacterised,'hypothetical' genes. interestingly, the only edge from this module into the rest of the network connected with the esat- virulence module described above. consistent with the network-based inference of virulence functions for these three genes, tmhmm indicated one transmembrane and two secreted/cell wall proteins, while database matches included conserved domains of unknown function and rtx toxin family. additionally, the most significantly enriched go term in the ranalexin downregulated gene set was'pathogenesis'. many genes annotated with this go term were strongly downregulated, for example: sar; sar; sar. indeed, several agr locus genes additional to hld were ranadown. ranalexin dependent repression of virulence genes agrees with the upregulation of sara attenuator sar, which inhibits sara-dependent virulence gene expression. furthermore, two ranadown genes encoded putative hth-type transcriptional regulators from the sara family, that activate virulence factor transcription. there is evidence linking traditional antibiotics to reduction of s. aureus virulence, however this had not previously been reported for a cationic amp. the observation that ranalexin results in repression of virulence factor expression has clear beneficial implications for potential clinical applications. prediction of additional s. aureus virulence factors was enabled by mapping go annotations for'pathogenesis' into the network. a total of ten modules were significant, and were manually inspected. these ten included the esat- and colonization-associated modules discussed above. interestingly a significant module had known two-component sensors or regulators, and a further four poorly characterised genes which may form novel virulence-associated two-component systems. manual inspection of these modules, with reference to literature and electronic resources, indicated eight genes not previously annotated as virulence factors. in summary, the above analysis indicated that ranalexin restricts s. aureus mrsa- pathogenicity, including the esat- system, and inferred twenty-two novel virulence factors. ranalexin induces cell wall stress the response to ranalexin included strong upregulation of several peptidoglycan synthesis genes, which suggests that ranalexin may act on the cell wall. indeed, the gene ontology term'peptidoglycan-based cell wall' was significantly enriched in genes upregulated by ranalexin. the vrar regulator protein of the vancomycin-resistance associated two-component system was ranaup. vrasr controls the expression of genes that are induced by vancomycin, many of which function in cell wall biosynthesis. several genes regulated by vrasr were found to be ranaup, including sar, sar, sar and sar. the above genes include members of a general cell wall stimulon induced in response to cell wall active agents. moreover, vrasr is upregulated in vancomycin-intermediate strains of s. aureus and induced by exposure to cell wall active antibiotics such as glycopeptides and β-lactams. a further ranaup protein, ftsh, had the highest betweenness centrality in the network, degree of, and did not fall into a module. therefore ftsh is an intermodular hub, which implies a key role in regulating system behaviour. indeed, ftsh is within the aaa family and acts as a chaperone required for incorporation of penicillin binding proteins into the cell membrane. as noted above, pbp was ranaup and is part of a cell wall stimulon, while pbps are upregulated in vancomycin-intermediate s. aureus. ftsh is also involved in response to osmotic stress and mutants are non-viable or have significantly attenuated virulence. considering the above, ftsh is proposed to be a key player in the mrsa response to antimicrobials such as ranalexin and a potential drug target. indeed, these results suggest that a drug targeting ftsh would be particularly effective in combination with cell wall active agents. the upregulation of ftsh in concert with the cell wall stimulon further supports action of ranalexin at the cell wall. the largest ranalexin dependent induction of transcription was observed for sar, which is not well characterised. however, vrax was over-expressed in isolates of vancomycin-intermediate s. aureus and showed >200-fold increased expression in vancomycin-sensitive s. aureus treated with vancomycin. overall, these data indicate that ranalexin caused cell wall stress similar to that produced by vancomycin - a cell wall active antibiotic. ranaup genes also included transcriptional regulatory proteins encoded by sar and sar that are known to be induced in the presence of cell wall active antibiotics. a transcriptional attenuator of sara, sar, was induced by ranalexin. significantly, msrr is induced upon exposure to cell wall active antibiotics, while its deletion results in sensitivity to methicillin and teicoplanin. an autolysis-deficient, teicoplanin-resistant strain of mrsa was found to upregulate fmta, sara, tcaa, msrr, vrar plus a large number of proteases. as noted above, these genes were found to be ranaup. indeed, inactivation of mrsa methicillin resistance genes has been reported to increase susceptibility to the β-defensin and ll amps. evidence supporting upregulation of cell wall anabolism also included enrichment of the go term'aspartate family amino acid biosynthesis' in the ranaup set. aspartate metabolism produces essential components for pepdidoglycan biosynthesis, such as diaminopimelic acid, and components of the aspartate pathway represent possible drug targets. in summary, the above results suggest that ranalexin exposure induced a cell wall stress response, similar to that associated with cell wall active antibiotics or glycopeptide intermediate-resistant s. aureus strains. follow-up laboratory experiments were performed to further investigate these findings. further investigation of ranalexin cell wall effects the physiological relevance of ranalexin induced cell wall related gene and protein expression changes was investigated by producing vrar and tcaa disruption mutants and subsequent examination for peptide sensitivity. prior to making mutants, ranalexin induced enhanced expression was verified by quantitative pcr for vrar and tcaa. in both cases, we again detected ranalexin dependent induction of expression after minute exposure that peaked after min and subsequently declined after min. the sigma targetron system was applied to generate disruption mutants, for which the s. aureus laboratory strain rn was recommended by the manufacturer. thus, the corresponding genes from mrsa- were identified in rn and disrupted. vrar disruption induced sensitivity to both ranalexin and vancomycin, compared to the parent strain. complementation experiments were attempted, but were inconclusive due to phenotypic effects induced by the empty cloning vector. growth of the vrar mutant was identical to the parent strain in the absence of ranalexin, but was completely inhibited when ranalexin was added. similarly, dose response experiments revealed dramatic viability loss for the vrar mutant both to increasing concentrations and to increased duration of exposure - contrasting with the parent strain which retained viability. considering that the vrars two-component system regulates a response to cell wall damage, these results support the view that ranalexin induces cell wall stress. tcaa disruption resulted in minor sensitivity to ranalexin. no significant differences in rate of loss of viability between the tcaa mutant and the parent strain exposed to ranalexin were detected. tcaa is a member of the cell wall stress stimulon known to be induced by exposure to antibiotics in a vrars dependent fashion. therefore, these results suggest that vrars may be a staphylococcal two-component response regulator involved in cationic peptide resistance. in s. aureus, vrars regulates the transcriptional induction of genes involved in cell wall peptidoglycan biosynthesis and is crucial for resistance to cell wall active antibiotics such as the glycopeptides vancomycin and teicoplanin. therefore, these results support the view that ranalexin's mode action includes disruption of the staphylococcal cell wall. to further examine ranalexin cell wall effects, osmotic fragility was measured for mrsa cells exposed to sub-lethal concentrations of antimicrobials. osmotic fragility is a hallmark of cell wall disruption. cultures were incubated in tsb, in the presence and absence of vancomycin or ranalexin, harvested and washed twice in sterile distilled water, followed by incubation in water for min. viability was determined throughout the experiment. control cells and vrar-disrupted cells showed negligible loss of viability or sensitivity to hypo-osmotic stress. as expected, cells pre-treated with a sub-lethal concentration of vancomycin were sensitive. notably, pre-treatment with a sub-lethal concentration of ranalexin also induced sensitivity to hypo-osmotic stress. furthermore, exposure to both ranalexin and vancomycin induced a similar degree of osmotic fragility compared to treatment with ranalexin; suggesting commonalities in modes of action. coupled with the induction of increased cell wall related gene/protein expression and hypersensitivity produced by vrar disruption, these results imply that the inhibitory action of ranalexin is not solely due to membrane disruption, but also involves significant effects at the staphylococcal cell wall. mrsa persister formation and drug tolerance exposure to ranalexin was found to produce strong upregulation of proteins encoded by the pstscab-phou operon psts, pstc, psta, phou and pstb. four of these formed a significant network module. sar did not fall into a module but had connections to all the above genes. phou acts as a persister switch in e. coli multidrug tolerance and impacts on widespread processes beyond inorganic phosphate transport. genetic disruption of the pstc transmembrane component of the pi transporter with the sigma targetron system did not have any effect on ranalexin sensitivity. combined with the proteome profiling and network analysis this data suggests that mrsa adopts a phou-mediated persister phenotype to acquire antimicrobial tolerance, and that upregulation of the pi transporter is not a major component of this response. indeed, the pstscab-phou operon was induced by streptococcus pneumoniae exposure to penicillin, and targeted disruption correlated with increased sensitivity to ciprofloxacin in mycobacterium smegmatis. a pyrimidine metabolism module was ranadown reflecting growth inhibition that is a hallmark of persister formation. additionally, several other cell division proteins were ranadown, including sar, sar, sar, sar, and the transcript sar was ranadown. these genes are therefore tentative candidates for negative regulation by phou in mrsa- the results presented in earlier sections, finding ranalexin exposure upregulated cell wall anabolism and reduced virulence, also align with persister formation. indeed, persister bacteria exhibit thickening of the cell wall and loss of virulence factors. these findings underline the importance of persister formation in mrsa drug tolerance. multiple actions in mrsa killing ranalexin is canonically associated with cell membrane permeabilisation. in agreement with this, a cation antiport module was significant upregulated. the ranaup genes in this module were subunits a-f of the na+/h+ antiporter encoded by the mnh operon, sar and subunit g of the na+/h+ antiporter encoded by the mnh operon. the module also included subunits a-f from the mnh operon, and three hypothetical proteins. the upregulation of these'electrogenic monovalent cation proton antiporter-3' operons implies that membrane permeabilisation leading to cation influx and possible dissipation of transmembane electrochemical gradient is a major effect of ranalexin exposure. the mnh and mnh operons are important for na+-dependent ph homeostasis in b. subtilis and are induced by alternative sigma factor b in s. aureus. further, the go term'ion transport' was significantly enriched with upregulated genes. additional to the above genes, these included sar, encoding a probable cadmium-effluxing p-type atpase that confers resistance to cd2+ toxicity; sar encoding a putative na+/pi-cotransporter; sar, encoding a putative tetracycline, k+ or na+/h+ antiporter; and sar, encoding a regulatory transcription factor induced in response to zn2+ stress. a significant module contained the opuc osmoprotectant transport operon; osmoprotectant transporters are thought to be strongly dependent on σb induction. upregulation of osmoprotectant transport is likely a product of σb induction by cation influx, especially given the observed upregulation of the mnh and mnh operons. interestingly, increased osmolarity would be expected to act in concert with the ranalexin cell wall effects presented above in producing hypo-osmotic fragility and disruption of cell integrity. therefore the membrane and cell wall actions of ranalexin may exert complementary effects in killing s. aureus mrsa- the response signature included upregulation of the dlt operon d-alanine and d-alanyl lipoteichoic acid synthesis, which leads to increased positive charge at the cell surface and reduced peptide binding. consistent with the expected ranalexin induced stress, a significant module contained chaperones and stress response genes. also, the chaperone proteins groel and ftsh were ranaup and intermodular hubs. another significant module represented genes for val/leu/ile synthesis, which utilizes pyruvate, and therefore may allow for greater glycolytic flux in powering ranalexin stressed cells. indeed, several glucose catabolism and uptake proteins were upregulated, including eiia-glc, pgk an intermodular hub, and pgi substantiating upregulation of glycolysis upon ranalexin exposure. interestingly, l-ldh was ranadown; in conjunction with upregulation of val/leu/ile synthesis and glycolysis this suggests repurposing of pyruvate into production of hydrophobic proteins for peptide sequestration. these results, and those presented in above sections, detail several lines of defence employed by mrsa to escape the effects of cationic amps. in summary, evidence supports multiple inhibitory actions for ranalexin including cell wall effects, increased cation influx and possible dissipation of transmembrane electrochemical gradient. mrsa attempts several survival strategies when faced with antimicrobial peptide stress, and evidence was found for complementary inhibitory actions in killing mrsa.  a global gene functional association network that covers 95% of s. aureus mrsa- genes was presented. careful benchmarking against blind test datasets found a false positive rate no greater than 3% in the final network. functional network analysis of drug response signatures enabled novel insights into the mode of action of a cationic antimicrobial peptide, ranalexin, as well as insights into mrsa- antimicrobial resistance mechanisms. evidence was presented for novel ranalexin effects on the bacterial cell wall, in addition to the previously characterised action at the cell membrane, and these actions were proposed to act in concert. these findings agree with previous observations that cationic amps exert complex inhibitory actions. the ftsh membrane chaperone was an intermodular hub, upregulated in response to ranalexin and suggested as a promising candidate drug target, particularly for combination therapy with cell wall active agents. indeed, combination therapy has clear advantages for treatment of resistant bacteria. additionally, the two-component response regulator vrar was suggested to be important for mediating tolerance to antimicrobial peptides. therefore, vrars may be a staphylococcal two-component response regulator involved in cationic peptide resistance, in addition to apsrs. ranalexin exposure produced mrsa- virulence reduction, and twenty-two novel virulence factors were inferred. these novel roles in virulence are supported by several additional lines of evidence, including predicted cellular localisation, domain matching and operon structure. finally, we present evidence to support phou-mediated persister switching as a mechanism of drug tolerance in mrsa; which merits further investigation as a route towards novel therapeutic targets. this work is informative to the development of therapeutic strategies against s. aureus, and demonstrates an elegant approach to study drug resistance and mode of action. 
antimicrobial peptides represent a source of potential novel antibiotics to combat resistant bacteria such as methicillin-resistant staphylococcus aureus. furthermore, twenty-two novel virulence factors were inferred, while the vrars two-component system and phou-mediated persister formation were implicated in mrsa tolerance to cationic antimicrobial peptides. we developed a systems-wide functional association network approach to integrate proteome and transcriptome profiles, enabling study of drug resistance and mode of action. this work demonstrates a powerful integrative approach to study drug resistance and mode of action. a promising antimicrobial peptide is ranalexin, which has potent activity against gram-positive bacteria, and particularly s. aureus. the functional association network was constructed by bayesian logistic regression, providing a framework for identification of antimicrobial peptide response modules from s. aureus mrsa- transcriptome and proteome profiling. these signatures of ranalexin treatment revealed multiple killing mechanisms, including cell wall activity. cell wall effects were supported by gene disruption and osmotic fragility experiments.
 staphylococcus aureus is a major human pathogen and strains resistant to existing treatments continue to emerge. development of novel treatments is therefore important. antimicrobial peptides represent a source of potential novel antibiotics to combat resistant bacteria such as methicillin-resistant staphylococcus aureus. a promising antimicrobial peptide is ranalexin, which has potent activity against gram-positive bacteria, and particularly s. aureus. understanding mode of action is a key component of drug discovery and network biology approaches enable a global, integrated view of microbial physiology, including mechanisms of antibiotic killing. we developed a systems-wide functional association network approach to integrate proteome and transcriptome profiles, enabling study of drug resistance and mode of action.  the functional association network was constructed by bayesian logistic regression, providing a framework for identification of antimicrobial peptide response modules from s. aureus mrsa- transcriptome and proteome profiling. these signatures of ranalexin treatment revealed multiple killing mechanisms, including cell wall activity. cell wall effects were supported by gene disruption and osmotic fragility experiments. furthermore, twenty-two novel virulence factors were inferred, while the vrars two-component system and phou-mediated persister formation were implicated in mrsa tolerance to cationic antimicrobial peptides.  this work demonstrates a powerful integrative approach to study drug resistance and mode of action. our findings are informative to the development of novel therapeutic strategies against staphylococcus aureus and particularly mrsa. 