We developed a technique of generating nonthermal atmospheric plasma-activated solution that had broad-spectrum antibacterial properties. Plasma-activated phosphate-buffered saline (PBS) causes rapid inactivation of bacteria following generation of oxidative stress. However, dose optimization requires understanding of cellular mechanisms. The objective of this study was to explore genome-wise response to develop gene expression profile of Escherichia coli using DNA microarray following exposure to plasma-activated PBS solution. Upon exposure to plasma-treated PBS solution, E. coli cells had differentially expressed genes involved in oxidative stress, and cell envelope and membrane associated porin and transporters. The genes involved in house-keeping and metabolism, energy generation, motility and virulence were conversely downregulated. This is the first report which demonstrates a severe oxidative stress induced in E. coli cells in response to an exposure to nonequilibrium nonthermal dielectric-barrier discharge plasma-activated PBS solution, and the genes that are responsive to reactive oxygen species appeared to play a role in cellular stress. Such studies are important to identify targets of inactivation, and to understand plasma-treated solution and bacterial cell interactions.
Nonthermal nonequilibrium atmospheric plasma (plasma) is being investigated for disinfection and sterilization processes in biology and medicine. Since last decade, several publications appeared which stressed the advantages of this technique over traditional disinfection techniques [
Recently, we have demonstrated a technique of applying plasma indirectly wherein the plasma generating probe doesn’t come in contact with such surface or the surface of skin or mucous membranes, and is highly portable and does not require gas or air cylinders and the associated assembly (unlike jet or afterglow plasma technique). We have developed plasma-treated solutions that retain strong antimicrobial property for up to two years of time period [
Genomic analysis is one of the most favored approaches of study underlying genetic mechanisms of inactivation and regulatory response of genes that govern cell death. Cellular defenses have their thresholds and beyond this limit cells cannot survive stress conditions. Escherichia coli is one of the suitable model bacteria for this study whose whole genome sequence is available and metabolic pathways are well studied [
Escherichia coli (ATCC-29522) was used for this study. The culture was developed by inoculation of a single isolated colony from overnight grown trypticase soy agar plate into 10 ml of trypticase soy broth (TSB) and incubated at 37˚C in a stationary incubator. On the following day, the culture was reinoculated and the growth monitored by taking optical density (OD600) of the culture aliquots.
Phosphate-buffered saline solution (PBS; Sigma; 150 mmol/L sodium chloride and 150 mmol/L sodium phosphate, pH 7.2, at 25˚C) was prepared using deionized water (MP Biomedicals Inc., Solon, OH). Solution was freshly prepared, 0.22 μ filter sterilized, aseptically handled; and aliquot either used fresh or stored at −20˚C for subsequent experiments. Similarly, α-tocopherol (vitamin E; 200 mM), thiourea and catalase (all from Sigma- Aldrich, St. Louis, MO, USA) were prepared as stock solutions in untreated PBS buffer, filter sterilized, and used at predetermined concentrations (α-tocopherol, 200 mM; thiourea, 50 mM; catalase, 200 units). Nonthermal plasma generator was used in this study, and the in-house built electrode and fluid chamber system was reported earlier by our laboratory [
Halfmilliliter (0.5 ml) of PBS solution treated with plasma for various duration of treatment was mixed with 0.5 ml of E. coli cell suspension (1 ml culture of 0.2 OD600 was centrifuged at 4000 revolutions per minute (RPM), cell pellet was washed twice with sterile PBS, and resuspended in 0.5 ml of PBS), and held for 15 min of contact time. The cells were harvested by centrifugation, and resuspended in untreated PBS to proceed for XTT assay using XTT reagents (Molecular Probes) as described earlier [
The amount of hydrogen peroxide retained in each sample was measured using Hydrogen Peroxide Detection Kit (National Diagnostics, Atlanta, GA) following manufacturer’s protocol. In brief, a working solution of 20 ml was prepared by combining two reagents supplied in the Kit, and the serial dilutions of standard hydrogen peroxide were tested to generate standard curve. In parallel, the undiluted or serially diluted plasma-activated PBS was prepared, and H2O2 detection assay performed in 96 wells plate in triplicate. The assay was repeated twice in triplicate and H2O2 concentrations determined as per manufacturer’s protocol.
A predetermined (60 seconds) dose of plasma treatment was used to activate PBS solution. One ml exponential E. coli culture (0.2 of OD600) was centrifuged at 4000 RPM to harvest cells and the cell pellet was washed twice with sterile PBS and resuspended in 500 μl of PBS. An equal amount (500 μl) of plasma-treated or untreated PBS solution or H2O2 solution was then added to this cell suspension and the reaction mix was held for 15 min (contact time), after which cells were suspended in RNA later reagent (Qiagen, Valencia, CA) to stabilize RNA, and then were pelleted again by centrifugation, and subjected to cell lysis using RNeasy mini kits (Qiagen) following the manufacturer’s protocol. RNA was isolated from E. coli cells exposed to either plasma-treated or untreated fluid or H2O2 solution, and quantified on a NanoDrop spectrophotometer (Thermo Scientific), followed by RNA quality assessment on an Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA, USA). Amplification and labeling was performed using the Ovation Pico WTA-system V2 RNA amplification system (NuGen Technologies, Inc.). Briefly, 50 ng of total RNA was reverse transcribed using a chimeric cDNA/mRNA primer, and a second complementary cDNA strand was synthesized. Purified cDNA was then amplified with ribo-SPIA enzyme and SPIA DNA/RNA primers (NuGen). Amplified c-DNA was purified with Qiagen MinElute reaction cleanup kit. The concentration of Purified cDNA was measured using the NanoDrop. The cDNAs (2.5 µg) were fragmented and chemically labeled with biotin to generate biotinylated cDNA using FL-Ovation cDNA biotin module V2 (NuGen). Affymetrix Genechip® E. coli Genome 2.0 array system (Affymetrix, Santa Clara, CA) was used. The product was hybridized with fragmented and biotin-labeled target (2.5 μg) in 110 μl of hybridization cocktail. Target denaturation was performed at 99˚C for 2 min and then 45˚C for 5 min, followed by hybridization for 18 h. Arrays were then washed and stained using Gene chip Fluidic Station 450, and hybridization signals were amplified using antibody amplification with goat IgG and anti-streptavidin biotinylated antibody. A hydrogen peroxide (0.3%) reagent and plasma untreated PBS solution were used as positive and negative controls respectively.
Gene-Chips were scanned on an Affymetrix Gene Chip Scanner 3000, using Command Console Software. Background correction and normalization were done using Iterative Plier 16 with Gene Spring V11.5 software (Agilent). A list of differentially expressed genes (in fold) was generated for the genes whose transcription is significantly influenced, and the list was loaded into Ingenuity Pathway Analysis (IPA) 5.0 software (http://www.ingenuity.com) to perform biological network and functional analyses. The transcript expression values of treated sample array (plasma-treated PBS) versus plasma untreated sample array were considered significant when the difference ratio was 1.2, and subsequently, we selected genes which were differentially expressed by >2 fold (against untreated samples). Experiments were repeated in triplicate, and a mean of fold expressions shown.
The plasma generating setup used in these studies is published earlier [
By whole genome approach, E. coli genes were analyzed for their expression profile against the treatment response of plasma-activated PBS solution. The plasma-activated PBS solution chemistry is not yet fully understood. Based on our preliminary observations of generation of reactive oxygen species [
Out of 412 genes differentially expressed upon plasma-activated PBS 230 were functionally defined genes, and rest 181 genes were either pseudo genes or hypothetical genes whose functional annotation is not defined. Total 120 genes were upregulated and 111 genes were downregulated. The findings of microarray assay of top
125 genes with their functional annotations which exhibited differential expression are grouped together and showed in
SOS response is a global response to DNA damage wherein cell cycle is arrested and DNA repair (and/or mutagenesis) is induced, and includes the proteins related to Rec family (RecA, RecB, RecD, RecN). The SOS regulon involves protein RecA, responsible for inactivation of LexA repressor (and thus negatively regulated by LexA repressor protein dimmers), and is a complex of several genes that are coordinately expressed and involved in DNA repair. The RecA protein is under control of recombination regulator RecX; and RecN is required in DNA recombination. In present study, overall SOS genes were moderately upregulated (<2 folds; recA, lexA, sulA, umuC, umuD). Only the genes, recN, nfo, nrdE and nrdF were upregulated by >2 folds (
We observed highest expression of oxyS transcript (28.8 fold upregulation), an indicator of a generation of severe oxidative stress. Hydrogen peroxide detoxifying genes such as katG (catalase), ahpC (peroxiredoxin), ahpF (hydroperoxidase) and yggP (putative oxidoreductase) were differentially expressed; respectively 19.3, 3.9, 7.0, and 3.1 folds (
Reactive nitrogen species [
Gene | Fold expression | Regulation | Function |
---|---|---|---|
Oxidative damage response | |||
oxyS | 28.8 | Up | Small regulatory RNA, responsive to oxidative stress |
katG | 19.3 | Up | Catalase/hydrogen peroxidase |
sodA | 2.1 | Up | Superoxide dismutase/detoxification of superoxide radicals |
soxS | 2.9 | Up | Transcriptional regulator of oxidative stress |
yhjA | 2.0 | Up | Cytochrome-C peroxidase activity |
ahpF | 7.0 | Up | Disulfide oxidoreductase activity/peroxidase |
ahpC | 3.9 | Up | Peroxidase activity/peroxiredoxin |
ybiX | −2.4 | Down | Fe(II)-dependent oxygenase, hydroxylase activity |
SOS response | |||
recN | 2.4 | Up | DNA recombination & repair |
dinL | 2.1 | Up | DNA-damage-inducible protein |
recD | 2.3 | Up | DNA repair, helicase activity, exonuclease V subunit |
recB | 2.1 | Up | Exonuclease V subunit (recBCD complex) |
nfo | 4.4 | Up | Endonuclease IV, DNA repair |
Nitrosative stress | |||
hda | −3.2 | Down | DNA replication initiation factor |
napA | −2.6 | Down | Nitrate reductase, periplasmic |
napD | −3.2 | Down | Assembly protein for periplasmic nitrate reductase |
napF | −3.5 | Down | Ferredoxin-type protein, Fe-Fe binding, electron transport |
Related to cell envelop | |||
tsx | −2.5 | Down | Porin activity, iron membrane-transporter |
ynbA | 2.2 | Up | Inner membrane, phosphotransferase |
yjcH | −2.2 | Down | Inner membrane protein, DUF485 family |
yagU | 2.7 | Up | Response to acidity, DUF1440 family |
yliE | 2.6 | Up | Hydrolase activity, inner membrane protein |
flgF | −2.0 | Down | Flagellar/motility, flagellum basal body |
ycfS | −3.5 | Down | Peptidoglycan synthetase activity, cell shape |
livJ | −2.8 | Down | Carbon starvation induced gene, cell wall |
Other stress-related response | |||
sufA | 2.4 | Up | Response to oxidative stress, Fe-S cluster protein |
acnA | 2.2 | Up | Aconitate hydratase/oxidative stress response/TCA cycle |
uspD | 5.1 | Up | Response to stress, cytoplamic protein |
cstA | −3.2 | Down | Response to stress, plasma membrane protein |
astD | −2.7 | Down | Succinylglutamic semialdehyde dehydrogenase, response to stress |
---|---|---|---|
bhsA | −2.6 | Down | Biofilm, cell surface and signaling protein, response to stress |
ymgC | 2.3 | Up | Biofilm formation, predicted protein |
yhcN | −2.7 | Down | Cellular response to hydrogen peroxide, periplasmic space |
ycfR | −2.6 | Down | Periplasmic protein, biofilm formation, response to stress |
Regulation/cell cycle/cell division | |||
glnL | 2.1 | Up | Histidine kinase activity, nitrogen regulation protein NR(II) |
ycfS | −3.5 | Down | Transferase, peptidoglycan synthetase, transcriptional regulator |
malT | −3.3 | Down | Transcriptional regulator MalT |
napD | −3.2 | Down | Assembly protein for periplasmic nitrate reductase |
Metabolism | |||
astA | −2.1 | Down | Acyltransferase, arginine N-succinyltransferase activity |
prpR | −2.1 | Down | Regulator for prp operon, propionate catabolism |
hcaR | −2.0 | Down | DNA-binding transcriptional regulator HcaR |
LipA | 2.0 | Up | Lipoate synthase, transferase activity |
CarB | −14.5 | Down | Arginine biosynthetic process, synthase activity |
putA | −6.6 | Down | Proline dehydrogenase transcription |
pyrD | −6.2 | Down | Dihydroorotate dehydrogenase/UMP biosynthetic process |
argH | 4.6 | Up | Argininosuccinate lyase activity |
poxB | 4.1 | Up | Pyruvate oxidase, thiamine-dependent, FAD-binding |
hemH | 4.1 | Up | Porphyrin biosynthetic process, ferrochelatase |
purD | −3.5 | Down | Purine base biosynthetic process, synthase |
ycfS | −3.5 | Down | Peptidoglycan synthetase activity |
carA | −3.3 | Down | Glutamine amidotransferase, synthase activity |
entC | −3.3 | Down | Isochorismate synthase, enterobactin biosynthesis |
entE | −3.2 | Down | Enterobactin synthase, siderophore biosynthesis |
pyrB | −3.1 | Down | Aspartate carbamoyltransferase activity |
leuB | −3.1 | Down | Isopropylmalate dehydrogenase, amino acid synthesis |
Pyrl | −2.7 | Down | Transferase activity, pyrimidine biosynthesis |
mdoD | −2.1 | Down | Osmoregulated glucan (OPG) biosynthesis, periplasmic protein |
alaC | 2.1 | Up | Valine-pyruvate aminotransferase activity |
yggF | −2.1 | Down | Fructose 1,6 bisphosphatase, glycerol metabolism |
aes | 3.9 | Up | Acetyl esterase, carboxylesterase activity |
rffH | −2.2 | Down | Extracellular polysaccharide biosynthetic process |
rffA | −2.2 | Down | Lipopolysaccharide biosynthetic process |
arnA | 2.0 | Up | Decarboxylase, lipid biosynthetic process |
mdoD | −2.1 | Down | Osmoregulated glucan (OPG) biosynthesis | |
---|---|---|---|---|
gabD | −2.4 | Down | Succinate-semialdehyde dehydrogenase activity | |
fumC | 2.4 | Up | Fumarate hydratase, TCA cycle | |
htrA | −2.2 | Down | Serine endoprotease, proteolysis | |
pncB | 2.2 | Up | Transferase activity, NAD biosynthetic process | |
Fe-S cluster assembly/cysteine synthesis | ||||
yfaE | 2.1 | Up | Ferredoxin metabolic process, 2Fe-2S ferredoxin | |
ygfT | 2.6 | Up | Glutamate biosynthetic process, oxidoreductase, Fe-S subunit | |
sufA | 2.4 | Up | Iron-sulfur cluster assembly scaffold protein | |
frdB | −2.1 | Down | Fumarate reductase, Fe-S subunit | |
hypC | −2.0 | Down | Hydrogenase assembly chaperone | |
grxA | 10.8 | Up | Glutaredoxin 1, electron carrier activity | |
sufD | 7.5 | Up | Cysteine desulfurase activator complex subunit SufD | |
sufC | 5.5 | Up | Cysteine desulfurase ATPase component, ABC superfamily | |
sufS | 8.4 | Up | Bifunctional cysteine desulfurase | |
Fpr | 7.0 | Up | Ferredoxin-NADP reductase | |
sufE | 3.5 | Up | Cysteine desufuration protein SufE, S-acceptor | |
sufB | 3.7 | Up | Iron-sulfur cluster assembly , SufBCD complex | |
fhuC | −2.1 | Down | Iron-hydroxamate transporter subunit | |
trxC | 6.1 | Up | Thioredoxin-2 | |
Transporters | ||||
mntH | 4.4 | Up | Manganese ion transmembrane transporter activity | |
fepA | −2.1 | Down | Iron-enterobactin outer membrane transporter | |
ftnB | −2.2 | Down | Ferritin-like protein, cellular iron ion homeostasis | |
lsrC | 2.1 | Up | Transport system permease protein | |
argH | 4.6 | Up | Cellular amino acid biosynthetic process | |
glnA | 2.3 | Up | Glutamine synthetase & transport regulation, N2-deprivation | |
putP | −3.7 | Down | Major sodium/proline symporter | |
cmtA | 2.6 | Up | Carbohydrate transport, sugar:hydrogen symporter | |
mngA | 2.4 | Up | Carbohydrate transporter, IIABC components system | |
malK | 2.0 | Up | Maltose/maltodextrin transporter | |
mdlB | 2.3 | Up | Multidrug transporter | |
nmpC | −4.8 | Down | Outer membrane porin protein, nmpC | |
dppC | −2.4 | Down | Dipeptide/heme transporter | |
narU | 5.1 | Up | Nitrate/nitrite transporter protein | |
araE | 2.3 | Up | Arabinose-proton symporter, carbohydrate transporter | |
dctA | −3.4 | Down | Sodium:dicarboxylate symporter activity | |
was less pronounced. Other stress-responsive universal genes which were differentially expressed include acnA (TCA cycle), uspD (cytoplasmic protein), cstA (plasma membrane protein), and yhcN (periplasmic space protein). The gene bhsA (formerly ycfR) that mediate biofilm formation was downregulated. Similarly, most of the genes encoding the proteins from biosynthesis and metabolic pathways were significantly downregulated (
The transporter of manganese (mntH) was upregulated >4 folds, while almost all iron mediating transporters and related protein transcription, such as fepA (iron-enterobactin) ftnB (ferritin-like protein involved in cellular iron ion homeostasis), fepC (iron-enterobactin, ATP-binding subunit), dppC (dipeptide, heme transporter), napF (ferredoxin type protein) were downregulated. This suggests a disrupted iron homeostasis. The transporters of glucarate, glutamine, sugar:hydrogen, carbohydrate, maltodextrin, multidrug, amino acid, nitrate-nitrite were all upregulated (
Despite of increasing reports on application of nonthermal plasma in disinfection and bacterial inactivation, relatively very little is known about the stress response of bacterial cell to it. In this study, we report gene expression profile and transcriptomic responses of E. coli to plasma-activated PBS solution which inactivates bacterial cell [
An analysis using global gene expression approach is recently used by Dr. Pruden’s Laboratory [
The SOS responses in E. coli are induced after DNA damage, and are dynamically regulated by interplay between Rec family protein, such as RecA, Lex protein, LexA, and Sul protein, SulA. RecN is a conserved SMC- like (structural maintenance of chromosomes) nucleoid-associated ATPase involved in the tethering of chromatids and in double strand break (DSB) repairs [
E. coli has several major regulators that are activated during oxidative stress and undergo conformational changes, but two regulon systems are important in oxidative stress management. These are OxyR and SoxR transcriptional regulators, sensitive to oxidation in presence of hydrogen peroxide (H2O2) and superoxide radicals
Cells under (ROS or RNS) stress often activate transcription factor OxyR which in turn regulates the expression of a large panel of genes, including a small regulatory RNA, oxyS, as mentioned above. Such observations are reported during severe oxidative as well as nitrosative stress conditions [
It can be predicted that the interaction of ROS generated by plasma-treated solution exposure to cellular iron may have potential detrimental effect on bacteria, either by unavailability of iron for bacterial cell (iron starvation) or leading to series of lethal reactions that further regenerate hydroxyl radical (such as Fenton reaction) [
Under oxidative stress in E. coli several transporters are differentially expressed such as iron transporters. Iron is required for many metabolic processes and plays a role in protection against oxidative damage. However, excess iron levels in cells contribute to oxidative damage through the generation of free radicals [
This transcriptomic study suggests that E. coli cells differentially express several important genes that are responsive to oxidative stress generated upon exposure to plasma-activated PBS solution. The genes responsive to hydrogen peroxide, superoxide and singlet oxygen, and reactive nitrogen species were observed, and might be collectively exerting their damaging effect on E. coli cell. The data presented here are predictive and for the guidance, and further detailed studies would be interesting to elucidate the exact nature of responses to different nonthermal plasma set-ups and their various parameters.
Adam Yost (graduate) and Siddharth Joshi (undergraduate) research students at School of Biomedical Engineering, Drexel University, and Suresh G. Joshi, Garth Ehrlich and Ari Brooks, the faculties at Center for Surgical Infection and Biofilm, Drexel University College of Medicine, and Sankar Addya, the in-charge of Microarray Core Facility at Thomas Jefferson University, Philadelphia, PA. All authors contributed to this manuscript, and approved the final version of it. SGJ designed the study; SGJ, AY, SSJ, and SA carried out the experiments; SGJ and SA analyzed preliminary data; SGJ, SA, GE and AB interpreted data from multiple ways; SGJ developed the manuscript draft; and SGJ, AB and GE evaluated the manuscript. This study was supported by the Department of Surgery through Surgical Infection Research Program. The authors thank Microarray Core Facility of Kimmel Cancer Center, Philadelphia, PA.
The authors declare “no conflict” and no competing interests.