Although many explanations have been proposed for drug resistance to isothiazolones, the scope of cellular and physiological changes associated with this resistance remains unclear. In this study, comparative proteomic profiles of Pseudomonas aeruginosa ATCC9027 (WT) and an induced strain of Pa-R, which showed resistance to Kathon (a type of isothiazolone), were characterized using two-dimensional electrophoresis and matrix-assisted desorption/ionization time-of-flight mass spectroscopy. The results showed that a total of 16 proteins were successfully identified, among which 5 proteins were upregulated and 11 proteins were found to be repressed in Pa-R. At the same time, there were 14 proteins that contributed to metabolic processes, 1 protein (ATP-binding component of ATP-binding cassette [ABC] transporter) was the cellular component, and 1 protein (LolA) exhibited a transporter activity. The respective gene expression patterns of all the identified proteins in both Pa-R and WT were also evaluated by quantitative real-time polymerase chain reaction and shown to consistently correlate with those deduced from the proteomic results. Moreover, the resistant levels of Pa-R and WT could be affected by temperature and pH. Additionally, Pa-R exhibited coresistance and cross-resistance to other types of antimicrobial agents. Our results suggest that the resistant levels of P. aeruginosa to isothiazolones could be affected by extracellular factors and the resistance features are a complex system.
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References
1.
AdibpourN., KhalajA., and RajabalianS.. 2010. Synthesis and antibacterial activity of isothiazolyl oxazolidinones and analogous 3(2H)-isothiazolones. Eur. J. Med. Chem., 45:19–24.
2.
BadgerM.R., and PriceG.D.. 1994. The role of carbonic anhydrase in photosynthesis. Annu. Rev. Plant Biol., 45:369–392.
3.
BakerM.D., WolaninP.M., and StockJ.B.. 2006. Signal transduction in bacterial chemotaxis. Bioessays, 28:9–22.
4.
BarmanB.1994. Influence of temperature on the degradation of isothiazolone biocides in aqueous media and in a metalworking fluid concentrate. Lubr. Eng., 50:351–355.
5.
BarmanB., and PrestonH.. 1992. The effects of pH on the degradation of isothiazolone biocides. Tribol. Int., 25:281–287.
6.
BokhoveM., JimenezP.N., QuaxW.J., and DijkstraB.W.. 2010. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket. Proc. Natl. Acad. Sci. U. S. A., 107:686–691.
7.
BrözelV.S., and CloeteT.E.. 1994. Resistance of Pseudomonas aeruginosa to isothiazolone. J. Appl. Bacteriol., 76:576–582.
8.
CarrS., AebersoldR., BaldwinM., BurlingameA., ClauserK., and NesvizhskiiA.. 2004. The need for guidelines in publication of peptide and protein identification data: working group on publication guidelines for peptide and protein identification data. Mol. Cell. Proteomics, 3:531–533.
9.
ChapmanJ.S., and DiehlM.A.. 1995. Methylchloroisothiazolone-induced growth inhibition and lethality in Escherichia coli. J. Appl. Microbiol., 78:134–141.
10.
ChapmanJ.S., DiehlM.A., and FearnsideK.B.. 1998. Preservative tolerance and resistance. Int. J. Cosmet. Sci., 20:31–39.
11.
ChengJ.J., ThanassiJ.A., ThomaC.L., BradburyB.J., DeshpandeM., and PucciM.J.. 2007. Dual targeting of DNA gyrase and topoisomerase IV: Target interactions of heteroaryl isothiazolones in Staphylococcus aureus. Antimicrob. Agents Chemother., 51:2445–2453.
12.
ClevengerK.D., WuR., ErJ.A., LiuD., and FastW.. 2013. Rational design of a transition state analogue with picomolar affinity for Pseudomonas aeruginosa PvdQ, a siderophore biosynthetic enzyme. ACS Chem. Biol., 8:2192–2200.
13.
CollierP.J., AustinP., and GilbertP.. 1990. Uptake and distribution of some isothiazolone biocides into Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. Int. J. Pharm., 66:201–206.
14.
CollierP.J., AustinP., and GilbertP.. 1991. Isothiazolone biocides: enzyme-inhibiting pro-drugs. Int. J. Pharm., 74:195–201.
15.
CollierP.J., RamseyA., WaighR.D., DouglasK.T., AustinP., and GilbertP.. 1990. Chemical reactivity of some isothiazolone biocides. J. Appl. Microbiol., 69:578–584.
16.
DavidsonA.L., and ChenJ.. 2004. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem., 73:241–268.
17.
DekkerF.J., GhizzoniM., van der MeerN., WisastraR., and HaismaH.J.. 2009. Inhibition of the PCAF histone acetyl transferase and cell proliferation by isothiazolones. Bioorg. Med. Chem., 17:460–466.
18.
DekkerF.J., and HaismaH.J.. 2009. Histone acetyl transferases as emerging drug targets. Drug Discov. Today, 14:942–948.
19.
FurdasS.D., ShekfehS., BissingerE.M., WagnerJ.M., SchlimmeS., ValkovV., HendzelM., JungM., and SipplW.. 2011. Synthesis and biological testing of novel pyridoisothiazolones as histone acetyltransferase inhibitors. Bioorg. Med. Chem., 19:3678–3689.
20.
GediV., MoonJ.Y., LimW.M., LeeM.Y., LeeS.C., KooB.S., GovindwarS., and YoonM.Y.. 2011. Identification and characterization of inhibitors of Haemophilus influenzae acetohydroxyacid synthase. Enzyme Microb. Technol., 49:1–5.
21.
GhizzoniM., HaismaH.J., and DekkerF.J.. 2009. Reactivity of isothiazolones and isothiazolone-1-oxides in the inhibition of the PCAF histone acetyltransferase. Eur. J. Med. Chem., 44:4855–4861.
22.
GorgA., ObermaierC., BoguthG., HarderA., ScheibeB., WildgruberR., and WeissW.. 2000. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 21:1037–1053.
23.
HancockR.E.1998. Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin. Infect. Dis. 27Suppl1:S93–S99.
KhalajA., AdibpourN., ShahverdiA.R., and DaneshtalabM.. 2004. Synthesis and antibacterial activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones. Eur. J. Med. Chem., 39:699–705.
30.
KingA., LodolaA., CarmiC., FuJ., MorM., and PiomelliD.. 2009. A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors. Br. J. Pharmacol., 157:974–983.
31.
KiselyovA.S., SemenovaM., and SemenovV.V.. 2009. 3,4-disubstituted isothiazoles: Novel potent inhibitors of VEGF receptors 1 and 2. Bioorg. Med. Chem. Lett., 19:1195–1198.
32.
KretzschmarU., RückertA., JeoungJ.H., and GörischH.. 2002. Malate: quinone oxidoreductase is essential for growth on ethanol or acetate in Pseudomonas aeruginosa. Microbiology, 148:3839–3847.
LambertP.2002. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J. R. Soc. Med., 95:22–26.
35.
LivakK.J., and SchmittgenT.D.. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods, 25:402–408.
36.
LotlikarS.R., HnatuskoS., DickensonN.E., ChoudhariS.P., PickingW.L., and PatrauchanM.A.. 2013. Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air. Microbiology, 159:1748–1759.
37.
MeloA.M., BandeirasT.M., and TeixeiraM.. 2004. New insights into type II NAD (P) H: quinone oxidoreductases. Microbiol. Mol. Biol. Rev., 68:603–616.
38.
MelocheH., and WoodW.. 1964. The mechanism of 6-phosphogluconic dehydrase. J. Biol. Chem., 239:3505–3510.
39.
MurahariP., AnishettyS., and PennathurG.. 2013. Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation. Comput. Biol. Chem., 47:71–80.
40.
NakamotoR.K., KetchumC.J., and Al-ShawiM.K.. 1999. Rotational coupling in the F0F1 ATP synthase. Annu. Rev. Biophys. Biomol. Struct., 28:205–234.
41.
Navarro-De la SanchaE., Coello-CoutiñoM.P., Valencia-TurcotteL.G., Hernández-DomínguezE.E., Trejo-YepesG., and Rodríguez-SotresR.2007. Characterization of two soluble inorganic pyrophosphatases from Arabidopsis thaliana. Plant Sci., 172:796–807.
42.
PorankiewiczJ., WangJ., and ClarkeA.K.. 1999. New insights into the ATP-dependent Clp protease: Escherichia coli and beyond. Mol. Microbiol., 32:449–458.
43.
PucciM.J., PodosS.D., ThanassiJ.A., LeggioM.J., BradburyB.J., and DeshpandeM.. 2011. In vitro and in vivo profiles of ACH-702, an isothiazoloquinolone, against bacterial pathogens. Antimicrob. Agents Chemother., 55:2860–2871.
44.
ReddyB.M., TanneeruK., MeeteiP.A., and GuruprasadL.. 2011. 3D-QSAR and molecular docking studies on substituted isothiazole analogs as inhibitors against MEK-1 kinase. Chem. Biol. Drug Des., 79:84–91.
45.
RemansK., PauwelsK., van UlsenP., ButsL., CornelisP., TommassenJ., SavvidesS.N., DecanniereK., and Van GelderP.. 2010. Hydrophobic surface patches on LolA of Pseudomonas aeruginosa are essential for lipoprotein binding. J. Mol. Biol., 401:921–930.
46.
SchneiderE., and HunkeS.. 1998. ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol. Rev., 22:1–20.
47.
StimsonL., RowlandsM.G., NewbattY.M., SmithN.F., RaynaudF.I., RogersP., BavetsiasV., GorsuchS., JarmanM., BannisterA., et al.2005. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol. Cancer Ther., 4:1521–1532.
48.
StockJ., SuretteM., McClearyW., StockA., QuehenbergerO., ProssnitzE., CochraneC., YeR., AmlaikyN., and RambozS.. 1992. Signal transduction in bacterial chemotaxis. Bioessays, 267:19753–19756.
49.
TanakaS.Y., NaritaS.I., and TokudaH.. 2007. Characterization of the Pseudomonas aeruginosa Lol system as a lipoprotein sorting mechanism. J. Biol. Chem., 282:13379–13384.
50.
TownsendD.M., and TewK.D.. 2003. The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene, 22:7369–7375.
51.
TsugeT., FukuiT., MatsusakiH., TaguchiS., KobayashiG., and IshizakiA.. 2000. Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiol. Lett., 184:193–198.
52.
TsugeT., TaguchiK., and DoiY.. 2003. Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid ß-oxidation. Int. J. Biol. Macromol., 31:195–205.
53.
VaraprasadC.V.N.S., BarawkarD., AbdellaouiH.E., ChakravartyS., AllanM., ChenH., ZhangW., WuJ.Z., TamR., HamatakeR., et al.2006. Discovery of 3-hydroxy-4-carboxyalkylamidino-5-arylamino-isothiazoles as potent MEK1 inhibitors. Bioorg. Med. Chem. Lett., 16:3975–3980.
54.
VicentiniC.B., RomagnoliC., ManfrediniS., RossiD., and MaresD.. 2011. Pyrazolo [3,4-c]isothiazole and isothiazolo [4,3-d]isoxazole derivatives as antifungal agents. Pharm. Biol., 49:545–552.
55.
WangC.C., and TsouC.. 1993. Protein disulfide isomerase is both an enzyme and a chaperone. FASEB J., 7:1515–1517.
56.
WilkinsonB., and GilbertH.F.. 2004. Protein disulfide isomerase. Biochim. Biophys. Acta, 1699:35–44.
57.
WilliamsT.M.2007. The mechanism of action of isothiazolone biocide. Power Plant Chem., 9:14–22.
58.
WinderC.L., Al-AdhamI.S., Abdel MalekS.M., BuultjensT.E., HorrocksA.J., and CollierP.J.. 2000. Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1. J. Appl. Microbiol., 89:289–295.
59.
WisastraR., GhizzoniM., MaarsinghH., MinnaardA.J., HaismaH.J., and DekkerF.J.. 2011. Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF. Org. Biomol. Chem., 9:1817–1822.
60.
XuF.L., LinQ., and HouB.R.. 2009. Synthesis and bioactivity of novel benzisothiazolone derivatives as potential microbiocides. J. Heterocycl. Chem., 46:320–323.
61.
ZhangR.G., AnderssonC.E., SavchenkoA., SkarinaT., EvdokimovaE., BeasleyS., ArrowsmithC.H., EdwardsA.M., JoachimiakA., and MowbrayS.L.. 2003. Structure of Escherichia coli ribose-5-phosphate isomerase: A ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle. Structure, 11:31–42.
62.
ZhouG., ShiQ., OuyangY., and ChenY.. 2014. Involvement of outer membrane proteins and peroxide-sensor genes in Burkholderia cepacia resistance to isothiazolone. World J. Microbiol. Biotechnol., 30:1251–1260.
63.
ZhouJ., HaoD., WangX., LiuT., HeC., XieF., SunY., and ZhangJ.. 2006. An important role of a “probable ATP-binding component of ABC transporter” during the process of Pseudomonas aeruginosa resistance to fluoroquinolone. Proteomics, 6:2495–2503.
