Bacterial uptake of octyl ethanolamine increases with pH

Bacterial uptake of octyl ethanolamine increases with pH

FEMS Microbiology Letters Ol (1992) 1-17-152 's:~ 1992 Federation of European Microbiologic~d Societies ()378-1t)97/92/$IISIHI Published by Elsevier ...

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FEMS Microbiology Letters Ol (1992) 1-17-152 's:~ 1992 Federation of European Microbiologic~d Societies ()378-1t)97/92/$IISIHI Published by Elsevier

147

FEMSLE 047t)3

Bacterial uptake of octyi ethanolamine increases with pH Michael Sandin, Stig AIIcnmark and Lars Edcbo Ih7~artment ~I" ( "linical Bacteriology, Unit erxtty ot (;iJt('horg. (ffJtehorg, Swedept Received 26 November 1091 Accepted 23 Decembcr It~t~l

Key words: Octyl ethanolaminc; Antibacterial; Uptake; I:~eu&nnonaspseudoak'aligenes

1. SUMMARY

enhance membrane interaction and toxicity at high pH.

The uplake of octyl ethanolamine (CsEA) by

Pseudommtas pse,~h~ah'aligem's was dctcrmincd at pH 7.1-10.0. At pH O.l the total uptake was nearly three times highcr, and at pH ll).0 four times higher than at pH 7.1. Also the initial rate of uptake was lowest at pH 7.1. At pH 7.1 five to ten times higher concentrations of CsEA were needed than at pH 9.1 to achieve the same degree of leakage of cytoplasmic constituents. The results support the hypt~the~is that penetration of the bacterial cytoplasmic membrane by CsEA in its uncharged form is favoured. This takes place particularly with high pH in the suspending medium. In the cytoplasm, thc pH is lower, and CsEA becomes more protonatcd. This will prevent back diffusion, promote accumulation and

('orr,,spondence to: M, Sandin, Department tff Clinical Baeteriolot.y. University of G~iteborg, Guldhedsgatan II1. S-413 4h Gfteborg. Sweden.

2. INTRODU(_q'ION In our search for alternative antimicrobial agents for metal working fluids (MWFs), alkyl ethanolamines (alkylEA) have been studied. These and similar substanccs, which often arc used as anticorrosive agents in MWFs. have been found to possess antimicrobial properties [I], especially when used at a pH around and above 9 [2,3]. AlkylEA with alkyl chains up to 10 carbon atoms all showed higher antibacterial activity at pH 9.1 as compared to pH 7.1 [4]. Thc related compound dodecyldiethanolamine has been shown to damage the cytoplasmic membrane of Escherichia colt [5]. A mechanism of alkylEA, suggesting that the uncharged form penetrates the membrane and becomcs protonated within thc cell, has bccn proposed [2]. To study the bacterial uptake at different pH values, wc synthesized a radioisotopically labelled octyl ethanolaminc (CsEA), This paper deals with

148 the accumulation of Cs EA in Pseudomonas pseudoalcaligenes and its capacity to damage the cellular membrane.

3. MATERIAL AND METHODS

3.1. Bacterial stra#t attd growth condition P. pseudoalcaligenes (CCUG 15284) was originally cultivated from used MWF. For uptake and leakage studies it was grown in nutrient broth [2]. After overnight incubation at 30°C on an orbital shaker it was centrifuged, washed once and then resuspended in 10 mM phosphate-buffered saline (PBS, pH 7.1) or 51} mM carbonate buffered saline (CBS, pH 9.1; 9.5; 10,0) respectively. 3.Z Synthesis of I~Hl-octyl ethanolamhte and radiochromatography [!-3 H]-ethan. 1-ol-2-amine hydrochloride (Amersham, U.K.), 1.0 mCi was diluted to 6.11 mmol by non-radioactive ethanolamine. [3H]-octyl ethanolamine (3H-CsEA) was then prepared essentially as described previously [6]. The product was purified by column chromatography (silica gel column, 28 × 300 ram, particle size 0.0630.200 mm, with 99% methanol and 1% concentrated ammonia as the mobile phase). A total yield of 58% was obtained with a specific activity of 170 nCi/gmol. The purity of [3H]-CsEA was confirmed by radiochromatography. The detection was made essentially as described previously [7] with a 100#1 injector loop and a 5(}0-/el liquid scintillation cell. The mobile phase was acetonitrile/water (9: 1; v/v) containing I).75% triethylamine, and a flow rate of 5 ml/min was used. The chromatogram showed a single peak at 7.2 rain containing 98.6% of the total radioactivity. 3.3. Uptake studies A microtitcr plate with 400-ttl wells was used for the uptake studies. The final concentration of [3H]-CsEA was 6.25-100 /.tM and the bacterial concentration was 0.6-0.9 mg dry weight/ml. A 25-#1 volume of a stock solution of CsEA (62.51000 gM) was added to 225 p,I of bacterial suspension and incubated up to 31} min at room

temperature (23-25°C). The bacteria were removed by filtration on a 0.22-p,m Durapore membrane using Multiscreen Filtration plates (Millipore, Bedford, MA). The filtration process took 1-2 min. The radioactivity in the filtrate was then determined in a liquid scintillation counter LKB 1217 (LKB Wallac, Finland), with Ecoscint A (National Diagnostics, Manville, N J) as scintillation medium. All determinations were made in triplicate.

3.4. Leakage and MBC-smdies Volumes of 5-22/.tl of a CsFA stock solution (0.4-400 raM) were added to 200 p,I of a prewarmed bacteria! :,uspension in a microtiter plate. The bact.e,rial concentration was 2-2.5 x I0" cfu/ml: After 31} min of incubation at 30°C, a 170-gl volume was translerrcd to the filters, and the absorbance at 2611 nm of the filtrate was determined on a Shimadzu recording spectropho. tometer UV-240 (Shimadzu Corporation, Kyoto, Japan). After the incubation, 10-gl samples were diluted 31) times and 10 #1 of the diluted sample then transferred to a nutrient agar plate [2], Minimum bactericidal concentration (MBC), was defined as 99.99% killing of the bacteria. All determinations of leakage and of MBC were made in triplicate.

4. RESULTS The bacterial uptake of CsEA by P. psemloal-, caligenes increased with pH of the medium, The uptake at pH 9.1 was nearly three times higher, and at pH 10.0 four times higher than at pH 7.1 (Fig. I). The uptake during the first two min at pH 7.1 was only 1% of the added CsEA, as compared to 29% at pH 10.0. After 10 min the uptake at pH 7.1 and 10.0 had risen to 9% and 31%, respectively (Table 1). No further uptake was seen during the first hour. At pH 9.5 (Fig. 2) approximately the same proportion of the CsEA was taken up by the bacteria (27-36%) regardless of the concentration added. Exposure of P. pseudoalcaligenes to

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Fig. I. Uptake of [JII]-C~EA (ILl M)by P. pseud¢~ak'aligene,~ in 30 rain at 25°C, pH 7.1 (13): pH tLI (1~): ptl tL5 (~): ptl IO.O( i ) .

Table I Uptake of [~Iti]-C~EA by P. pscudoalcaligem~ at 23"C in rl of added substance Incubation time

pH

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10-12 rain

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cone. CxEA (/~M) Fig. 2. [~H].CsEA not taken up by P. I,wudout,'alieent~ in 15 min at pH t).5 and 23°C. Total radioltcti';i~v (t2); radioactivity after filtration (e); radioactivity after incubatitla with b~tt'teri~ and after filtratk~a (e).

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conc. CsEA (mM) Fig. 3. Leakage of cytoplasmic materials from P. pseudoah'uligenes al'ter 311 rain incubation with CxEA at 31FC. p}! 7.1 (e): pH 9.1 (o),

CsEA led to leak~:ge of cytoplasmic material (Fig. 3). At pH 7.1 a 5-Ill-times higher concentration was needed for the same degree of leakage as at pH 9.1. The MBC values at pH 7.1 and 9.1 were 20 mM and 4 mM respectively. At both MBCs about the same degree of leakage was found ( A.,,~ = 0.3).

5. DISCUSSION Low molccular mass amines, like methyl-, ethyl-, benzyl-, hc.'cylaminc and ethanolamine have been used as pH-probes for intracellular pH determ|tuition. The principle behind the method is lhe easier membrane penetration shown by the unprotonated amines [8-11]. The same principle accounts for thc ai,fimicrobial action of benzoic acid. whcrc the um'harged form has a higher ant|microbial effect [12], Another example is the enhanced antifungal effect of ammonium salts at higher pH values. This effect is duc to higher concentrations of flee ammonia in the solution [13]. The toxic mechanism of lysosomotropic agents is similar. Such agents, which are often long-chain amines, are tt~ought to penetrate the membranes of eukaryotic cells ie fi'~ir uncharged state into the acidic Jysosomes. Within the lysosotues thc amines become protonated and accumulate, evcntually killing the cell [14-16].

1511

Earlier we have shown a close connection between antibacterial activity and the concentration of the uncharged torm of alkanolamincs [2]. As hypothesized this is accompanied by enhanced uptake and accumulation of Ihe alkanolamine within the cell at higher pH (Fig. I). Since the amount of the added ethanolamine did not affect the proportion of bacterial uptake (Fig. 2), C~EA seems to form an equilibrium between the suspending medium and the bacterial cells. Calculations based on a bacterial dry weight of 25% of its wet weight [17] and a bacterial density of 1.1 mg//.tl [18], givc a bactcrial volumc of 0.3% of the total volume, and a concentration of CsEA which is 511-2(1tl times higher in the bacteria than in the suspending medium. The initial uptake was also more rapid at the higher pH values than at pH 7.1 (Table I), which fits with the model of high membrane permeability for the uncharged form of CsEA. When the C~EA has pcnctrated the cytoplasmic membrane in its uncharged state, it will face a more proton-rich environment within the cell [19,20]. This means that the C~EA becomes positively charged which prevents back-diffusion and promotes further accumulation. The positive charge renders cationic amphiphilic properties to the compound, which may act as a cationic detergent and bind to the cytoplasmic membrane from within (Fig. 4, path A). This results in membrane damage, leakage of cytoplasmic constituen' • rod finally death to the bacterial cell. The leakage at pH 9.1 increased with increasing concentration of CxEA, causing cell death when the leakage was great enough (Fig. 3). Despite the fact that less than 1% of CsEA is unprotonated at pH 7.1 there is a substantial uptake (Fig. !, Table 1). This may be due to binding of the uncharged species to the bacteria, leading to more unprotonated C~EA being formed in the suspending medium duc to shift of the equilibrium. However, also the protonated f~rm may accumulate in the cells, possibly by adhering to the membrane from the outside (Fig. 4, path B). Further, the protonated species of CxEA may accumulate in the cell in response to a membrane potential (Fig. 4, path C) which has recently been shown for dibucainc in E coli cclls

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Fig. 4, Proposed mechanism fi~r('sEA accumulation in bacterial cells.

[21]. All these mechanisms may contribute to the heavy leakage of cytoplasmic constituents at pit 7.1. The proportion of the uncharged species increases nearly three times when raising the pH from 9.1 to 10.0 (pK, of CsEA = 9.6), while the bacterial uptake increased only i.7 times. This may be duc to accumulation also of the protonatcd substance as well as changes in bacterial cell membrane(s) due to the high pH. The high toxicity and high bacterial uptake of CsEA at pH above 9 as compared to pH 7 makes the compound an attractive alternative as a preservative in alkaline fluids, e.g., MWFs. Selective toxicity seems to be achievable, since the pH-dcpendcnt cellular uptake" and toxicity will kill the microorganisms in the fluid without harmful effects to the more acid skin of th,: metalworkers.

ACKNOWLEDGEMENT This work was supported by the Swedish Work Environment Fund.

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151 [5] Lambert, P.A. and Smith, A.R.W. (10761 Microbios 15. 191-202. [6] Cope, A.C. and Hancock. E.M. 0942) J. Am. Chem. Soc. 64, 1503-1506. [7] Thompson, R.A., Lindstedt, M. and AIIcnmark. S. (19901 Anal. Lett. 23, 787-798. [8] Rottenberg, M. (19751 Bioenergetics 7, 01-74. [9] Azzone, G.F., Pietrobon, D. and Zoratti, M. (19841 in: Determination of the proton electrochemical gradient across biological membranes: Current topics in biocnergetics. Vol. 13, (Lee. C.P., Ed.), pp. 41-44. Academic Press. NY. [10] Kashkel, E.R. 0985) Ann. Rev. Microbiol. 39, 219-242. [11] Padan, E. and Schuldinen, S. (19861 In: Methods in Enzymology, Vol. 125, 337-352. [12] Albert, A. (19851 Selective Toxicity, pp 423-42,5. Chapman and Hall, London and New York. [13] DePasquale, D.A and Montville, TJ. (19901 Appl. Env. Mierobiol. 56, 3711-3717.

[14] deDuve. C., de Barsy, T., Pt~fle, B., Trouel, A., Tulkens. P. and Van I~h~)f. F. (1974). Biochem. Pharmacol. 23. 2405-2531. [15] Miller. D.K., Griftiths, E., Lcnard. J. and Firestone. R.A. (10831J, Cell Biol. 97, 1841-1851. [Ib] Hussain, M., Lcibowitz, M.J, and Lenard, J. (19871 Antimicrob. Agents Chemother. 31,512-517. [17] Lucia, S.E. (1060) In: The bacterial protoplasm: composition and organizalion: The Bacteria, Vol. h Structure (Gunsalus, I.C. and S~anier. R.Y., Ed.), [18] Baldwin, W.W., Shen, M.J.-T., Bankstone, P.W. and Woldlringh. C.L. (1988) J. Bact. 17(I. 452-455. [101 B~)th, I.R. (10851 Mi=robiol. Rev. 49, 350-378. [20] Krulwich. T.A. and Guffanti, A.A. (10891 Ann. Roy. Microhiol. 43, 435-463. [21] Collura. V. and Lelcllier, L. {199~1) Biochim Biophys. Acta. 11127,238-244.