efflux j. antimicrob. chemother. 2005 bredin 1013 5
TRANSCRIPT
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Propyl paraben induces potassium efflux in Escherichia coli
Jerome Bredin, Anne Davin-Regli and Jean-Marie Pages*
Enveloppe Bacterienne, Permeabilite et Antibiotiques, IFR 48, Faculte de Medecine,
Universite de la Mediterranee, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
Received 27 October 2004; returned 22 December 2004; revised 15 February 2005; accepted 27 February 2005
Objectives: Parabens are currently used as antibacterial preservatives in pharmaceutical, cosmetic and
food products but there are no precise data concerning their activity on bacterial membranes.
Methods: We analysed the cytoplasmic potassium release during propyl paraben addition by using a
selective electrode. Various conditions were assayed to investigate the bacterial paraben suscepti-
bility. We compared the activity of propyl paraben with the activities of colicin A and polymyxin B.
Results: Propyl paraben induced potassium efflux that was related to the porin expression in the
bacterial outer membrane. In addition, the presence of spermine, previously described as an efficient
OmpF channel-blocker, protected susceptible cells against paraben activity.
Conclusions: Propyl paraben induced potassium release in susceptible Escherichia coli cells similar to
that observed with polymyxin B. Moreover, this efflux depended on porin channel activity. This
permeabilizing effect is probably related to antibacterial properties of paraben molecules.
Keywords: outer membrane proteins, membrane channels, porins, pore forming molecules
Introduction
Parabens are commonly added in pharmaceutical, cosmetic and
food products according to their wide antibacterial properties,
low toxicity, inertness and chemical stability.1 Although this
family of molecules is largely used for its antibacterial capacity,
our knowledge about its activity and bacterial target is quite
unclear.2 Some authors reported that paraben molecules could
induce an alteration of cell membrane properties.1,2 Moreover, at
the moment only few data report the existence of bacterial resist-
ance against this compound: Valkova et al.3 recently reported
the characterization of an esterase that is involved in the
hydrolysis of parabens in Enterobacter cloacae and Enterobacter
gergoviae. Despite these results, the first steps of parabenactivity, i.e. penetration and targeting, remain quite unclear and
the aim of this study is to decipher these key points.
In the present work, we used an in vivo approach based on K+
efflux measurements as a reporter of membrane alteration after
paraben addition. We established that propyl paraben alters the
integrity of bacterial membranes, favouring potassium release,
and that this mechanism is accelerated by the presence of func-
tional OmpF porin in the outer membrane. In addition, this pot-
assium release caused by propyl paraben was compared with the
effect of colicin A and polymyxin B, two antibacterial agentsthat induce bacterial membrane permeabilization.4
Materials and methods
Bacterial strains and susceptibility tests
The strain used in this work was Escherichia coli SM1005 strain
(F
, DlacU169, rpsL, relA, thiA, fibB, gyrA, ompC, ompF14) which
does not express OmpF or OmpC, and the derivative strain contain-
ing the plasmid pNLF encoding OmpF.4 Immunodetection assays
with specific porin antiserum were used to check the level of porin
synthesis in the strain as previously described. The standard disc
diffusion method on MuellerHinton (MH) agar was used. Approxi-
mately 104 cells were inoculated onto plates of MH agar. Variousamounts of propyl paraben or polymyxin B were loaded and the
corresponding zones of inhibition were measured after 18 h at 378C.
Evaluation of potassium efflux
The potassium efflux measurements were carried out after addition
of propyl paraben, colicin A or polymyxin B as previously
described.4 Briefly, cells grown to an A600 of 0.6 in Luria-Bertani
broth were collected by centrifugation and washed with 100mM
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*Corresponding author. Tel: +33-4-91-32-45-87; Fax: +33-4-91-32-46-06; E-mail: [email protected]..........................................................................................................................................................................................................................................................................................................................................................................................................................
Journal of Antimicrobial Chemotherapy (2005) 55, 10131015
doi:10.1093/jac/dki110
Advance Access publication 11 April 2005JAC
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q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
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sodium phosphate buffer, pH 7. After washing, the pellet was resus-
pended in 1/100 of culture volume in the same buffer containing 5%
glycerol. Cells (5 109
) were suspended in a glass cup filled with6 mL of the same buffer at 378C and under agitation. Propyl
paraben was injected when temperature and potassium fluxes were
equilibrated. Potassium concentration measurements were performed
with a K+-specific electrode and recorded. Colicin A was obtained
from our laboratory stock.4 The plotted values, presented as extra-
cellular K+ increases as previously described,4 are the means of
three independent experiments.
Results
Effect of propyl paraben on bacterial membranes
Potentiometric measurements performed with an ion-selective
electrode have been developed to follow membrane permeabili-
zation in turbid bacterial suspensions; the potassium electrode,
allowing fast, easy and continuous measurements, is especially
appropriate for analysing the action of antibacterial compounds
on the membrane.4,5 To investigate the effect of propyl paraben
on bacterial membranes, we added various concentrations to
OmpF-producing E. coli. A major concern with propyl paraben
solubility is an appropriate medium showing no adverse activity
on bacterial membranes. We thus tested propyl paraben concen-
trations up to 0.5 mg/mL. The results are presented in Figure 1(a).
At this concentration, the addition of propyl paraben induced a
significant linear K+ release. No lag time was observed after the
addition of paraben and the curve was quite linear with a slope of
about 10mM K+
/min for 0.5 mg/mL, whereas no significantrelease was obtained with lower amounts of propyl paraben.
It has been previously reported that polymyxin and colicin
induce an intracellular potassium efflux from E. coli cells.4,5 We
compared the K+ release induced by propyl paraben with the
curves resulting from polymyxin B or colicin A addition. Under
the same conditions, we observed propyl paraben-induced pot-
assium release kinetics that could be compared to those gener-
ated by polymyxin B (Figure 1a). These two release kinetics
contrasted with the kinetics obtained with colicin A. Concerning
this pore-forming protein, we observed a short lag time followed
by a linear rapid release, a typical curve of colicin activity, as
previously reported.4 This suggests that propyl paraben may
induce a K+ release with a profile and an efficiency close to that
obtained with polymyxin B.To correlate these results with the antibacterial activity of
propyl paraben, we measured the inhibition of E. coli growth.
The results presented in Table 1 indicate that only the porin-
producing cells were susceptible to propyl paraben under the
conditions used. In addition, the propyl paraben MIC for this
OmpF-producing strain was about 1 mg/mL. In contrast, no
significant change was observed for polymyxin B susceptibility
whatever the strain tested (Table 1).
OmpF channel participation in propyl paraben activity
A major concern with agents that destabilize the membrane
is the involvement of membrane proteins during the uptake and
travel to the inner target, e.g. the role of OmpF porin in colicinA activity.4 To clarify this point, we analysed the outer mem-
brane proteins that can modulate the activity of propyl paraben
by two independent methods. We measured the potassium leak-
age in: (i) an OmpF strain and in OmpF-producing cells; and
(ii) the presence or absence of spermine with the OmpF-produ-
cing cells. This polyamine has previously been demonstrated to
jam the OmpF channels and generate a protection against b-
lactams and fluoroquinolones using this penetration route
through the outer membrane.6
Under these conditions, a weak K+ release was observed after
the addition of propyl paraben to E. coli cells devoid of porin
while the expression of OmpF porin restored a significant potass-
ium efflux (Figure 1b). In OmpF-producing bacteria, the K+
release was severely decreased by the addition of spermine.
These results indicate that the level of propyl paraben activity
on the bacterial membrane depended on the presence of func-
tional porin channels in the outer membrane and are in accord-
ance with the inhibition measurements.
Discussion
The widespread nature of multidrug bacterial resistance mechan-
isms strongly affects the activity of all antibiotic families.
Consequently, it is interesting to investigate the effect of propyl
Figure 1. Effect of propyl paraben on intrabacterial potassium concentration. (a) Measurements of potassium release induced by colicin A (0.1 mg/mL, open
squares), polymyxin B (0.8 mg/mL, open triangles) and propyl paraben (0.5 mg/mL, open circles; 0.2 mg/mL, crosses). (b) Role of functional OmpF porin in
paraben activity: experiments were carried out on OmpF cells (open squares), on OmpF+ cells (open circles) and on OmpF+ cells in the presence of spermine
(1 mM, open triangles) with propyl paraben (0.5 mg/mL). Values were obtained from three independent measurements ( SD).
Bredin et al.
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paraben on bacterial membranes since this product is widely
used as an antimicrobial preservative.1 Despite various studies
carried out on eukaryotic and prokaryotic cells, there has been
no clear evidence to explain its antibacterial properties. This is a
key point since at this moment, several bacterial resistance
mechanisms against these products have been reported,1,3,7
including enzymic degradation or efflux pumps.
In this study, we report that propyl paraben can induce a
destabilization of bacterial membranes. The profile of potassium
release is similar to that obtained with polymyxin B, a drug
inducing outer membrane permeabilization.5 In addition, the
OmpF porin participates in the activity of propyl paraben. In
OmpF mutant cells or in the presence of spermine, an efficient
channel blocker,4 a drastic reduction in potassium release is
observed. These results are close to those previously reported
with colicin A: pore-forming colicin uses the OmpF channel to
penetrate and this process is altered by spermine.4 However, the
absence of OmpF only reduced the potassium efflux induced bypropyl paraben. The contribution of OmpF porin in propyl para-
ben activity is especially important taking into account the wide-
spread use of parabens in cosmetics. This molecule family may
favour the selection of low-level resistant Gram-negative bac-
teria with a very low porin expression as previously mentioned
for cefepime and imipenem, two b-lactam molecules: Gram-
negative bacteria expressing an altered porin pattern have a
noticeable level of b-lactam resistance.8 In addition, recent
studies described that the treatment of E. coli with p-hydroxy-
benzoic acid (pHBA), a compound structurally related to propyl
paraben, induced an overexpression of AaeAB, an efflux system
involved in pHBA resistance.9
It has been proposed that the cytotoxic action of parabens may
be linked to mitochondrial failure dependent on induction of
membrane permeability transition accompanied by the mitochon-
drial depolarization and depletion of cellular ATP through uncou-
pling of oxidative phosphorylation.1,2 These observations in
eukaryotic cells are in accordance with the activity of paraben on
bacterial membranes reported in this study. The parabens may
mimic the activity of pore-forming proteins or polymyxins,
which induce potassium release from the target cell. This potass-
ium efflux is the first clue of bacterial membrane leakage induced
by propyl paraben. In this context, it is worth considering the
paraben content in various standard cosmetic products. Rastogi
et al.10 have determined that a maximum of 0.32% propyl paraben
was detected in paraben-containing cosmetics, an amount signifi-
cantly above the concentration used in this study.
We can hypothesize that propyl paraben firstly induces the
permeabilization of bacterial membranes causing the release of
intracellular molecules and the de-energization of membranes.
This cascade of processes may support the antibacterial activity
of this family of preservatives.
Acknowledgements
We thank C. Bollet and J. Chevalier for discussions. J. B. was
funded by the Fondation pour la Recherche Medicale. This study
was supported by the Universite de la Mediterranee.
References1. Soni, M. G., Taylor, S. L., Greenberg, N. A. et al. (2002).
Evaluation of the health aspects of methyl paraben: a review of the
published literature. Food and Chemical Toxicology40, 133573.
2. Maillard, J. Y. (2002). Bacterial target sites for biocide action.
Symposium Series Society for Applied Microbiology31, 16S27S.
3. Valkova, N., Lepine, F., Labrie, L. et al. (2003). Purification and
characterization of PrbA, a new esterase from Enterobacter cloacae
hydrolyzing the esters of 4-hydroxybenzoic acid (parabens). Journal of
Biological Chemistry 278, 1277985.
4. Bredin, J., Simonet, V., Iyer, R. et al. (2003). Colicins, spermine
and cephalosporins: a competitive interaction with the OmpF eyelet.
Biochemical Journal 376, 24552.
5. Katsu, T., Nakagawa, H. & Yasuda, K. (2002). Interaction
between polyamines and bacterial outer membranes as investigatedwith ion-selective electrodes. Antimicrobial Agents and Chemotherapy
46, 10739.
6. Chevalier, J., Mallea, M. & Pages, J.-M. (2000). Comparative
aspects of norfloxacin, cefepime and spermine diffusion through the F
porin channel of Enterobacter cloacae. Biochemical Journal 348,
2237.
7. Chuanchuen, R., Karkhoff-Schweizer, R. R. & Schweizer, H. P.
(2003). High-level triclosan resistance in Pseudomonas aeruginosa is
solely a result of efflux. American Journal of Infection Control 31,
1247.
8. Pages, J.-M. (2004). Role of bacterial porins in antibiotic
susceptibility of Gram-negative bacteria. In Bacterial and Eukaryotic
Porins (Benz, R., Ed.), pp. 41 59. Wiley-VCH Verlag, Weinheim,
Germany.
9. Van Dyk, T. K., Templeton, L. J., Cantera, K. A. et al. (2004).Characterization of the Escherichia coli AaeAB efflux pump: a
metabolic relief valve? Journal of Bacteriology186, 7196204.
10. Rastogi, S. C., Schouten, A., de Kruijf, N. et al. (1995). Content
of methyl-, ethyl-, propyl-, butyl- and benzylparaben cosmetic products.
Contact Dermatitis 32, 2830.
Table 1. Susceptibilities of E. coli to propyl paraben and
polymyxin B
Inhibition diameter (mm)
Compound SM1005 SM1005, pNLF10
Propyl paraben0.1 mg 6a 6a
0.5 mg 6a 111 mg 6a 12
Polymyxin B1 mg 21 225 mg 25 25
E. coli cells, producing OmpF (SM1005, pNLF10) or devoid of porins(SM1005) were used. Values are means of two independent determinations.aNo inhibition, corresponds to disc/well diameter.
Propyl paraben alters bacterial membranes
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