AAC Accepts, published online ahead of print on 7 April 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.01043-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Bactericidal activity of human skin fatty acid
1
Analysis of the Bactericidal Activity of the Human Skin Fatty Acid, Cis-6-Hexadecanoic Acid on
2
Staphylococcus aureus
3 4
Michaël L. Cartron1, Simon R. England2, Alina Iulia Chiriac3, Michaele Josten3, Robert
5
Turner1, Yvonne Rauter1, 4, Alexander Hurd1, Hans-Georg Sahl3, Simon Jones2 and Simon J
6
Foster1#
7
1
The Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield,
8 9
Western Bank, Sheffield S10 2TN, United Kingdom 2
Department of Chemistry, University of Sheffield, Dainton Building, Brook Hill, Sheffield. S3 7HF.
10
United Kingdom 3
11
Institute of Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn,
12 13 14
Sigmund-Freud-Str. 25, 53105 Bonn, Germany 4
Present address: Institute for Molecular and Clinical Immunology, Helmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
15 16
Running title: Bactericidal activity of human skin fatty acid
17 18
# Correspondent footnote: Simon J. Foster, the Krebs Institute, Department of Molecular Biology and
19
Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. Tel.:
20
0044 114 2224411, E-mail: [email protected]
21 22
1
Bactericidal activity of human skin fatty acid
23
Abstract
24
Human skin fatty acids are a potent aspect of our innate defences, giving surface protection
25
against potentially invasive organisms. They provide an important parameter in determining
26
the ecology of the skin microflora and alterations can lead to increased colonisation by
27
pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new
28
avenue to explore in the generation of control measures against drug resistant organisms.
29
Despite their importance the mechanism(s) whereby skin fatty acids kill bacteria have remained
30
largely elusive. Here we describe an analysis of the bactericidal effects of the major human skin
31
fatty acid, cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen, S. aureus.
32
Several, C6H concentration dependent, mechanisms were found. At high concentrations C6H
33
swiftly kills cells associated with a general loss of membrane integrity. However at lower
34
concentrations C6H is still able to kill, acting via disruption of the proton motif force, an
35
increase in membrane fluidity and effects on electron transfer. The design of analogues, with
36
altered bactericidal effects has begun to determine the structural constraints on activity and
37
paves the way for rational design of new antistaphylococcal agents.
38
2
Bactericidal activity of human skin fatty acid
39
Introduction
40
The human skin is an important protection against microbial infection. The epidermis provides a
41
physical barrier via an external cross-linked keratin layer upon a scaffold of keratinocytes. Most
42
importantly the skin also has a broad range of innate immunity components including antimicrobial
43
peptides and fatty acids ((1), (2) and (3)). Despite these properties the skin is host to a large and
44
complex microflora of over 1000 species (4).
45
commensals but there are also opportunist pathogens.
46
potentially dangerous organisms is thus of great interest as harnessing our innate defences may lead to
47
novel control regimes.
Many of these microorganisms are harmless How the skin is able to defend against
48
Staphylococcus aureus is an extremely versatile bacterium, whose primary niche is as a
49
commensal in the human nose. Despite being carried harmlessly by 30% of the human population this
50
organism is an opportunist pathogen causing many infections and deaths worldwide (5). The problem
51
is exacerbated by the alarming spread of antibiotic resistance, in particular methicillin resistant S.
52
aureus (MRSA) is prevalent in hospitals and is beginning to spread in the wider community.
53
ability of S. aureus to survive in the nares and on the skin is an important facet of its capacity to spread
54
from host to host. In particular skin fatty acids in sebum have been found to be potent staphylocidal
55
agents ((6), (3) and (7)). Fatty acids are an important facet of our innate defences and in fact a toll-like
56
receptor mediated pathway in mice leads to increased fatty acid production and protection against S.
57
aureus skin infections (8). The most important antistaphylococcal, human, skin fatty acid is cis-6-
58
hexadecanoic acid (C16:1Δ6, C6H) ((3) and (7)). Patients with atopic dermatitis show reduced C6H
59
levels and increased colonisation with S. aureus and topical treatment with C6H results in a decrease
60
in S. aureus levels (7). We have also shown that purified C6H can be used to treat both cutaneous and
61
systemic models of S. aureus disease (9). Fatty acids are also able to kill S. aureus within abscesses
62
(10). As well as being bactericidal, at sublethal concentrations human sebum and C6H can inhibit the
63
production of virulence determinants and the induction of antibiotic resistance by S. aureus and other
64
important pathogens (9). Thus fatty acids can debilitate potentially harmful bacteria at several levels.
The
65
In response to such a potent molecule, S. aureus possesses a number of resistance mechanisms,
66
which allow it to withstand skin fatty acids ((11) and (9)). We have found that the major S. aureus 3
Bactericidal activity of human skin fatty acid
67
surface protein IsdA is produced in response to the lack of available iron associated with the human
68
host and is required for nasal colonisation (9). IsdA contributes to skin fatty acid resistance by
69
rendering the cells more hydrophilic via its C-terminal domain. It is also this domain that is required
70
for survival of S. aureus on human skin. Thus the interaction between S. aureus and human skin fatty
71
acids is a crucial factor in its ability to colonize a host. Despite this importance it is still unknown as
72
to the bactericidal mechanism of action of skin fatty acids on S. aureus. The surfactant nature of these
73
compounds likely results in membrane perturbation. Fatty acids also inhibit many central metabolic
74
processes but this may occur indirectly via uncoupling of ATP synthesis ((12) and (13)). Also
75
accumulation and incorporation of linoleic acid ((12)
76
hydroperoxides ((15) and (16)).
77
staphylocidal skin fatty acid, C6H, on S. aureus. This revealed multiple mechanisms of killing via the
78
ability of C6H to disrupt essential membrane functions.
and (14)) may result in toxic lipid
This study aimed to elucidate the mode of action of the major
79 80
Materials and Methods
81
Bacterial strains and chemicals - S. aureus strain SH1000 was used in all assays except otherwise
82
stated. Inverted vesicles and membranes were prepared from Micrococcus flavus and Escherichia coli
83
K12. All chemicals were purchased from Sigma Aldrich except as otherwise stated. Radiolabelled
84
[14C]-UDP-GlcNAc was from Hartmann Analytic (Germany) and undecaprenyl phosphate (C55-P)
85
from Larodan Fine chemicals, (Sweden). Bacteria were routinely grow in BHI (Brain Heart Infusion)
86
medium aerobically, for anaerobic growth cells were grown in air tight anaerobic jars.
87
C6H Killing Assays - Bacteria were grown to an OD600 of c. 0.6 in iron limited, tryptic soy broth
88
(TSB), or chemically defined medium (CDM) (17). Cells were harvested by centrifugation at 3000 g
89
(4oC, 5 min) washed twice in sterile dH2O by centrifugation and resuspension. Cell suspensions (c.
90
2×108cfu/ml in appropriate buffers) were incubated at 37°C with and without C6H (and other
91
chemicals). Except when stated otherwise, all experiments were done in 20mM Mes pH 5.5 with 3
92
µg/ml C6H. Cell viability was determined by plating on TSB agar (9).
93
MICs - MICs were determined as previously described (9).
4
Bactericidal activity of human skin fatty acid
94
Assessment of membrane integrity - Membrane integrity was assayed by determination of the
95
permeability of the cells to propidium iodide (P-io). Nisin served as positive control for membrane
96
disruption. Bacteria were prepared as for the C6H killing assay (in 20 mM Mes) and P-io was added to
97
the cell suspension to a final concentration of 13 μM and fluorescence of the mixture was followed
98
with excitation at 535nm and emission at 617nm (18). After 1 min equilibration time C6H was added
99
to the assay (at 3 or 5µg/ml).
100
Effect of C6H on lipid II polymerization – Lipid II was purified as described by Schneider et al.
101
(19). The enzymatic activity of S. aureus PBP2 was determined by incubating 2.5 nmol lipid II in 100
102
mM Mes, 10 mM MgCl2 , pH 5.5, and 0, 2, 4, 20 and 40nM C6H in a total volume of 50 µl. The
103
reaction was initiated by the addition of 7.5 µg PBP2-His6 and incubated for 1.5 h at 30°C. Residual
104
lipid II was extracted from the reaction mixtures with n-butanol/pyridine acetate, pH 4.2 (1:1; v/v),
105
and analysed by TLC (silica plates, 60F254; Merck) using chloroform–methanol–water–ammonia
106
(88:48:10:1 v/v) as the solvent. (20). Spots were visualized by PMA staining reagent
107
(phosphomolybdic acid 2.5%, ceric-sulfate 1%, sulphuric acid 6%). After drying the TLC plate, spots
108
are developed by heating at 150°C (21).
109
Inhibition of the in vitro lipid II synthesis
110
Inhibition of lipid II synthesis was performed in vitro using membrane preparations of
111
Micrococcus luteus DSM 1790 as described by Schneider et al., (2004) (19) with the addition of
112
radiolabelled [14C]-UDP-GlcNAc. Membranes were isolated from lysozyme-treated cells by
113
centrifugation (40.000 x g, 60 min, 4oC), washed twice in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5,
114
and stored under liquid nitrogen until use. Reaction mixtures were carried out in a final volume of 75
115
µl containing 400 µg of membrane protein, 5 nmol C55-P, 50 nmol UDP-N-acetylmuramyl
116
pentapeptide (UDP-MurNAc-PP), 50 nmol [14C]-UDP-GlcNAc in 60 mM Tris-HCl pH 8, 5 mM
117
MgCl2 and 0.5% (w/v) Triton X-100. UDP-MurNAc-PP was purified as described previously (22).
118
C6H was added to the reaction mixture in molar ratios of 2:1 referring to the total amount of C55-P (5
119
nmol). After 1 h at 30°C, the lipids were extracted with 1 volume of n-butanol/6 M pyridine-acetate
120
(2:1, v/v), pH 4.2. The reaction products were separated by TLC (silica plates, 60F254; Merck) using
121
chloroform-methanol-water-ammonia (88:48:10:1) as solvent (20). Radiolabelled spots were 5
Bactericidal activity of human skin fatty acid
122
visualized using a biomolecular imager for radioisotope detection (Storm 820 PhosphorImager,
123
Amersham Biosciences) and the image was analyzed with ImageQuant TL v2005 (Nonlinear
124
Dynamics Ltd) software.
125
Determination of membrane potential (ΔΨ) using TPP+ - To 5 ml of cell suspension (prepared as
126
above), at 37oC, 5 μl of 3H-TPP (3H tetraphenylphosphonium; final concentration 1μCi/ml (0.74-1.48
127
TBq/mmol) was added. 100 μl samples were removed, washed with PBS by filtration and used to
128
calculate total emission. Where appropriate, valinomycin and/or C6H were added (20 and 3 μg/ml
129
final concentration, respectively) and samples prepared as above. Calculation of the membrane
130
potential was as previously described (23).
131 132
Assessment of pHin- The experiment and controls were performed as previously described by Breeuwer et al. (24) using cell prepared as above.
133
ATP assays– Cell suspensions were prepared as above, samples (200μl) were taken and 800μl
134
DMSO added. Cells were recovered by centrifugation (10,000 g, 5 min, RT) and the supernatant
135
removed for analysis of extracellular ATP. ATP levels were measured using a bioluminescence assay
136
(Sigma).
137
Respiration - Cells grown as above for the killing assays were resuspended to an OD600c. 1.0 in
138
iron limited TSB transferred to a Clark-type oxygen electrode chamber and oxygen consumption
139
recorded.
140
Membrane fluidity - Cell membrane fluidity was determined by measuring fluorescence
141
polarization with 1,6-diphenyl-1,3,5-hexatriene (DPH; Sigma), as described by Bayer et al. (25). A
142
1ml S. aureus cell suspension was prepared as above (OD600 of c. 1.0) and DPH (2 μM final
143
concentration) was added. Fluorescence polarization was measured immediately after addition of
144
inhibitor to the labelled sample (1ml) using an Edinburgh Instruments 199 spectrometer. The
145
excitation and emission wavelengths were 360 and 426 nm, respectively. The degree of fluorescence
146
polarization, or the polarization index (PI), was calculated from the following formula [IV − IH
147
(IHV/IHH)]/[IV + IH (IHV/IHH)], where I is the corrected fluorescence intensity and subscripts V and H
6
Bactericidal activity of human skin fatty acid
148
indicate the values obtained with vertical or horizontal orientation of the analyzer, respectively. The
149
lower that the PI value is, the more fluid is the membrane ((25), (26) and (27)).
150
INT reduction assay - Reduction of the iodonitrotetrazolium chloride (INT) by the components of the
151
electron transport chain (ETC) was performed as described by Smith & McFeters, (28) in 250 µl
152
volume reaction.
153
Preparation of inverted vesicles - Inverted vesicle were prepared using a method adapted from
154
Burstein et al. (29). Cells from a 1l TSB post-exponential phase culture (OD600 3-4) were harvested
155
by centrifugation (3,000 xg, 10 min, 4°C) washed with 100 ml buffer containing 50 mM Tris-HCl pH
156
8.0, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA. The pellet was resuspended with 5 ml of the
157
same buffer, the pH was adjusted to 8.0 and 10 µg/ml DNAse and 10 µg/ml RNAse added. The cells
158
were disrupted by mechanical lysis using a microorganism lysing kit (0.1 mm glass beads in 2 mL
159
standard tubes) in a Precellys®24 homogenizer. The crude homogenate was centrifuged at 30,000 xg,
160
(20 min, 4°C). The resulting supernatant was centrifuged at 175,000 xg (120 min, 4°C) and the pellet
161
containing the vesicles was finally resuspended in 2 ml of the above buffer supplemented with 10%
162
(v/v) glycerol and stored in liquid nitrogen.
163
Liposomes-. Liposomes were prepared and quantified according to Bonelli et al. (30). 2µmol of
164
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti polar lipid) was dried in a glass tube. 300µl
165
of a 50mM carboxyfluorescin, 50mM NaCl or KCl solution was added to the dried DOPC. The glass
166
tube was vortexed thoroughly, freeze thawed 10 times and then extruded through a 400nm pore
167
membrane. Liposomes were loaded on a Sephadex G50 column pre-equilibrated with the appropriate
168
buffer and harvested by centrifugation (13000 g,1 min, RT).
169
Production of C6H analogues - The synthesis of C6H analogues and BODIPY dyes were carried
170
out by sequential alkylation of alkynyl anions with appropriate alkyl and tetrahydropyranyl alkyl
171
bromides, followed by dichromate oxidation to the carboxylic acid. Comprehensive details will be
172
reported in due course. A method describing a similar strategy to produce related compounds has
173
recently been described in the literature (31).
174 175
Transmission Electron Microscopy - Samples were prepared and analysed as previously described (32). 7
Bactericidal activity of human skin fatty acid
176
Fluorescence Microscopy - 17 μM Bodipy 1(in DMSO) and 10 μl of a 1μg/ml solution of
177
BODIPY labelled vancomycin (Vancomycin, BODIPY® FL Conjugate, InvitrogenTM) were added to
178
1 mL of S. aureus (grown as above) and incubated for 5 minutes at 37 °C on a rotary wheel (20 rpm).
179
The cells were harvested by centrifugation (10,000 g, 1 min, 4°C) and washed, 3 times, by
180
resuspension in dH2O, fixed, mounted and examined by deconvolution microscopy as previously
181
described (33).
182
Ion level determination – Cells were grown and treated as for a killing assay described earlier. At
183
each time point required cells were harvested by centrifugation (10,000 g, 1 min, 4°C) and ion levels
184
within the supernatant were monitored via the ion chromatography service (Kroto Centre, University
185
of Sheffield).
186
Statistics – The students T-test was used where appropriate.
187 188
Results
189
Characterisation of the bactericidal effects of C6H - Previous work has demonstrated that C6H is
190
able to kill S. aureus ((9) and (7)). It is well established that the pH of the skin is slightly acidic (34)
191
and so the effect of pH on C6H sensitivity was tested (Fig. 1A). S. aureus shows greatest survival at
192
pH 5.5 (~skin pH) without C6H losing only 50% viability after 2hr. Paradoxically 5μg/ml C6H has its
193
greatest activity at pH 5.5 with only 0.5% survival after 2 hr treatment in MES buffer (Fig. 1B). There
194
is also a clear C6H dose dependence in S. aureus survival at pH 5.5 (Fig. 1C). At 10μg/ml C6H
195
viability is quickly lost (>99% death after 30min). At 3 and 5μg/ml C6H, 60 and 99% of cells were
196
nonviable after 30 min respectively.
197
In order to begin to determine the molecular mechanism of C6H bactericidal activity, propidium
198
iodide (P-io) was used. P-iofluorescence greatly increases upon binding to nucleic acids and so it can
199
be used as a marker for membrane integrity, when added to the extracellular milieu. At pH 5.5, only
200
concentrations of C6H above 3μg/ml led to a rapid loss of membrane integrity as shown by an
201
increase in fluorescence (Fig. 2A,B). However at 3μg/ml C6H the cells still die. This suggests that
202
death can occur independently of drastic membrane permeabilization.
8
Bactericidal activity of human skin fatty acid
203
Inhibition of C6H activity by salts- Potassium ions have previously been shown to affect fatty
204
acid activity on mitochondria (35). Addition of 50mM KCl to 50mM Mes pH 5.5 gave a significant
205
reduction in the rate of killing by 5μg/ml C6H (p value 0.03 at 60min) but to a lesser extent by 3μg/ml
206
C6H (p value 0.19 at 60min) (Fig. 1C). Addition of NaCl or CaCl2 also had a similar inhibitory effect
207
(data not shown). Salt inhibition of C6H activity further suggests more than one mode of killing,
208
disruption of membrane integrity and an uncharacterised mechanism at lower concentrations
209
(≤3μg/ml). The binding of P-io to nucleic acid was inhibited in the presence of salt hence preventing
210
the fluorescence assay.
211
Does C6H affect peptidoglycan synthesis? – Previously the lantibiotic nisin has been shown at low
212
concentrations to associate with lipid II (a precursor in the peptidoglycan biosynthesis pathway)
213
which leads to simultaneous inhibition of cell wall biosynthesis and formation of defined pores in the
214
cell membrane whilst at high concentration it has several other effects such as induction of autolysis
215
and destabilization of the lipid bilayer (36). Initially, using M. luteus isolated membranes, C6H was
216
found not to inhibit the synthesis of lipid II (data not shown). The effect of C6H on peptidoglycan
217
synthesis via binding to lipid II was then tested. Using nisin as a positive control, C6H was found to
218
have no apparent capacity to bind lipid II or to inhibit the activity of S. aureus PBP2, which has the
219
ability to polymerise lipid II (data not shown).
220
C6H affects the proton motive force (PMF)- Figure 1B demonstrates that S. aureus is more
221
susceptible to C6H at low pH. At pH 5.5 C6H, 3μg/ml leads to cell death (Fig. 1C), but without
222
apparent loss of membrane integrity (Fig. 2A). The action of C6H may involve movement of protons
223
across the membrane with C6H acting as a protonophore. The PMF has two components: membrane
224
potential (ΔΨ) and proton gradient (ΔpH) (PMF,Δp = ΔΨ − 59 ΔpH). The cells use both to generate
225
ATP using the flow of protons through the ATPase. A protonophore affects both components of the
226
PMF.
227
polarised (data not shown). Lower concentrations of C6H (3μg/ml) led to an apparent membrane
228
depolarisation (Fig. 2C) and then slower repolarisation, compared to valinomycin. Hence C6H affects
229
the ΔΨ component of the PMF.
At high concentrations of C6H (≥5μg/ml), the membrane depolarises rapidly to become un-
9
Bactericidal activity of human skin fatty acid
230
The ΔpH was measured using the fluorophore, cFDASE. Once intracellular, the ratio in the
231
emission at 530 nm from excitation at 500 and 440nm is a marker of pH. By calibrating that ratio it is
232
then possible to assess the intracellular pH. High concentrations of C6H (≥5μg/ml) led to a rapid drop
233
of internal pH within minutes resulting in an equilibration of the internal pH with the outside pH of 5.5
234
(data not shown). With 3μg/ml C6H, the internal pH drops rapidly by around 0.1 pH unit in 2min and
235
then stabilises (Fig. 3). It appears that at 3μg/ml C6H, protons are being carried into the cell even
236
though the membrane is still intact. Thus C6H has protonophore activity and at high concentrations
237
can act as a likely surfactant.
238
C6H reduces internal ATP levels -2 min after the addition of a high concentration of C6H
239
(≥5μg/ml) the ATP level inside of the cell drops by 85% whilst the percentage of ATP outside of cells
240
increases by ~40% (data not shown). This is consistent with the damage to the membrane, which
241
allows ATP to be released. With 3μg/ml C6H the cells lose around 93% ATP within 2 min incubation
242
(Table 1) whilst the ATP outside of the cell only increases ~10% compared to the untreated sample
243
(data not shown). The drop in ATP levels could be due to the use of energy by the cell to restore the
244
ΔΨ and ΔpH and/or cessation of ATP synthesis due to loss of the proton gradient required for its
245
production.
246
Effect of C6H in combination with other inhibitors-
In order to further elucidate the potential
247
mechanism of C6H activity its effects were determined in combination with inhibitors of known
248
function. The protonophore CCCP (carbonyl cyanide m-chloro phenyl hydrazone) is a very powerful
249
uncoupling agent that carries protons across the membrane resulting in loss of both ΔpH and ΔΨ (37).
250
C6H and CCCP have an additive effect on S. aureus killing (Table 2), with an associated drop in ATP
251
levels (Table 1).
252
Valinomycin is a potassium carrier, which equilibrates intra- and extracellular levels. It affects
253
ΔΨ and can either depolarise or hyperpolarise the cells dependent on extracellular K+ levels. When
254
valinomycin was used alone, this resulted in only a small loss of ATP and when used in combination it
255
prevents the drastic loss seen by C6H alone (Table 1).
256
hyperpolarisation in contrast to C6H (Fig. 2C) and in combination, the membrane becomes transiently
10
Valinomycin leads to membrane
Bactericidal activity of human skin fatty acid
257
hyperpolarised for 5 min prior to a slow return to control levels. With the combination of inhibitors
258
there is also an additive effect in terms of S. aureus killing (Table 2). Addition of KCl significantly
259
reduces the bactericidal affect of both C6H and valinomycin (Table 2).
260
DCCD (dicyclohexylcarbodiimide) is a direct inhibitor of the ATPase and affects the ΔpH.
261
DCCD causes significant death of S. aureus (Table 2) but when used in combination with C6H there
262
is no increase in killing. In addition DCCD alone only results in a small loss of ATP and in
263
combination with C6H more ATP is maintained than with C6H alone (Table 1). DCCD inhibits the
264
activity of the ATPase thus preventing the use of ATP whilst extruding protons. As DCCD treated
265
cells do not lose a significant amount of ATP under the conditions used in 20 mM Mes pH 5.5 (Table
266
1, P value 0.58) it would appear that the cells are not metabolically active in these conditions.
267
C6H inhibits the electron transport chain (ETC) - The effect of C6H on the ETC was studied
268
using inverted vesicles and iodonitrotetrazolium chloride (INT; (28)). INT is reduced by almost all the
269
components of the ETC forming iodonitrotetrazolium violet formazan (INF), which can be
270
colorimetrically quantified. NADH or succinate substrate was added to generate the electron flow.
271
Inhibition of the ETC downstream the site of INT reduction, i.e. closer to the terminal oxidase
272
(complex IV) should either increase or not affect the amount of formazan compared to control (28).
273
The effect of C6H on the ETC with Escherichia coli inverted vesicles is shown in Fig. 4 and was
274
confirmed with Micrococcus flavus inverted vesicles (data not shown). Inhibition of the first
275
components of the ETC, complex I by rotenone in the NADH reaction and complex II by malonate
276
when succinate was used as substrate, resulted in a decrease of INF formed whereas inhibition of
277
complex IV by sodium azide resulted in increased amount of INF (Fig 4).
278
produced higher amounts of formazan. However, no effect of the C6H was seen in the absence of an
279
electron flow, for instance when used in combination with rotenone or malonate. Moreover a
280
cumulative effect in combination with NaN3 was observed suggesting that C6H interferes with the
281
electron flow through the ETC. Confirmation will require the establishment of the assays using the
282
native S. aureus system.
283
Effect of C6H on external ion levels - Ion chromatography (IC) was used to determine the effects of
284
inhibitors (C6H and CCCP) on ion levels. Of the extracellular ions tested all were unchanged (data not 11
Similar to NaN3, C6H
Bactericidal activity of human skin fatty acid
285
shown) apart from K+, in the presence of inhibitors. As expected CCCP leads to K+ extrusion (6.49
286
+/-0.02 and 2.67+/-0.15 ppm for the CCCP treated and control respectively ), which most likely occurs
287
during re-establishment of the membrane potential. C6H also results in significant release of K+
288
(4.47+/-0.14 ppm; P value
Bactericidal activity of human skin fatty acid
1
Analysis of the Bactericidal Activity of the Human Skin Fatty Acid, Cis-6-Hexadecanoic Acid on
2
Staphylococcus aureus
3 4
Michaël L. Cartron1, Simon R. England2, Alina Iulia Chiriac3, Michaele Josten3, Robert
5
Turner1, Yvonne Rauter1, 4, Alexander Hurd1, Hans-Georg Sahl3, Simon Jones2 and Simon J
6
Foster1#
7
1
The Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield,
8 9
Western Bank, Sheffield S10 2TN, United Kingdom 2
Department of Chemistry, University of Sheffield, Dainton Building, Brook Hill, Sheffield. S3 7HF.
10
United Kingdom 3
11
Institute of Microbiology, Immunology and Parasitology, Medical Faculty, University of Bonn,
12 13 14
Sigmund-Freud-Str. 25, 53105 Bonn, Germany 4
Present address: Institute for Molecular and Clinical Immunology, Helmholtz Center for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
15 16
Running title: Bactericidal activity of human skin fatty acid
17 18
# Correspondent footnote: Simon J. Foster, the Krebs Institute, Department of Molecular Biology and
19
Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. Tel.:
20
0044 114 2224411, E-mail: [email protected]
21 22
1
Bactericidal activity of human skin fatty acid
23
Abstract
24
Human skin fatty acids are a potent aspect of our innate defences, giving surface protection
25
against potentially invasive organisms. They provide an important parameter in determining
26
the ecology of the skin microflora and alterations can lead to increased colonisation by
27
pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new
28
avenue to explore in the generation of control measures against drug resistant organisms.
29
Despite their importance the mechanism(s) whereby skin fatty acids kill bacteria have remained
30
largely elusive. Here we describe an analysis of the bactericidal effects of the major human skin
31
fatty acid, cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen, S. aureus.
32
Several, C6H concentration dependent, mechanisms were found. At high concentrations C6H
33
swiftly kills cells associated with a general loss of membrane integrity. However at lower
34
concentrations C6H is still able to kill, acting via disruption of the proton motif force, an
35
increase in membrane fluidity and effects on electron transfer. The design of analogues, with
36
altered bactericidal effects has begun to determine the structural constraints on activity and
37
paves the way for rational design of new antistaphylococcal agents.
38
2
Bactericidal activity of human skin fatty acid
39
Introduction
40
The human skin is an important protection against microbial infection. The epidermis provides a
41
physical barrier via an external cross-linked keratin layer upon a scaffold of keratinocytes. Most
42
importantly the skin also has a broad range of innate immunity components including antimicrobial
43
peptides and fatty acids ((1), (2) and (3)). Despite these properties the skin is host to a large and
44
complex microflora of over 1000 species (4).
45
commensals but there are also opportunist pathogens.
46
potentially dangerous organisms is thus of great interest as harnessing our innate defences may lead to
47
novel control regimes.
Many of these microorganisms are harmless How the skin is able to defend against
48
Staphylococcus aureus is an extremely versatile bacterium, whose primary niche is as a
49
commensal in the human nose. Despite being carried harmlessly by 30% of the human population this
50
organism is an opportunist pathogen causing many infections and deaths worldwide (5). The problem
51
is exacerbated by the alarming spread of antibiotic resistance, in particular methicillin resistant S.
52
aureus (MRSA) is prevalent in hospitals and is beginning to spread in the wider community.
53
ability of S. aureus to survive in the nares and on the skin is an important facet of its capacity to spread
54
from host to host. In particular skin fatty acids in sebum have been found to be potent staphylocidal
55
agents ((6), (3) and (7)). Fatty acids are an important facet of our innate defences and in fact a toll-like
56
receptor mediated pathway in mice leads to increased fatty acid production and protection against S.
57
aureus skin infections (8). The most important antistaphylococcal, human, skin fatty acid is cis-6-
58
hexadecanoic acid (C16:1Δ6, C6H) ((3) and (7)). Patients with atopic dermatitis show reduced C6H
59
levels and increased colonisation with S. aureus and topical treatment with C6H results in a decrease
60
in S. aureus levels (7). We have also shown that purified C6H can be used to treat both cutaneous and
61
systemic models of S. aureus disease (9). Fatty acids are also able to kill S. aureus within abscesses
62
(10). As well as being bactericidal, at sublethal concentrations human sebum and C6H can inhibit the
63
production of virulence determinants and the induction of antibiotic resistance by S. aureus and other
64
important pathogens (9). Thus fatty acids can debilitate potentially harmful bacteria at several levels.
The
65
In response to such a potent molecule, S. aureus possesses a number of resistance mechanisms,
66
which allow it to withstand skin fatty acids ((11) and (9)). We have found that the major S. aureus 3
Bactericidal activity of human skin fatty acid
67
surface protein IsdA is produced in response to the lack of available iron associated with the human
68
host and is required for nasal colonisation (9). IsdA contributes to skin fatty acid resistance by
69
rendering the cells more hydrophilic via its C-terminal domain. It is also this domain that is required
70
for survival of S. aureus on human skin. Thus the interaction between S. aureus and human skin fatty
71
acids is a crucial factor in its ability to colonize a host. Despite this importance it is still unknown as
72
to the bactericidal mechanism of action of skin fatty acids on S. aureus. The surfactant nature of these
73
compounds likely results in membrane perturbation. Fatty acids also inhibit many central metabolic
74
processes but this may occur indirectly via uncoupling of ATP synthesis ((12) and (13)). Also
75
accumulation and incorporation of linoleic acid ((12)
76
hydroperoxides ((15) and (16)).
77
staphylocidal skin fatty acid, C6H, on S. aureus. This revealed multiple mechanisms of killing via the
78
ability of C6H to disrupt essential membrane functions.
and (14)) may result in toxic lipid
This study aimed to elucidate the mode of action of the major
79 80
Materials and Methods
81
Bacterial strains and chemicals - S. aureus strain SH1000 was used in all assays except otherwise
82
stated. Inverted vesicles and membranes were prepared from Micrococcus flavus and Escherichia coli
83
K12. All chemicals were purchased from Sigma Aldrich except as otherwise stated. Radiolabelled
84
[14C]-UDP-GlcNAc was from Hartmann Analytic (Germany) and undecaprenyl phosphate (C55-P)
85
from Larodan Fine chemicals, (Sweden). Bacteria were routinely grow in BHI (Brain Heart Infusion)
86
medium aerobically, for anaerobic growth cells were grown in air tight anaerobic jars.
87
C6H Killing Assays - Bacteria were grown to an OD600 of c. 0.6 in iron limited, tryptic soy broth
88
(TSB), or chemically defined medium (CDM) (17). Cells were harvested by centrifugation at 3000 g
89
(4oC, 5 min) washed twice in sterile dH2O by centrifugation and resuspension. Cell suspensions (c.
90
2×108cfu/ml in appropriate buffers) were incubated at 37°C with and without C6H (and other
91
chemicals). Except when stated otherwise, all experiments were done in 20mM Mes pH 5.5 with 3
92
µg/ml C6H. Cell viability was determined by plating on TSB agar (9).
93
MICs - MICs were determined as previously described (9).
4
Bactericidal activity of human skin fatty acid
94
Assessment of membrane integrity - Membrane integrity was assayed by determination of the
95
permeability of the cells to propidium iodide (P-io). Nisin served as positive control for membrane
96
disruption. Bacteria were prepared as for the C6H killing assay (in 20 mM Mes) and P-io was added to
97
the cell suspension to a final concentration of 13 μM and fluorescence of the mixture was followed
98
with excitation at 535nm and emission at 617nm (18). After 1 min equilibration time C6H was added
99
to the assay (at 3 or 5µg/ml).
100
Effect of C6H on lipid II polymerization – Lipid II was purified as described by Schneider et al.
101
(19). The enzymatic activity of S. aureus PBP2 was determined by incubating 2.5 nmol lipid II in 100
102
mM Mes, 10 mM MgCl2 , pH 5.5, and 0, 2, 4, 20 and 40nM C6H in a total volume of 50 µl. The
103
reaction was initiated by the addition of 7.5 µg PBP2-His6 and incubated for 1.5 h at 30°C. Residual
104
lipid II was extracted from the reaction mixtures with n-butanol/pyridine acetate, pH 4.2 (1:1; v/v),
105
and analysed by TLC (silica plates, 60F254; Merck) using chloroform–methanol–water–ammonia
106
(88:48:10:1 v/v) as the solvent. (20). Spots were visualized by PMA staining reagent
107
(phosphomolybdic acid 2.5%, ceric-sulfate 1%, sulphuric acid 6%). After drying the TLC plate, spots
108
are developed by heating at 150°C (21).
109
Inhibition of the in vitro lipid II synthesis
110
Inhibition of lipid II synthesis was performed in vitro using membrane preparations of
111
Micrococcus luteus DSM 1790 as described by Schneider et al., (2004) (19) with the addition of
112
radiolabelled [14C]-UDP-GlcNAc. Membranes were isolated from lysozyme-treated cells by
113
centrifugation (40.000 x g, 60 min, 4oC), washed twice in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5,
114
and stored under liquid nitrogen until use. Reaction mixtures were carried out in a final volume of 75
115
µl containing 400 µg of membrane protein, 5 nmol C55-P, 50 nmol UDP-N-acetylmuramyl
116
pentapeptide (UDP-MurNAc-PP), 50 nmol [14C]-UDP-GlcNAc in 60 mM Tris-HCl pH 8, 5 mM
117
MgCl2 and 0.5% (w/v) Triton X-100. UDP-MurNAc-PP was purified as described previously (22).
118
C6H was added to the reaction mixture in molar ratios of 2:1 referring to the total amount of C55-P (5
119
nmol). After 1 h at 30°C, the lipids were extracted with 1 volume of n-butanol/6 M pyridine-acetate
120
(2:1, v/v), pH 4.2. The reaction products were separated by TLC (silica plates, 60F254; Merck) using
121
chloroform-methanol-water-ammonia (88:48:10:1) as solvent (20). Radiolabelled spots were 5
Bactericidal activity of human skin fatty acid
122
visualized using a biomolecular imager for radioisotope detection (Storm 820 PhosphorImager,
123
Amersham Biosciences) and the image was analyzed with ImageQuant TL v2005 (Nonlinear
124
Dynamics Ltd) software.
125
Determination of membrane potential (ΔΨ) using TPP+ - To 5 ml of cell suspension (prepared as
126
above), at 37oC, 5 μl of 3H-TPP (3H tetraphenylphosphonium; final concentration 1μCi/ml (0.74-1.48
127
TBq/mmol) was added. 100 μl samples were removed, washed with PBS by filtration and used to
128
calculate total emission. Where appropriate, valinomycin and/or C6H were added (20 and 3 μg/ml
129
final concentration, respectively) and samples prepared as above. Calculation of the membrane
130
potential was as previously described (23).
131 132
Assessment of pHin- The experiment and controls were performed as previously described by Breeuwer et al. (24) using cell prepared as above.
133
ATP assays– Cell suspensions were prepared as above, samples (200μl) were taken and 800μl
134
DMSO added. Cells were recovered by centrifugation (10,000 g, 5 min, RT) and the supernatant
135
removed for analysis of extracellular ATP. ATP levels were measured using a bioluminescence assay
136
(Sigma).
137
Respiration - Cells grown as above for the killing assays were resuspended to an OD600c. 1.0 in
138
iron limited TSB transferred to a Clark-type oxygen electrode chamber and oxygen consumption
139
recorded.
140
Membrane fluidity - Cell membrane fluidity was determined by measuring fluorescence
141
polarization with 1,6-diphenyl-1,3,5-hexatriene (DPH; Sigma), as described by Bayer et al. (25). A
142
1ml S. aureus cell suspension was prepared as above (OD600 of c. 1.0) and DPH (2 μM final
143
concentration) was added. Fluorescence polarization was measured immediately after addition of
144
inhibitor to the labelled sample (1ml) using an Edinburgh Instruments 199 spectrometer. The
145
excitation and emission wavelengths were 360 and 426 nm, respectively. The degree of fluorescence
146
polarization, or the polarization index (PI), was calculated from the following formula [IV − IH
147
(IHV/IHH)]/[IV + IH (IHV/IHH)], where I is the corrected fluorescence intensity and subscripts V and H
6
Bactericidal activity of human skin fatty acid
148
indicate the values obtained with vertical or horizontal orientation of the analyzer, respectively. The
149
lower that the PI value is, the more fluid is the membrane ((25), (26) and (27)).
150
INT reduction assay - Reduction of the iodonitrotetrazolium chloride (INT) by the components of the
151
electron transport chain (ETC) was performed as described by Smith & McFeters, (28) in 250 µl
152
volume reaction.
153
Preparation of inverted vesicles - Inverted vesicle were prepared using a method adapted from
154
Burstein et al. (29). Cells from a 1l TSB post-exponential phase culture (OD600 3-4) were harvested
155
by centrifugation (3,000 xg, 10 min, 4°C) washed with 100 ml buffer containing 50 mM Tris-HCl pH
156
8.0, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA. The pellet was resuspended with 5 ml of the
157
same buffer, the pH was adjusted to 8.0 and 10 µg/ml DNAse and 10 µg/ml RNAse added. The cells
158
were disrupted by mechanical lysis using a microorganism lysing kit (0.1 mm glass beads in 2 mL
159
standard tubes) in a Precellys®24 homogenizer. The crude homogenate was centrifuged at 30,000 xg,
160
(20 min, 4°C). The resulting supernatant was centrifuged at 175,000 xg (120 min, 4°C) and the pellet
161
containing the vesicles was finally resuspended in 2 ml of the above buffer supplemented with 10%
162
(v/v) glycerol and stored in liquid nitrogen.
163
Liposomes-. Liposomes were prepared and quantified according to Bonelli et al. (30). 2µmol of
164
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti polar lipid) was dried in a glass tube. 300µl
165
of a 50mM carboxyfluorescin, 50mM NaCl or KCl solution was added to the dried DOPC. The glass
166
tube was vortexed thoroughly, freeze thawed 10 times and then extruded through a 400nm pore
167
membrane. Liposomes were loaded on a Sephadex G50 column pre-equilibrated with the appropriate
168
buffer and harvested by centrifugation (13000 g,1 min, RT).
169
Production of C6H analogues - The synthesis of C6H analogues and BODIPY dyes were carried
170
out by sequential alkylation of alkynyl anions with appropriate alkyl and tetrahydropyranyl alkyl
171
bromides, followed by dichromate oxidation to the carboxylic acid. Comprehensive details will be
172
reported in due course. A method describing a similar strategy to produce related compounds has
173
recently been described in the literature (31).
174 175
Transmission Electron Microscopy - Samples were prepared and analysed as previously described (32). 7
Bactericidal activity of human skin fatty acid
176
Fluorescence Microscopy - 17 μM Bodipy 1(in DMSO) and 10 μl of a 1μg/ml solution of
177
BODIPY labelled vancomycin (Vancomycin, BODIPY® FL Conjugate, InvitrogenTM) were added to
178
1 mL of S. aureus (grown as above) and incubated for 5 minutes at 37 °C on a rotary wheel (20 rpm).
179
The cells were harvested by centrifugation (10,000 g, 1 min, 4°C) and washed, 3 times, by
180
resuspension in dH2O, fixed, mounted and examined by deconvolution microscopy as previously
181
described (33).
182
Ion level determination – Cells were grown and treated as for a killing assay described earlier. At
183
each time point required cells were harvested by centrifugation (10,000 g, 1 min, 4°C) and ion levels
184
within the supernatant were monitored via the ion chromatography service (Kroto Centre, University
185
of Sheffield).
186
Statistics – The students T-test was used where appropriate.
187 188
Results
189
Characterisation of the bactericidal effects of C6H - Previous work has demonstrated that C6H is
190
able to kill S. aureus ((9) and (7)). It is well established that the pH of the skin is slightly acidic (34)
191
and so the effect of pH on C6H sensitivity was tested (Fig. 1A). S. aureus shows greatest survival at
192
pH 5.5 (~skin pH) without C6H losing only 50% viability after 2hr. Paradoxically 5μg/ml C6H has its
193
greatest activity at pH 5.5 with only 0.5% survival after 2 hr treatment in MES buffer (Fig. 1B). There
194
is also a clear C6H dose dependence in S. aureus survival at pH 5.5 (Fig. 1C). At 10μg/ml C6H
195
viability is quickly lost (>99% death after 30min). At 3 and 5μg/ml C6H, 60 and 99% of cells were
196
nonviable after 30 min respectively.
197
In order to begin to determine the molecular mechanism of C6H bactericidal activity, propidium
198
iodide (P-io) was used. P-iofluorescence greatly increases upon binding to nucleic acids and so it can
199
be used as a marker for membrane integrity, when added to the extracellular milieu. At pH 5.5, only
200
concentrations of C6H above 3μg/ml led to a rapid loss of membrane integrity as shown by an
201
increase in fluorescence (Fig. 2A,B). However at 3μg/ml C6H the cells still die. This suggests that
202
death can occur independently of drastic membrane permeabilization.
8
Bactericidal activity of human skin fatty acid
203
Inhibition of C6H activity by salts- Potassium ions have previously been shown to affect fatty
204
acid activity on mitochondria (35). Addition of 50mM KCl to 50mM Mes pH 5.5 gave a significant
205
reduction in the rate of killing by 5μg/ml C6H (p value 0.03 at 60min) but to a lesser extent by 3μg/ml
206
C6H (p value 0.19 at 60min) (Fig. 1C). Addition of NaCl or CaCl2 also had a similar inhibitory effect
207
(data not shown). Salt inhibition of C6H activity further suggests more than one mode of killing,
208
disruption of membrane integrity and an uncharacterised mechanism at lower concentrations
209
(≤3μg/ml). The binding of P-io to nucleic acid was inhibited in the presence of salt hence preventing
210
the fluorescence assay.
211
Does C6H affect peptidoglycan synthesis? – Previously the lantibiotic nisin has been shown at low
212
concentrations to associate with lipid II (a precursor in the peptidoglycan biosynthesis pathway)
213
which leads to simultaneous inhibition of cell wall biosynthesis and formation of defined pores in the
214
cell membrane whilst at high concentration it has several other effects such as induction of autolysis
215
and destabilization of the lipid bilayer (36). Initially, using M. luteus isolated membranes, C6H was
216
found not to inhibit the synthesis of lipid II (data not shown). The effect of C6H on peptidoglycan
217
synthesis via binding to lipid II was then tested. Using nisin as a positive control, C6H was found to
218
have no apparent capacity to bind lipid II or to inhibit the activity of S. aureus PBP2, which has the
219
ability to polymerise lipid II (data not shown).
220
C6H affects the proton motive force (PMF)- Figure 1B demonstrates that S. aureus is more
221
susceptible to C6H at low pH. At pH 5.5 C6H, 3μg/ml leads to cell death (Fig. 1C), but without
222
apparent loss of membrane integrity (Fig. 2A). The action of C6H may involve movement of protons
223
across the membrane with C6H acting as a protonophore. The PMF has two components: membrane
224
potential (ΔΨ) and proton gradient (ΔpH) (PMF,Δp = ΔΨ − 59 ΔpH). The cells use both to generate
225
ATP using the flow of protons through the ATPase. A protonophore affects both components of the
226
PMF.
227
polarised (data not shown). Lower concentrations of C6H (3μg/ml) led to an apparent membrane
228
depolarisation (Fig. 2C) and then slower repolarisation, compared to valinomycin. Hence C6H affects
229
the ΔΨ component of the PMF.
At high concentrations of C6H (≥5μg/ml), the membrane depolarises rapidly to become un-
9
Bactericidal activity of human skin fatty acid
230
The ΔpH was measured using the fluorophore, cFDASE. Once intracellular, the ratio in the
231
emission at 530 nm from excitation at 500 and 440nm is a marker of pH. By calibrating that ratio it is
232
then possible to assess the intracellular pH. High concentrations of C6H (≥5μg/ml) led to a rapid drop
233
of internal pH within minutes resulting in an equilibration of the internal pH with the outside pH of 5.5
234
(data not shown). With 3μg/ml C6H, the internal pH drops rapidly by around 0.1 pH unit in 2min and
235
then stabilises (Fig. 3). It appears that at 3μg/ml C6H, protons are being carried into the cell even
236
though the membrane is still intact. Thus C6H has protonophore activity and at high concentrations
237
can act as a likely surfactant.
238
C6H reduces internal ATP levels -2 min after the addition of a high concentration of C6H
239
(≥5μg/ml) the ATP level inside of the cell drops by 85% whilst the percentage of ATP outside of cells
240
increases by ~40% (data not shown). This is consistent with the damage to the membrane, which
241
allows ATP to be released. With 3μg/ml C6H the cells lose around 93% ATP within 2 min incubation
242
(Table 1) whilst the ATP outside of the cell only increases ~10% compared to the untreated sample
243
(data not shown). The drop in ATP levels could be due to the use of energy by the cell to restore the
244
ΔΨ and ΔpH and/or cessation of ATP synthesis due to loss of the proton gradient required for its
245
production.
246
Effect of C6H in combination with other inhibitors-
In order to further elucidate the potential
247
mechanism of C6H activity its effects were determined in combination with inhibitors of known
248
function. The protonophore CCCP (carbonyl cyanide m-chloro phenyl hydrazone) is a very powerful
249
uncoupling agent that carries protons across the membrane resulting in loss of both ΔpH and ΔΨ (37).
250
C6H and CCCP have an additive effect on S. aureus killing (Table 2), with an associated drop in ATP
251
levels (Table 1).
252
Valinomycin is a potassium carrier, which equilibrates intra- and extracellular levels. It affects
253
ΔΨ and can either depolarise or hyperpolarise the cells dependent on extracellular K+ levels. When
254
valinomycin was used alone, this resulted in only a small loss of ATP and when used in combination it
255
prevents the drastic loss seen by C6H alone (Table 1).
256
hyperpolarisation in contrast to C6H (Fig. 2C) and in combination, the membrane becomes transiently
10
Valinomycin leads to membrane
Bactericidal activity of human skin fatty acid
257
hyperpolarised for 5 min prior to a slow return to control levels. With the combination of inhibitors
258
there is also an additive effect in terms of S. aureus killing (Table 2). Addition of KCl significantly
259
reduces the bactericidal affect of both C6H and valinomycin (Table 2).
260
DCCD (dicyclohexylcarbodiimide) is a direct inhibitor of the ATPase and affects the ΔpH.
261
DCCD causes significant death of S. aureus (Table 2) but when used in combination with C6H there
262
is no increase in killing. In addition DCCD alone only results in a small loss of ATP and in
263
combination with C6H more ATP is maintained than with C6H alone (Table 1). DCCD inhibits the
264
activity of the ATPase thus preventing the use of ATP whilst extruding protons. As DCCD treated
265
cells do not lose a significant amount of ATP under the conditions used in 20 mM Mes pH 5.5 (Table
266
1, P value 0.58) it would appear that the cells are not metabolically active in these conditions.
267
C6H inhibits the electron transport chain (ETC) - The effect of C6H on the ETC was studied
268
using inverted vesicles and iodonitrotetrazolium chloride (INT; (28)). INT is reduced by almost all the
269
components of the ETC forming iodonitrotetrazolium violet formazan (INF), which can be
270
colorimetrically quantified. NADH or succinate substrate was added to generate the electron flow.
271
Inhibition of the ETC downstream the site of INT reduction, i.e. closer to the terminal oxidase
272
(complex IV) should either increase or not affect the amount of formazan compared to control (28).
273
The effect of C6H on the ETC with Escherichia coli inverted vesicles is shown in Fig. 4 and was
274
confirmed with Micrococcus flavus inverted vesicles (data not shown). Inhibition of the first
275
components of the ETC, complex I by rotenone in the NADH reaction and complex II by malonate
276
when succinate was used as substrate, resulted in a decrease of INF formed whereas inhibition of
277
complex IV by sodium azide resulted in increased amount of INF (Fig 4).
278
produced higher amounts of formazan. However, no effect of the C6H was seen in the absence of an
279
electron flow, for instance when used in combination with rotenone or malonate. Moreover a
280
cumulative effect in combination with NaN3 was observed suggesting that C6H interferes with the
281
electron flow through the ETC. Confirmation will require the establishment of the assays using the
282
native S. aureus system.
283
Effect of C6H on external ion levels - Ion chromatography (IC) was used to determine the effects of
284
inhibitors (C6H and CCCP) on ion levels. Of the extracellular ions tested all were unchanged (data not 11
Similar to NaN3, C6H
Bactericidal activity of human skin fatty acid
285
shown) apart from K+, in the presence of inhibitors. As expected CCCP leads to K+ extrusion (6.49
286
+/-0.02 and 2.67+/-0.15 ppm for the CCCP treated and control respectively ), which most likely occurs
287
during re-establishment of the membrane potential. C6H also results in significant release of K+
288
(4.47+/-0.14 ppm; P value
