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. 2022 Oct 3;4(10):acmi000397. doi: 10.1099/acmi.0.000397

Development of skin sebum medium and inhibition of lipase activity in Cutibacterium acnes by oleic acid

Keisuke Nakase 1,*, Misato Momose 1, Tomoko Yukawa 1, Hidemasa Nakaminami 1
PMCID: PMC9675171  PMID: 36415741

Abstract

Cutibacterium acnes is associated with the exacerbated inflammation of acne vulgaris, which occurs through the immune induction and pathogenicity factor production. Sebum, which is not present in the growth medium currently used to study acne, is present in acne pustules in differing concentrations among the pathological stages, such as the initial formation and inflammatory phase. To evaluate the effect of C. acnes on inflammation exacerbation in acne pustules in vitro, we developed an skin sebum medium containing artificial sebum and studied the growth and pathogenicity factor production of C. acnes in the skin sebum medium.

The growth and lipase activity of C. acnes ATCC11828 were tested using skin sebum medium containing different sebum concentrations. Only lipase activity decreased in the skin sebum medium culture containing 0.5 % sebum when compared with that without sebum, while both growth and lipase activity decreased in cultures with 1.0 % sebum. Therefore, the growth and lipase activity of C. acnes changed in the presence of sebum. Furthermore, when the growth and lipase activity of C. acnes were tested in skin sebum medium containing sebum components, unsaturated fatty acids, such as oleic acid and triolein, led to a decrease in lipase activity without inducing a change in growth. In the presence of oleic acid, C. acnes lipase activity decreased noncompetitively in a concentration-dependent manner.

Our data showed that C. acnes growth and lipase activity changed upon sebum addition to the skin sebum medium, and acne inflammation caused by C. acnes needs to be studied under conditions similar to those in acne pustules.

Keywords: Acne vulgaris, Cutibacterium acnes, lipase, oleic acid, sebum

Introduction

Cutibacterium acnes , a skin bacterium, exacerbates acne vulgaris. C. acnes , which overgrows as a result of follicular occlusion, is associated with inflammation and cytotoxicity because it induces the production of inflammatory cytokines and produces enzymes such as lipase, hyaluronidase and protease [1–3]. Notably, C. acnes lipase has been studied as a target for acne treatment because it produces fatty acid-inducing inflammatory cytokines by hydrolysing triglycerides in the sebum [4]. Acne pustules, which store sebum, show different conditions than healthy follicles [5]. Acne pathology is known to involve comedo formation, sebum storage in follicles, inflammation induction and cicatrization by cellular necrosis [6]. C. acnes in acne pustules have also been reported to produce biofilms, which are related to the avoidance of antimicrobials and to the excessive production of the haemolysin Christie–Atkins–Munch–Peterson (CAMP) factor [7].

Human sebum consists of squalene, triglycerides, fatty acids and wax esters [5]. Although the sebum contributes to maintaining moisture on the skin surface, excessive fatty acids, especially oleic acid, lead to the development of acne by hyperkeratinization [8]. C. acnes strains with lipase activity metabolize triglycerides, producing fatty acids that play a role in inducing inflammation. Therefore, C. acnes pathogenicity has been evaluated as a contributory factor to acne vulgaris, was used to evaluate C. acnes pathogenicity [9, 10]. However, the in vitro culture medium is rich in nutrients, whereas the skin environment is poor in nutrients and sebum. Additionally, the sebum concentration in acne pustules is unknown and is estimated to change according to the pathological stage. Sebum was digested by C. acnes lipase to free fatty acids, which causes a failure of the skin barrier function by hyperkeratinization and induces an increase in inflammatory cytokines [11]. Although C. acnes has multiple genes encoding extracellular lipases, gehA is most related to C. acnes lipase activity [12]. Its expression differs among C. acnes strains classified into subspecies or by phylogenetic typing [9].

To understand the C. acnes phenotypes in acne pustules at each pathological stage, we developed a skin sebum medium as an in vitro acne pustule model, which is poor in nutrients and sebum, and used it to study C. acnes growth and lipase activity. Here, we studied the growth and pathogenicity factor production of C. acnes in our skin sebum medium.

Methods

Development of the skin sebum medium

Skin sebum medium was developed from low-nutrient broth (LNB) and artificial sebum, because acne pustules are poor in nutrients, including sebum. LNB [15.0 g/L tryptone (Oxoid, Hampshire, UK), 3.0 g/L yeast extract (Oxoid), and 2.0 g/L NaCl] was modified from the minimal medium used by Kishishita et al. for a sugar metabolism test [13]. Artificial sebum [12.5 w/w % squalene, 2.5 w/w % jojoba oil, 45.0 w/w % triolein, and 17.0 w/w % oleic acid] was prepared based on a report by Wertz et al. [14]. Jojoba oil (used as wax ester), squalene, oleic acid, and triolein were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Artificial sebum was added at 0.05–20 % (final concentration) in the skin sebum medium; there is currently no report on the optimum sebum concentration that should be used in culture media.

Analysis of growth and lipase activity in C. acnes

C. acnes subsp. defendens ATCC11828 (single-locus sequence typing [SLST], K9, type strain), which showed elevated lipase activity, was used in all of these studies. C. acnes subsp. acnes ATCC6919 (SLST A1, type strain), which is frequently isolated from healthy skin and acne pustules, and Staphylococcus aureus JCM2874 as type strains, were used in a portion of these studies [15]. C. acnes was grown in brain heart infusion broth (Oxoid) and inoculated at approximately 105 c.f.u./ml in skin sebum medium or LNB-containing sebum components. After 72 hr of anaerobic incubation, the bacterial population and lipase activity were measured. For the sebum components, palmitic acid and linoleic acid were purchased from FUJIFILM Wako Pure Chemical, while stearic acid and palmitoleic acid were purchased from Tokyo Chemical Industries (Tokyo, Japan).

Quantification of biofilm formation

Biofilm formation was quantified using a microtitre plate assay, as described in our previous study [16]. C. acnes was incubated anaerobically in brain heart infusion broth (BHI, Oxoid) for 72 hr. Bacterial cultures were adjusted to an OD of 0.2 in 150 µl of skin sebum medium containing a prepared sebum concentration, and inoculated into a 96-well flat-bottom plate (Thermo Fisher Scientific, MA, USA). After incubation for 72 hr at 37℃, supernatant was removed, and biofilms were washed with PBS and was stained with 0.5 % crystal violet. Planktonic crystal violet was washed with PBS, and biofilms were subsequently extracted with 160 µl of 30 % acetic acid. Biofilm quantification was performed by reading OD at 600 nm using a MULTISKAN microplate reader (Thermo Fisher Scientific). Results are shown as means±sd of three independent tests.

Measurement of lipase activity and quantification of mRNA for gehA

Lipase activity was measured using the method reported by Nakase et al. [10]. 4-Methylumbelliferyl oleate (4-MUO, Sigma-Aldrich, St. Louis, USA) was used as a lipase fluorescent substrate, and fluorescence intensity was measured using Varioskan Flash 2.4 (Thermo, MS, USA) with an excitation filter of 320 nm and an emission filter of 450 nm. The expression of the gehA gene encoding extracellular lipase was analysed using the method reported by Nakase et al. [10]. After anaerobic incubation of C. acnes ATCC11828 for 72 hr, total RNA was extracted using a High Pure RNA Isolation Kit (Roche). Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Gene expression was analysed by real-time PCR using THUNDERBIRDTM SYBR qPCR MIX (TOYOBO). qPCR analysis was conducted using the StepOne Real-Time PCR System (Thermo) set to 40 cycles of 95 °C for 15 sec and 60 °C for 40 sec. The amplification primers for gehA and endogenous control genes oxc were as follows: gehA-F, ACAGCCCCGACGGTATTC gehA-R;, GCTTGACCTTGCAGGAGAA oxc-F;, CTTGTCATCGGCGTATTCG; and oxc-R, CACTTCAAGCGGAAGGTGA. The results are averages of three independent experiments.

Enzyme inhibition assay on bacterial lipase

Bacterial lipase solution was used as the supernatant, and the bacterial culture was centrifuged at 3000 × g for 10 min and sterilized using a 0.45 µm pore size filter (ADVANTEC, Tokyo, Japan). C. acnes and Staphylococcus aureus were cultured in Gifu anaerobic (GAM) broth, modified Nissui (Nissui Pharmaceutical, Tokyo, Japan) containing sodium thioglycolate, hemin, and vitamin K1 in addition to basic nutrients under anaerobic conditions for 72 h, and tryptone soya broth (Oxoid, Hampshire, UK) under aerobic conditions for 24 hr, respectively. S. aureus strain JCM2874 was used as the strain type in this study. Bacterial lipase solutions of C. acnes and S. aureus (10 mM final concentration) were added to sebum components and incubated for 30 min at 37 °C. After centrifugation at 15,000 r.p.m. for 1 min, the lipase activity was measured [10]. The fatty acids elaidic acid (Cayman Chemical, Michigan, USA), erucic acid (Tokyo Chemical Industries), and sodium oleate (Tokyo Chemical Industries) were used as compounds similar to oleic acid and as sebum components. CaCl2 and BaCl2 (10 mM final concentration) were used as suppressors of lipase inhibitors [17].

Determination of the reaction rate of lipase in C. acnes

The inhibition pattern of oleic acid was evaluated using a Michaelis–Menten plot and a Lineweaver–Burk plot. Fivefold diluted lipase solution containing oleic acid was incubated for 30 min at 37 °C. The reacted solution was mixed with 0.6–80 µM 4-MUO, and the lipase activity was measured. Lipase for Biochemistry (FUJIFILM Wako Pure Chemical) was used to create a standard curve to calculate the reaction rate. Lipase activity values (fluorescence intensity) were converted to enzyme amounts (units) in the region of concentrations showing linearity. Data are shown as the mean±sem from three experimental replicates.

Statistical analysis

All data (figures and tables) are presented as means±sem of three experimental replicates. Two different groups were compared using Welch’s t-test as a parametric test.

Results

Lipase activity and growth of C. acnes in the presence of sebum and sebum components

The skin and acne pustules are low in nutrients and contain sebum, but limited quantities of sugar [18]. To evaluate C. acnes -induced exacerbated inflammation under the same conditions found in acne pustules in vitro, we developed skin sebum medium containing modified minimum medium and artificial sebum. C. acnes growth and lipase activity were tested in skin sebum medium containing different concentrations of sebum, because it is predicted that the sebum concentration changes based on the acne vulgaris pathology stages (Fig. 1a and b). In the culture grown in skin sebum medium containing 0.05 % sebum, no change in growth of C. acnes ATCC11828 was observed, while bacterial lipase activity was significantly increased relative to that in skin sebum medium without sebum (P<0.05) (Fig. 1a). Similarly, in medium containing 0.5–10% sebum, only lipase activity was significantly decreased. Furthermore, in medium containing 20 % sebum, both growth and lipase activity decreased. By contrast, no change in lipase activity was observed in C. acnes ATCC6919, which showed lower lipase activity than ATCC 11828, and growth was decreased in medium containing 20 % sebum (Fig. 1b). Therefore, C. acnes growth and lipase activity varied with sebum concentration and differed among strains.

Fig. 1.

Fig. 1.

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Growth and lipase activity of C. acnes in skin sebum medium containing different sebum concentrations. The bacterial population and lipase activity were analysed using bacterial solutions and supernatants incubated for 72 h in LNB containing different sebum concentrations in (a) ATCC11828 and (b) ATCC6919. Bars and error bars represent means and sem, respectively. The 0.05 % artificial sebum contained 0.15 mM squalene, 0.02 mM jojoba oil, 0.25 mM triolein and 0.3 mM oleic acid. * and † indicate that lipase activity and bacterial population, respectively, showed statistically significant differences compared to their controls using Welch’s t-test (P<0.05).

The sebum components causing changes in growth and lipase activity of C. acnes ATCC11828 were evaluated using C. acnes cultured in skin sebum medium containing sebum components (Fig. 2a to h). Growth and lipase activity did not vary in the culture on skin sebum medium containing jojoba oil and stearic acid compared to those without sebum components. However, adding 10 mM (highest tested concentration) palmitic acid, palmitoleic acid or linoleic acid to cultures decreased both growth and lipase activity. By contrast, adding oleic acid or triolein (10 mM) to cultures decreased lipase activity, but not growth.

Fig. 2.

Fig. 2.

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Growth and lipase activity of C. acnes ATCC11828 in the presence of sebum components. (a) Jojoba oil, (b) Squalene, (c) Triolein, (d) Palmitic acid (C16 : 0), (e) Stearic acid (C18 : 0), (f) Palmitoleic acid (C16 : 1)(n-7), (g) Oleic acid (C18 : 1)(n-9), (h) Linoleic acid (C18 : 2)(n-6). Bacterial populations and lipase activity were analysed using bacterial solutions and supernatants incubated for 72 h in LNB containing different concentrations of sebum components. Bars and error bars represent means and sem, respectively. * and † indicate that lipase activity and bacterial populations, respectively, showed statistically significant differences compared to their controls using Welch’s t-test (P<0.05).

Biofilm formation was evaluated using C. acnes ATCC11828 in skin sebum medium, and varied with varying sebum concentrations (Fig. 3). Biofilm formation was enhanced in the presence of sebum relative to its rate of formation in its absence (P<0.05). The greatest amounts of biofilm were observed in skin sebum medium containing 1 % sebum.

Fig. 3.

Fig. 3.

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Biofilm quantification in skin sebum medium. Bars and error bars represent means and sem, respectively. * indicates a significant difference (P<0.05) compared to media without sebum (0%) using Welch’s t-test.

Inhibition of C. acnes lipase activity by oleic acid

An exacerbation of acne vulgaris by C. acnes may be prevented by the development of novel medicines that selectively inhibit lipase activity. It is suggested that triolein acts as an oleic acid because it is decomposed to oleic acid and glycerol. To analyse the mechanism by which oleic acid decreases lipase activity, the expression of gehA encoding extracellular lipase was measured (Fig. 4). No difference in the expression level of this gene upon the addition of oleic acid and triolein was found.

Fig. 4.

Fig. 4.

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Quantitation of C. acnes gehA gene expression in the presence of oleic acid and triolein. NS, not significant. Total RNA was extracted from C. acnes cells incubated for 72 h in skin sebum medium with or without 10 mM oleic acid or triolein. Bars and error bars represent means and sem, respectively. Statistical analysis was performed using Welch’s t-test.

Subsequently, the direct reaction of C. acnes lipase and oleic acid led to a significant decrease in lipase activity (Table 1). Similarly, lipase activity decreased when triolein, palmitic acid, palmitoleic acid, and linoleic acid were added. In contrast, no change was observed in lipase activity of S. aureus, which is the most populous pathogenic skin bacterium, upon the addition of oleic acid (P=0.443). Therefore, we suggest that oleic acid directly reacts with C. acnes lipase and decreases its activity (Fig. 5). Oleic acid analogues were further tested to clarify the structures of compounds capable of inhibiting C. acnes lipase activity (Table 1). C. acnes lipase activity decreased upon addition of elaidic acid, a trans-isomer of oleic acid; erucic acid, a longer unsaturated fatty acid; and sodium oleate. Because sodium oleate mediated lipase inhibitory activity against Pseudomonas fraji decreases in the presence of bivalent metallic iron [17], when the reactivation experiment was conducted in the presence of bivalent metallic iron, reactivation of C. acnes lipase was not observed in the presence of CaCl2 and BaCl2. Therefore, oleic acid apparently inhibits C. acnes lipase via a different mechanism than that for the lipases of P. fraji and S. aureus .

Table 1.

C. acnes lipase inhibition by sebum components and fatty acids

Compound

Relative lipase activity (%)

C. acnes

S. aureus

Jojoba oil

126.7±9.6*

97.5±5.5

Squalene

116.2±16.7

97.5±3.3

Triolein

67.7±6.1*

102.9±1.5

Palmitic acid (C16 : 0)

90.4±2.9*

100.2±5.3

Stearic acid (C18 : 0)

96.4±15.2

99.3±6.0

Palmitoleic acid (C16 : 1)(n-7)

87.7±13.4*

108.4±6.6

Oleic acid (C18 : 1)(n-9)

36.1±12.9*

100.6±6.3

Linoleic acid (C18 : 2)(n-6)

72.0±8.3*

104.8±3.1

Elaidic acid (C18 : 1)(n-9)

73.3±11.8*

103.7±4.5

Erucic acid (C22 : 1)(n-9)

46.0±12.1*

102.8±5.6

Sodium oleate

3.4±1.1*

6.5±3.6*

Sodium oleate + BaCl2

6.9±3.3*

103.5±5.9

Sodium oleate + CaCl2

4.3±0.2*

101.8±4.8
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Lipase activity was evaluated relative to activity in the absence of compound, which was defined as 100%. Elaidic acid and erucic acid, which do not exist on the skin, were used as structural isomers of oleic acid. BaCl2 and CaCl2 were used to inhibit lipase and oleic acid.

∗ P < 0.05 vs. without any compound (positive control, 100%).

Fig. 5.

Fig. 5.

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Inhibition of C. acnes lipase activity by different concentrations of oleic acid. Bars and error bars represent means and sem, respectively. Lipase activity was analysed in bacterial supernatants directly mixed with oleic acid. * indicates a significant difference (P<0.05) compared with the control using Welch’s t-test.

Mode of inhibition of C. acnes lipase by oleic acid

The inhibition of lipase activity by oleic acid showed Michaelis–Menten kinetics. C. acnes lipase activity showed concentration-dependent inhibition (Fig. 6a, b). The inhibition levels were observed to be the same between 1 mM and 10 mM oleic acid, and 10 mM oleic acid was considered an excess amount. When the C. acnes lipase solution was prepared for the measurement of enzyme inhibition activity, a fivefold dilution solution that showed a linear correlation plot was determined as the appropriate concentration. The measured C. acnes lipase activity was converted to a unit of lipase based on a standard curve obtained from the activity of the lipase reference standard. From Michaelis–Menten kinetics, lipase activity (Vmax, 2.51 mUnit/min; Km, 3.55) was calculated in the absence of oleic acid (no inhibitor) (Fig. 6a). By contrast, lipase activity was calculated in the analysis with 0.01 (Vmax, 2.07 mUnit/min; Km, 3.09) and 10 mM (Vmax, 1.07 mUnit/min; Km, 3.17) of oleic acid. Vmax values decreased with oleic acid addition in a concentration-dependent manner, while no change in Km values was observed either with or without oleic acid. Similarly, in the analysis of Lineweaver–Burk kinetics, C. acnes lipase activity was also inhibited in a non-competitive and concentration-dependent manner upon oleic acid addition (Fig. 6b).

Fig. 6.

Fig. 6.

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Inhibition of C. acnes lipase activity by oleic acid. Enzyme activity analysis for lipase of C. acnes in a (a) Michaelis–Menten plot and (b) Lineweaver–Burke plot. A curve was created from the calculated values using the mean from three experimental replicates.

Discussion

There are many experimental models of acne vulgaris. Although models using keratinocytes or sebaceous cells are known in vitro, they have conditions different from the actual conditions in acne pustules [19, 20]. Similarly, anaerobic growth media, such as reinforced clostridial medium and Brucella broth, are very nutrient rich. Animal models developed for assessing C. acnes-induced exacerbated inflammation in vivo are difficult to evaluate, because the method causing acne vulgaris in animals has not been established, and there are no reports of animals that have C. acnes as a skin microbe. Therefore, to study the phenotype of C. acnes in acne pustules, we developed a skin sebum medium that models the nutrient conditions of acne pustules. C. acnes biofilm formation and pathogenicity factor production were evaluated using an in vitro acne model by Spittales et al. [21]. However, it is difficult to evaluate C. acnes phenotypes other than biofilm formation in the semi-solid medium used. Our developed medium allows variation in the sebum concentration, and evaluation of phenotypes including growth, lipase activity, and biofilm formation.

Using skin sebum medium, C. acnes growth, lipase activity and biofilm formation were observed to change based on the different sebum concentrations added. When C. acnes was cultured in skin sebum medium containing 0.5–10% sebum, a range which is assumed to reflect the sebum concentration of acne during the initial formation stage, C. acnes growth was not inhibited, but lipase activity significantly decreased. Furthermore, upon addition of 20 % sebum, which is assumed to mimic the later stage of acne, both C. acnes growth and lipase activity were significantly inhibited. Consequently, C. acnes is related to the exacerbation of inflammation by actively growing during the initial stages of acne vulgaris. Our data suggest that it is possible to use skin sebum medium to evaluate the C. acnes phenotype found in acne pustules in vitro. To evaluate C. acnes phenotypes in acne pustules, we considered that skin sebum medium containing 5 % sebum, which does not inhibit growth or lipase activity, is preferential. The pathogenicity of C. acnes in acne pustules can be investigated and clarified by using different phylogenetic types of C. acnes strains in this medium. One limitation of this study is that only C. acnes ATCC6919 and ATCC11828, which were classified as subsp. acnes and subsp. defendens, respectively, were selected as candidates for this study. Because the lipase activity of C. acnes ATCC11828 was higher than that of ATCC6919 in BHI broth, this strain was used in this study. For further study, phenotypic changes in clinical isolates need to be studied.

Pantothenic acid (vitamin B5) inhibits lipase activity without causing growth inhibition; however, its mechanism is not understood. Our data are the first to show that oleic acid directly inhibits C. acnes lipase activity noncompetitively. Similarly, triolein inhibited C. acnes lipase. The active component within triolein is postulated to be oleic acid digested by C. acnes lipase; however, triolein itself may inhibit C. acnes lipase. Sebum composition is different for each report, but it is mainly composed of triglycerides, fatty acids, wax esters and squalene [5, 22, 23]. In fatty acids, palmitic acid, palmitoleic acid and oleic acid are abundant in sebum [24]. Both oleic acid and other unsaturated fatty acids decreased lipase activity in our study. We tested both cis- and trans-unsaturated fatty acids to identify which chemical structure inhibited C. acnes lipase and both decreased lipase activity. In the study of the epidermal model, oleic acid caused a failure of the skin barrier function by hyperkeratinization and induced an increase in inflammatory cytokines [11]. Excessive unsaturated fatty acids, including oleic acid, are considered to be related to the development of acne vulgaris, because a similar pathology is observed in acne lesions. Therefore, topical application of unsaturated fatty acids may adversely affect the skin. Topical and oral antimicrobials are used for acne treatment, and the emergence of antimicrobial-resistant C. acnes has become a critical problem [15]. We believe that the development of novel medicines that inhibit only C. acnes lipase activity without antimicrobial activity, based on unsaturated fatty acids, may lead to decreased antimicrobial use and contribute to preventing the emergence of antimicrobial-resistant bacteria. Therefore, identification of the lipase inhibitory active site in oleic acid warrants further study.

Our data showed that the C. acnes phenotype changes in the presence of sebum and suggest that the effect of acne inflammation by C. acnes can be satisfactorily evaluated using skin sebum medium.

Funding information

This work was supported by the Matching Fund Subsidy for Private Schools in Japan.

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Footnotes

Abbreviations: LNB, low-nutrient broth; 4-MUO, 4-methylumbelliferyl oleate; SLST, single-locus sequence typing.

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