611 Pentapeptide to Control Acne
the pilosebaceous zones that are invaginated, sebum-rich and oxygen-poor.1,2 It has been
largely documented that dysbiosis of microbiota in relation to physiological, environmental,
or behavioral modifications favor pathologies.3
Acne is one of the primary causes of dermatological consultations due to the potential
psychological distress caused by the associated scarring stigma it leaves on the skin. A
2020 study conducted on 4,000 people demonstrated that 78% of them claim to have
acne breakouts on a regular basis—on the cheeks, chin, forehead, nose, or on the upper
part of the body.4 In addition, more than 90% of teenagers undergo strong changes in
the appearance of their skin linked to an overproduction of sebum and Cutibacterium acnes
(C. acnes) proliferation.
C. acnes, formerly called Propionibacterium acnes and Corynebacterium acnes, is an anaerobic
gram-positive rod-shaped bacterium. It lives both at the upper part of the stratum corneum,
in contact with oxygen, and deeper into hair follicles, where it faces an environment rich in
sebum and proteins, but that is poor in oxygen.5–7 C. acnes represents 90% of the microbial
flora of the pilosebaceous follicle, yet C. acnes growth and virulence vary according to its
ribotype and the environment it lives in. This could explain why this commensal bacterium
is described with or without pro-acne properties.8
Bacteria sense environmental modifications which alter their behavior.9 They can
cluster together and reach a critical quantity, a quorum, that triggers more cell-to-cell
communications, thanks to increasing concentrations of secreted molecules or enzymes.
A consequence of bacterial clustering is the production of a biofilm. Biofilm isolates
and protects these cells from environmental insults such as antibiotics and host defense
mechanisms.10 Hence, preventing quorum from being reached helps to control biofilm
production.
C. acnes can switch from an isolated form to dense colonies, forming a biofilm in the
depression of hair follicles.11,12 In hair follicle sheath sections of acne-prone volunteers,
C. acnes generated more biofilms than in non-acne prone volunteer samples. The
biofilm therefore appears to contribute to the virulence of this bacterium. Both C. acnes
multiplication and biofilm formation disturb hair follicle sheath physiology through the
increasing of C. acnes lipase activities. Lipase is an extracellular enzyme responsible for
production of irritant-free fatty acids that cause local irritation. Consequences are the
increase of proinflammatory mediators, of matrix protease activities, and of pilosebaceous
ducts clogging,13–14 all damaging neighboring tissues at various depths. Acne exists in
several forms, with or without inflammatory features, redness, blemishes, comedones, or
scars. This creates significant skin damage and psychological distress, leading a significant
portion of people with acne to seek medical advice.
Several prescription-based treatments are used to moderate C. acnes activities and to restore
skin surface smoothness by reducing blemishes. However, these treatments have severe
side effects: facial erythema, hair loss, coronary heart disease, fetal development issues,
depression, clothes staining, and irritation. Peptides are molecules that can be tailored
to achieve desirable selective and potent biological activity. More than 60 peptide-based
therapeutics already exist on the market and hundreds are currently in developmental
stages for treatment of various diseases (type 2 diabetes, irritable bowel syndrome,
acromegaly), as selective or broad spectrum microbicidal agents, and anticancer agents.15
Since pioneering works on peptides years ago, the cosmetic industry widely and safely uses
small and well characterized peptides with anti-aging properties: dermal compartment

612 JOURNAL OF COSMETIC SCIENCE
quality improvement, skin barrier property reinforcement, skin soothing, or hair follicle
pigmentation triggering.16–20 Pentapeptide Palmitoyl-Lysyl-Threonyl-Seryl-Lysyl-Serine
(pKTSKS) was selected among 30 candidates, designed and synthesized by our chemistry
department, that went under a first screening of their biological activities on both
normal human keratinocytes and fibroblasts (NHK, NHF). In our findings, pKTSKS
selectively acts on C. acnes growth, adhesion, and biofilm formation without significant
modulation of S. epidermidis population. It also reinforces epidermal barrier functions,
modulates interleukin-6 (IL-6) and -1Ra (IL-1Ra) production by skin cells, and improves
extracellular matrix protein synthesis such as collagens. pKTSKS significantly reduced
skin inflammatory marks, pockmark volume, and skin roughness. It is therefore the first
verified peptide acting specifically on C. acnes, preserving skin homeostasis, and in parallel
promoting extracellular matrix protein synthesis.
METHODS
PEPTIDE SYNTHESIS
Both peptides pKTSKS and KTSKS were synthesized using solid-phase peptide synthesis
with non-CMR (carcinogenic, mutagenic and reprotoxic) solvents. Peptides were made
using a modified standard fluorenylmethylcarbonyl (Fmoc) technology.21 Fmoc-L-Serine-
resin with sequential coupling of L-lysine, L-serine, L-threonine, and L-lysine (Iris Biotech,
Marktredwitz, Germany) derivatives using coupling agents followed by Fmoc-deprotection
steps were used. All amino acids used were of L-stereochemistry and of non-animal origin.
Finally, coupling of palmitic acid of RSPO (roundtable on sustainable palm oil) quality
(Stéarinerie Dubois, Ciron, France) was performed using a coupling agent followed by
removal of the resin part and of the lateral protecting groups. Pure peptides were obtained as
hydrochloride salts their purity was assessed by mass spectrometry and high-performance
liquid chromatography (MS/HPLC) (HPLC Agilent 1200 Agilent, Les Ulis, France), and
were above 90%.
BACTERIAL GROWTH ASSAYS
C. acnes strain ribotype-1 (RT-1 CIP53.117T-ATCC 6919) was obtained from the Pasteur
Institute (Paris, France) whereas RT-4 and RT-5 strains (HL045PA1 and HL043PA2
respectively) were obtained from BEI Resources (National Institute of Allergy and
Infectious Diseases and National Institutes of Health as part of the Human Microbiome
Project). All strains were routinely cultivated in modified medium-20 (3% tryptone,
0.05% L-cysteine hydrochloride, 0.1% triethanolamine (all Sigma Aldrich, Germany),
0.5% glucose (Cooper), and 2% yeast extract (Oxoid™ Thermo Fisher Scientific, Illkirch-
Graffenstaden, France). For growth studies, bacteria were seeded at 106 CFU (colony-
forming units)/mL in the same medium ± pKTSKS (6–12 ppm) or its solvent (same
culture medium +0.1% dimethyl sulfoxide [DMSO]). As these strains are anaerobic,
the plates were incubated for 1 week with BD GasPack™ (Thermo Fischer Scientific,
Illkirch-Graffenstaden, France) under anoxic conditions at 37°C. To monitor the growth
kinetic, samples were collected every day and medium turbidity (optical density: OD)
was measured at 600 nm to get growth curves. In parallel, bacteria were treated with