1. Introduction
Staphylococcus aureus, a Gram-positive bacterium, can reside as a normal friendly commensal in up to two-thirds of healthy individuals [
1]. However,
S. aureus may be better known for its sinister side as one of the ESKAPEE opportunistic pathogens causing serious life-threatening infections, most notably those infections with methicillin-resistant
S. aureus (MRSA) [
2,
3]. This “Jekyll and Hyde” personality of
S. aureus may play a role in non-infectious disease as well. Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by recurring flares of erythema, edema, scaling, and itch. It affects 15–30% of children and approximately 5% of adults in industrialized countries [
4,
5]. More than 90% of AD patients are colonized on the lesional skin by
S. aureus [
6] and
S. aureus colonization and biofilm formation are directly associated with disease flares and remission [
7,
8]. Although treatment with lukewarm bleach baths and steroid creams or immunosuppressive, anti-histamine or anti-IL-4R drugs are approved for AD patients [
9,
10,
11,
12], most of these treatments have limited effectiveness and negative side effects. An alternative treatment approach is to specifically target the mechanism of AD pathogenesis which should lead to more effective prophylactic and therapeutic solutions without the negative side effects.
Quorum sensing is the mechanism by which bacteria sense, communicate and respond to their own cell density. This cell-to-cell communication system is used by bacteria to regulate a variety of physiological functions via activation of genes involved in virulence and biofilm formation [
13,
14].
S. aureus uses quorum sensing to invade tissues of the human body including skin [
8]. The operon controlling quorum sensing in
S. aureus is called the accessory gene regulator or
agr [
15,
16].
Agr might be thought of as the “Jekyll and Hyde gene”, regulating the dual personalities of
S. aureus. Activation of
agr causes the transformation of the friendly and commensal “Dr. Jekyll”
S. aureus into the evil and virulent “Mr. Hyde”
S. aureus.
The
agr system of
S. aureus contains two transcriptional units, RNAII and RNAIII that are transcribed in opposite orientation [
17]. In the
agr pathway, the cyclic thiolactone peptide pheromone called autoinducing peptide (AIP) generated from the AgrD precursor is released by the export protein AgrB and activates the AgrC/AgrA two-component signal transduction system. Upon binding of the AIP to the receptor kinase AgrC, the response regulator AgrA is activated and binds to the promoter regions in the
agr system (P2 for RNAII and P3 for RNAIII) [
18]. The production of RNAII and RNAIII leads to the production of virulence factors including the phenol soluble modulins (PSMs), a group of cytotoxic peptides that are important for the outcome of infections by community-associated MRSA and skin inflammation in a feedback loop [
19].
δ-toxin is a 26-amino acid PSM that stimulates rapid mast cell degranulation and release of histamine [
20]. Another PSM produced by
S. aureus during quorum sensing, PSMα, can promote skin inflammation in vitro in human keratinocytes [
21]. Using a mouse model of epicutaneous
S. aureus infection, Nakamura et al. found that δ-toxin and PSMα promote AD-like skin inflammation [
20]. Finally, expression of RNAIII is increased in the skin lesions of children with AD [
20]. The potential role of
agr expression and the quorum sensing system in AD etiology makes it an attractive mechanistic target for treatment intervention.
Quorum sensing inhibition or QSI has been proposed as an alternative therapeutic approach to conventional antibiotics. Since the target of QSI is bacterial virulence, not viability, there is less chance that the bacteria will develop resistance to the quorum sensing inhibitor [
22,
23]. The therapeutic potential of targeted anti-virulence therapy was demonstrated using Solonamide B. Solonamide B is a cyclodepsipeptides isolated from a marine bacterium
Photobacterium spp. with a tertiary structure remarkably like AIP. Due to this structural similarity, Solonamide B is a competitive inhibitor of AIP and interferes with AIP binding to AgrC, the sensor kinase. Solonamide B blocks δ-toxin synthesis and reduces
S. aureus-induced skin inflammation in an epicutaneous mouse model [
24]. More recently, Williams et al. showed that certain commensal strains of
S. hominis produce an AIP that interferes with
S. aureus agr toxin production and inflammation in murine skin [
25].
The mechanisms of bacterial virulence can be shared across plants and animals [
26,
27]. As such, plants have evolved sophisticated host defense mechanisms to prevent bacterial infection, many of which may have utility in human medicine. These include the terpenes and flavonoids present in plant essential oils [
28,
29]. Carnosic acid is a diterpene present in high levels in
Salvia and
Rosmarinus plant species [
30]. Certain
Rosmarinus officinalis L. (rosemary) cultivars can carry as much as 10% carnosic acid in the air-dried leaves. In the plant, carnosic acid helps to protect chloroplasts and chloroplastic membranes from oxidative stress [
31,
32]. Extracts of rosemary have a long history of medicinal use [
33]. For example, topically applied rosemary extract accelerates wound healing in a mouse model [
34] and is used in the treatment of androgenic alopecia [
35]. Rosemary extract contains in addition to carnosic acid, carnosol and rosmarinic acid both of which have antimicrobial, antioxidant and anti-inflammatory properties [
33,
36] (
Figure 1).
Nunez et al. used a high throughput in vitro assay to screen over 4000 compounds for their ability to inhibit
S. aureus agr expression [
37]. Carnosic acid at 10 μM was identified as one of the most potent compounds. Here we extend that observation to characterize carnosic acid and rosemary extracts for inhibiting
agr expression.
3. Discussion
Bacterial pathogens have numerous virulence mechanisms to allow them to enter, replicate within, and persist at the host sites [
38] but with only a few common mechanistic themes [
39]. The virulence factors produced by pathogenic organisms such as proteases, toxins, and biofilms, are largely responsible for the damage caused to the host tissue. As such, inhibiting these virulence factors, also known as anti-virulence technology, has emerged as an attractive therapeutic alternative to antibiotics [
17,
19,
40]. It is supposed that by specifically targeting the mechanisms of bacterial virulence, and not viability, the pathogenicity of the bacteria can be controlled without increasing the chance of developing a resistant phenotype. Moreover, by specifically targeting the virulence mechanisms, there is less chance of disrupting the composition of the commensal or beneficial microorganisms in and on the host.
Bacterial quorum sensing is a key mechanism of bacterial virulence. Quorum sensing is the cell-cell communication system used by bacteria to sense their population density and adapt to the environment [
13,
15]. There are several approaches to the design of effective therapeutics based on quorum sensing inhibition. Many directly and indirectly target inhibition of the amount or function of the peptide auto-inducers. Such inhibitors can be found in nature with Solonamide B as a perfect example [
41]. Since many host defense responses to bacterial pathogens are common to plants, insects and animals, it becomes obvious to look at plant-based small molecules as a rich source for new therapeutic agents based on quorum sensing inhibition [
27,
29].
The use of flavonoids and other small molecules from plants as anti-virulence agents has been reported previously and reader is directed to the excellent review by Wu et al. [
42]. Dong et al. showed that the flavonoid morin, used in traditional Chinese medicine, directly inhibits the hemolytic activity of aerolysin, the primary virulence factor of
A. hydrophila strains and protects catfish from
A. hydrophila infection [
43]. Eugenol, a constituent of clove oil, can inhibit quorum sensing in methicillin-resistant
S. aureus (MRSA) clinical strains [
44]. Low concentrations of tea tree oil (from M. alternifolia) containing terpinen-4-ol inhibit MRSA biofilm formation [
45]. Essential oil from
S. hortensis, containing carvacrol, terpinene and thymol, significantly down-regulates the
S. aureus hld gene expression at concentrations below the bacterial minimal inhibitory concentration [
46]. The triterpene saponin glycyrrhetinic acid isolated from liquorice is a potent inhibitor of
S. aureus alpha-haemolysin (Hla) at 1-8 μg/mL, concentrations well below its minimal inhibitory concentration (MIC) of >512 μg/mL and protects mice from
S. aureus pneumonia [
47]. Hydroalcoholic extracts of rosemary, similar to the extracts used in our study, have been shown to exhibit anti-MRSA biofilm activity as low as 20 μg/mL [
48]. De Olivira et al. have also found that rosemary extracts to be a safe and effective in controlling not only
S. aureus, but also
Candida albicans,
Enterococcus faecalis,
Streptococcus mutans, and
Pseudomonas aeruginosa in both mono- and polymicrobial biofilms. They suggested that extracts of rosemary should have potential as a therapeutic agent when added to formulations such as toothpastes, skin creams and soaps [
49].
In the present study, we have extended our understanding for how rosemary extracts may impart anti-virulence biological activity. We have shown that two primary constituents of rosemary leaves, in pure form, carnosic acid and carnosol, are potent and specific inhibitors of S. aureus RNAIII and psmα gene expression. Rosmarinic acid, a water-soluble rosemary phytochemical, was shown to be inactive in these assays. Nine different hydroalcoholic extracts of rosemary, with analytically determined amounts of carnosic acid and carnosol, also inhibited S. aureus virulence expression. Importantly, we observed this anti-virulence activity both in luciferase reporter strains of S. aureus as well as in clinical isolates from atopic dermatitis patients using direct qPCR quantification of gene expression.
The concentration of pure carnosic acid or carnosol required to specifically inhibit
S. aureus RNAIII and
psmα gene expression is in the low μM range (≥5 μM). The rosemary extracts tested were also potent RNAIII and
psmα gene expression inhibitors showing significant inhibition in the low μg/mL range (≥5 μg/mL). At 5 μg/mL, the total molar concentration of carnosic acid plus carnosol in Extract C2, is 2 μM, consistent with the results observed with the pure compounds. At these concentrations, there is little to no inhibition of bacterial growth. Endo et al. measured the minimal inhibitory concentration (MIC) of a hydroalcoholic extract of
R. officinalis for inhibition of various strains of MRSA in both planktonic and biofilm states [
48]. Rosemary extracts showed MICs ranges from 15.6–62.5 μg/mL for planktonic growth and as low 45-250 μg/mL for preformed biofilms, depending on the MRSA isolate.
In theory, the S. aureus bacteria present in and on AD skin lesions should come into direct contact with the topically applied formulation. That is, little if any penetration of carnosic acid through the stratum corneum barrier (compromised or not) should be necessary. This would imply that the concentration of carnosic acid in a topical formulation required to specifically inhibit agr, without killing the bacteria, should be similar to those observed in the in vitro experiments presented in this paper. This is an important consideration as we try to translate our lab results to clinical application.