Enhancement of hypericin nanoparticle-mediated sonoinduced disruption of biofilm and persister cells of Streptococcus mutans by dermcidin-derived peptide DCD-1L.

BACKGROUND: Streptococcus mutans is considered a major significant contributor to dental caries and its effective removal is difficult due to the formation of biofilm. Therefore, the development of adjuvant therapeutic strategies with anti-biofilm properties is a promising approach. In the present study, we examined the effect of dermcidin-derived peptide DCD-1 L on the antibacterial activity of hypericin nanoparticle (HypNP)-mediated antimicrobial sonodynamic therapy (aSDT) against persister cells growing- and biofilm cultures of S. mutans. MATERIALS AND METHODS: Following synthesis and confirmation of HypNP, the fractional inhibitory concentration (FIC) index of HypNP and DCD-1 L was determined by checkerboard assay. Cellular uptake of HypNP-DCD-1 L and generation of endogenous reactive oxygen species (ROS) were assessed and followed by the determination of antimicrobial sonoactivity of HypNP-DCD-1 L against persister cells growing- and biofilm cultures of S. mutans. The water-insoluble extracellular polysaccharide (EPS) and expression of the gtfD, comDE, and smuT genes were then evaluated in persister cells growing- and biofilm cultures of S. mutans. RESULTS: There was a synergistic activity in the combination of HypNP and DCD-1 L against S. mutans with an FIC index value of 0.37. The HypNP-DCD-1L-mediated aSDT also displayed the highest cellular uptake and endogenous ROS generation by bacterial cells. When biofilm and persister cells of S. mutans were treated with HypNP-DCD-1 L and subsequently exposed to ultrasound waves, 5.1 log and 3.8 log reductions, respectively, in bacterial numbers were observed (P<0.05). According to the data, EPS in both persister cells growing- and biofilm cultures of S. mutans were significantly decreased after exposure to the HypNP-DCD-1L-mediated aSDT (P<0.05). In addition, the quantitative real-time PCR data illustrated the high level of similarities in very low-expression profiles of the gtfD before and after all treated groups for persister cells. While, following HypNP-DCD-1L-mediated aSDT treatment, the expression levels of gtfD, comDE, and smuT were significantly lower in treated persister cells growing- and biofilm cultures of S. mutans in comparison with control groups (P<0.05). CONCLUSIONS: Combined, the results of this study indicate that ultrasound waves-activated HypNP-DCD-1 L can sonoinactivate S. mutans biofilms and persister cells, as well as reduce effectively pathogenicity potency of S. mutans. Hence, HypNP-DCD-1L-mediated aSDT may be proposed as a promising adjunctive therapeutic approach for dental caries.

Application of Topical Sandalore® Increases Epidermal Dermcidin Synthesis in Organ-Cultured Human Skin ex vivo.

INTRODUCTION: Several olfactory receptors (ORs) are expressed in human skin, where they regulate skin pigmentation, barrier function, wound healing, and hair growth. Previously, we found that the selective activation of OR family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® differentially stimulates the expression of antimicrobial peptides (AMPs) in human scalp hair follicle epithelium ex vivo. As OR2AT4 is also expressed by epidermal keratinocytes, we hypothesized that it may modulate intraepidermal AMP synthesis, thereby contributing to skin microbiome management. METHODS: We investigated this hypothesis in organ-cultured human skin in the presence of Sandalore® and antibiotics and evaluated epidermal production of two AMPs, LL37 (cathelicidin) and dermcidin (DCD), as well as OR2AT4, by quantitative immunohistomorphometry. Moreover, we quantified DCD secretion into the culture medium by ELISA and studied the effect of culture medium on selected bacterial and fungal strains. RESULTS: Topical application of Sandalore®to organ-cultured human skin increased OR2AT4 protein expression, the number of DCD-positive intraepidermal cells, and DCD secretion into culture media, without significantly affecting epidermal LL37 expression. In line with the significantly increased secretion of DCD into the culture medium, we demonstrated, in a spectrophotometric assay, that application of conditioned media from Sandalore®-treated skin promotes Staphylococcus epidermidis, Malassezia restricta, and, minimally, Cutibacterium acnes and inhibits Staphylococcus aureus growth. CONCLUSION: In addition to demonstrating for the first time that DCD can be expressed by epidermal keratinocytes, our pilot study suggests that topical treatment of human skin with a cosmetic odorant (Sandalore®) has the potential to alter the composition of the human skin microbiome through the selective upregulation of DCD. If confirmed, Sandalore® could become an attractive adjuvant, nondrug treatment for dermatoses characterized by dysbiosis due to overgrowth of S. aureus and Malassezia, such as atopic dermatitis and seborrheic dermatitis.

Aptamer decorated emodin nanoparticles-assisted delivery of dermcidin-derived peptide DCD-1L: Photoactive bio-theragnostic agent for Enterococcus faecalis biofilm destruction.

BACKGROUND: Despite the high success rate of root canal treatment, failures are observed in a broad range of cases. Therefore, the need for novel approaches with the development of new generations of antimicrobial agents and intracellular drug delivery systems as adjunctive therapy is undeniable. In this study, we investigated the antimicrobial effects of antimicrobial photodynamic therapy (aPDT) using dermcidin­derived peptide DCD­1L loaded onto aptamer-functionalized emodin nanoparticles (Apt@EmoNp-DCD-1L) against Enterococcus faecalis as one of the most common bacteria involved in recurrent root canal treatment failures. MATERIALS AND METHODS: Following preparation of EmoNp-DCD-1L, the binding of selected labeled Apt to EmoNp-DCD-1L was performed, followed by the specificity of Apt@EmoNp-DCD-1L to E. faecalis was determined. The antimicrobial potential of aPDT was then assessed after the determination of the minimum inhibitory concentration (MIC) of Apt@EmoNp-DCD-1L. The molecular docking analysis was conducted to evaluate the potential binding modes of EmoNp to the proteins involved in E. faecalis pathogenesis. Eventually, the anti-virulence capacity of Apt@EmoNp-DCD-1L-mediated aPDT was investigated via quantitative real-time PCR (qRT-PCR) assay following measurement of intracellular reactive oxygen species (ROS) generation. RESULTS: The binding specificity of Apt@EmoNp-DCD-1L to E. faecalis was confirmed by flow cytometry. The results showed that the cell viability of E. faecalis exposed to aPDT groups employing the sub-MIC doses of Apt@EmoNp-DCD-1L (7.8 and 15.6 µM) was significantly reduced compared to the control group (P < 0.05). Also, Apt@EmoNp-DCD-1L in combination with a blue laser light was capable of enhancing the anti-biofilm activity of aPDT against E. faecalis biofilm. Data obtained from the qRT-PCR analysis showed significant downregulation in the expression level of genes involved in bacterial biofilm formation after exposure to aPDT (P < 0.05). CONCLUSIONS: This in vitro study highlights that aPDT with the minimum concentration of Apt@EmoNp-DCD-1L can be considered as a targeted bio-theragnostic agent for the detection and elimination of E. faecalis in the dispersed and biofilm states.

Human Dermcidin Protects Mice Against Hepatic Ischemia-Reperfusion-Induced Local and Remote Inflammatory Injury.

Background: Hepatic ischemia and reperfusion (I/R) injury is commonly associated with surgical liver resection or transplantation, and represents a major cause of liver damage and graft failure. Currently, there are no effective therapies to prevent hepatic I/R injury other than ischemic preconditioning and some preventative strategies. Previously, we have revealed the anti-inflammatory activity of a sweat gland-derived peptide, dermcidin (DCD), in macrophage/monocyte cultures. Here, we sought to explore its therapeutic potential and protective mechanisms in a murine model of hepatic I/R. Methods: Male C57BL/6 mice were subjected to hepatic ischemia by clamping the hepatic artery and portal vein for 60 min, which was then removed to initiate reperfusion. At the beginning of reperfusion, 0.2 ml saline control or solution of DCD (0.5 mg/kg BW) or DCD-C34S analog (0.25 or 0.5 mg/kg BW) containing a Cys (C)→Ser (S) substitution at residue 34 was injected via the internal jugular vein. For survival experiments, mice were subjected to additional resection to remove non-ischemic liver lobes, and animal survival was monitored for 10 days. For mechanistic studies, blood and tissue samples were collected at 24 h after the onset of reperfusion, and subjected to measurements of various markers of inflammation and tissue injury by real-time RT-PCR, immunoassays, and histological analysis. Results: Recombinant DCD or DCD-C34S analog conferred a significant protection against lethal hepatic I/R when given intravenously at the beginning of reperfusion. This protection was associated with a significant reduction in hepatic injury, neutrophilic CXC chemokine (Mip-2) expression, neutrophil infiltration, and associated inflammation. Furthermore, the administration of DCD also resulted in a significant attenuation of remote lung inflammatory injury. Mechanistically, DCD interacted with epidermal growth factor receptor (EGFR), a key regulator of liver inflammation, and significantly inhibited hepatic I/R-induced phosphorylation of EGFR as well as a downstream signaling molecule, protein kinase B (AKT). The suppression of EGFR expression by transducing Egfr-specific shRNA plasmid into macrophages abrogated the DCD-mediated inhibition of nitric oxide (NO) production induced by a damage-associated molecular pattern (DAMP), cold-inducible RNA-binding protein, CIRP. Conclusions: The present study suggests that human DCD and its analog may be developed as novel therapeutics to attenuate hepatic I/R-induced inflammatory injury possibly by impairing EGFR signaling.

Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus.

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.

Antimicrobial peptides in human skin disease.

The skin continuously encounters microbial pathogens. To defend against this, cells of the epidermis and dermis have evolved several innate strategies to prevent infection. Antimicrobial peptides are one of the primary mechanisms used by the skin in the early stages of immune defense. In general, antimicrobial peptides have broad antibacterial activity against gram-positive and negative bacteria and also show antifungal and antiviral activity. The antimicrobial activity of most peptides occurs as a result of unique structural characteristics that enable them to disrupt the microbial membrane while leaving human cell membranes intact. However, antimicrobial peptides also act on host cells to stimulate cytokine production, cell migration, proliferation, maturation, and extracellular matrix synthesis. The production by human skin of antimicrobial peptides such as defensins and cathelicidins occurs constitutively but also greatly increases after infection, inflammation or injury. Some skin diseases show altered expression of antimicrobial peptides, partially explaining the pathophysiology of these diseases. Thus, current research suggests that understanding how antimicrobial peptides modify susceptibility to microbes, influence skin inflammation, and modify wound healing, provides greater insight into the pathophysiology of skin disorders and offers new therapeutic opportunities.