Inhibitory and anti‐inflammatory effects of two antimicrobial peptides moronecidin and temporin‐1Dra against Propionibacterium acnes in vitro and in vivo

Proliferation of Propionibacterium acnes (P. acnes) is one of the main pathogenetic mechanisms of acne. Antimicrobial peptides with low‐drug resistance and nonresidual are potential anti‐acne agents. In this study, two antimicrobial peptides named temporin‐1Dra and moronecidin were synthesized and tested their antimicrobial activity against P. acnes in vitro and in vivo. These two peptides inhibited the growth of Escherichia coli, Staphylococcus aureus, Candida albicans, and P. acnes. The minimal inhibitory concentrations (MICs) of temporin‐1Dra and moronecidin to P. acnes were 30 and 10 μM, respectively. Both peptides exhibited strong resistance to heat and pH, but no obvious cytotoxicity to HaCaT cells. They also displayed persistent antimicrobial activities in the microbial challenge test. In the P. acnes‐induced inflammation mouse model, moronecidin significantly decreased the ear swelling thickness in a concentration‐dependent manner. At the 14th day after injection, 20 μg/day moronecidin reduced the ear swelling thickness to 46.15 ± 5.23% compared with the normal cream group. Tissue staining showed that moronecidin effectively reduced abscess and thickness of the dermis layer. Our results indicate that the antimicrobial peptide moronecidin could be developed as a potential natural anti‐acne agent in the cosmetics or pharmaceutical industries.     

Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole

The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic‐resistant P. acnes strains has been increasing.     

A Small Peptide with Therapeutic Potential for Inflammatory Acne Vulgaris

A designed peptide named LZ1 with 15 amino acid residues containing strong antimicrobial activity against bacteria pathogens of acne vulgaris including Propionibacterium acnes, Staphylococcus epidermidis and S. aureus. Especially, it exerted strong anti-P. acnes ability. The minimal inhibitory concentration against three strains of P. acnes was only 0.6 µg/ml, which is 4 times lower than that of clindamycin. In experimental mice skin colonization model, LZ1 significantly reduced the number of P. acnes colonized on the ear, P. acnes-induced ear swelling, and inflammatory cell infiltration. It ameliorated inflammation induced by P. acnes by inhibiting the secretion of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. LZ1 showed little cytotoxicity on human keratinocyte and hemolytic activity on human blood red cells. Furthermore, LZ1 was very stable in human plasma. Combined with its potential bactericidal and anti-inflammatory properties, simple structure and high stability, LZ1 might be an ideal candidate for the treatment of acne.     

Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris

Background

Acne vulgaris afflicts more than fifty million people in the United State and the severity of this disorder is associated with the immune response to Propionibacterium acnes (P. acnes). Systemic therapies for acne target P. acnes using antibiotics, or target the follicle with retinoids such as isotretinoin. The latter systemic treatment is highly effective but also carries a risk of side effects including immune imbalance, hyperlipidemia, and teratogenicity. Despite substantial research into potential new therapies for this common disease, vaccines against acne vulgaris are not yet available.

Methods and Findings

Here we create an acne vaccine targeting a cell wall-anchored sialidase of P. acnes. The importance of sialidase to disease pathogenesis is shown by treatment of a human sebocyte cell line with recombinant sialidase that increased susceptibility to P. acnes cytotoxicity and adhesion. Mice immunized with sialidase elicit a detectable antibody; the anti-sialidase serum effectively neutralized the cytotoxicity of P. acnes in vitro and P. acnes-induced interleukin-8 (IL-8) production in human sebocytes. Furthermore, the sialidase-immunized mice provided protective immunity against P. acnes in vivo as this treatment blocked an increase in ear thickness and release of pro-inflammatory macrophage inflammatory protein (MIP-2) cytokine.

Conclusions

Results indicated that acne vaccines open novel therapeutic avenues for acne vulgaris and other P. acnes-associated diseases.     

Propionibacterium acnes CAMP factor and host acid sphingomyelinase contribute to bacterial virulence: potential targets for inflammatory acne treatment

Background

In the progression of acne vulgaris, the disruption of follicular epithelia by an over-growth of Propionibacterium acnes (P. acnes) permits the bacteria to spread and become in contact with various skin and immune cells.

Methodology/Principal Findings

We have demonstrated in the present study that the Christie, Atkins, Munch-Peterson (CAMP) factor of P. acnes is a secretory protein with co-hemolytic activity with sphingomyelinase that can confer cytotoxicity to HaCaT keratinocytes and RAW264.7 macrophages. The CAMP factor from bacteria and acid sphingomyelinase (ASMase) from the host cells were simultaneously present in the culture supernatant only when the cells were co-cultured with P. acnes. Either anti-CAMP factor serum or desipramine, a selective ASMase inhibitor, significantly abrogated the P. acnes-induced cell death of HaCaT and RAW264.7 cells. Intradermal injection of ICR mouse ears with live P. acnes induced considerable ear inflammation, macrophage infiltration, and an increase in cellular soluble ASMase. Suppression of ASMase by systemic treatment with desipramine significantly reduced inflammatory reaction induced by intradermal injection with P. acnes, suggesting the contribution of host ASMase in P. acnes-induced inflammatory reaction in vivo. Vaccination of mice with CAMP factor elicited a protective immunity against P. acnes-induced ear inflammation, indicating the involvement of CAMP factor in P. acnes-induced inflammation. Most notably, suppression of both bacterial CAMP factor and host ASMase using vaccination and specific antibody injection, respectively, cooperatively alleviated P. acnes-induced inflammation.

Conclusions/Significance

These findings envision a novel infectious mechanism by which P. acnes CAMP factor may hijack host ASMase to amplify bacterial virulence to degrade and invade host cells. This work has identified both CAMP factor and ASMase as potential molecular targets for the development of drugs and vaccines against acne vulgaris.     

Surfactin inhibits the growth of Propionibacterium acnes by destroying the cell wall and membrane

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 μg ml⁻¹ effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 μg ml⁻¹ of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K⁺, and Ca²⁺, membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes–induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.     

Macrophages largely contribute to heterologous anti‐Propionibacterium acnes antibody‐mediated protection from Actinobacillus pleuropneumoniae infection in mice

Actinobacillus pleuropneumoniae is the causative agent of acute and chronic pleuropneumonia. Propionibacterium acnes is a facultative anaerobic gram‐positive corynebacterium. We have previously found that anti‐P. acnes antibodies can prevent A. pleuropneumoniae infections in mice. To investigate the role of macrophages in this process, affinity‐purified anti‐P. acnes IgG and anti‐A. pleuropneumoniae IgG were used in opsonophagocytosis assays. Additionally, the efficacy of passive immunization with P. acnes serum against A. pleuropneumoniae was tested in macrophage‐depleted mice. It was found that anti‐P. acnes IgG had an effect similar to that of anti‐A. pleuropneumoniae IgG (P > 0.05), which significantly promotes phagocytosis of A. pleuropneumoniae by macrophages (P < 0.01). It was also demonstrated that, after passive immunization with anti‐P. acnes serum, macrophage‐replete mice had the highest survival rate (90%), whereas the survival rate of macrophage‐depleted mice was only 40% (P < 0.05). However, macrophage‐depleted mice that had been passively immunized with naïve serum had the lowest survival rate (20%), this rate being lower than that of macrophage‐replete mice that had been passively immunized with naïve serum. Overall, anti‐P. acnes antibodies did not prevent A. pleuropneumoniae infection under conditions of macrophage depletion (P > 0.05). Furthermore, in mice that had been passively immunized with anti‐P. acnes serum, macrophage depletion resulted in a greater A. pleuropneumoniae burden and more severe pathological features of pneumonia in lung tissues than occurred in macrophage‐replete mice. It was concluded that macrophages are essential for the process by which anti‐P. acnes antibody prevents A. pleuropneumoniae infection in mice.     

Inducible Siphoviruses in superficial and deep tissue isolates of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>

Background  

Propionibacterium acnes is a commensal of human skin but is also known to be involved in certain diseases, such as acne vulgaris and infections of orthopaedic implants. Treatment of these conditions is complicated by increased resistance to antibiotics and/or biofilm formation of P. acnes bacteria. P. acnes can be infected by bacteriophages, but until recently little has been known about these viruses. The aim of this study was to identify and characterize inducible phages from P. acnes on a genetic and morphological basis.     

Antimicrobial activity of enterocins from Enterococcus faecalis SL-5 against Propionibacterium acnes, the causative agent in acne vulgaris, and its therapeutic effect

A lactic acid bacterial strain was isolated from human fecal specimen and identified as Enterococcus faecalis SL-5. The isolated strain showed antimicrobial activity against Gram-positive pathogens assayed, especially the highest activity against Propionibacterium acnes. The antimicrobial substance was purified and verified as a bacteriocin (named ESL5) of E. faecalis SL-5 by activity-staining using P. acnes as an indicator. N-terminal sequence of ESL5 was determined (MGAIAKLVAK) and sequence analysis revealed that it is almost identical to the some of enterocins including L50A/B of E. faecium L50 and MR10A/B of E. faecalis MRR 10-3. From the sequencing data of L50A/B structural genes, the nucleotide sequence showed 100% identity with that of the MR10A/B structural genes, implying that ESL5 is an equivalent of enterocin MR10. Meanwhile, we also tested the therapeutic effect of anti-P. acnes activity in patients with mild to moderate acne because of its pathogenic role to acne vulgaris. For this purpose, a concentrated powder of CBT SL-5 was prepared using cell-free culture supernatant (CFCS) of E. faecalis SL-5 and included in a lotion for application in the patients. The study showed that CBT SL-5 lotion significantly reduced the inflammatory lesions like pustules compared to the placebo lotion. Therefore our results indicate that the anti-P. acnes activity produced by E. faecalis SL-5 has potential role to the treatment of acne as an alternative to topical antibiotics. These authors contributed equally to this work.     

Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: implications of probiotics in acne vulgaris

Increasing evidence demonstrates that commensal microorganisms in the human skin microbiome help fight pathogens and maintain homeostasis of the microbiome. However, it is unclear how these microorganisms maintain biological balance when one of them overgrows. The overgrowth of Propionibacterium acnes (P. acnes), a commensal skin bacterium, has been associated with the progression of acne vulgaris. Our results demonstrate that skin microorganisms can mediate fermentation of glycerol, which is naturally produced in skin, to enhance their inhibitory effects on P. acnes growth. The skin microorganisms, most of which have been identified as Staphylococcus epidermidis (S. epidermidis), in the microbiome of human fingerprints can ferment glycerol and create inhibition zones to repel a colony of overgrown P. acnes. Succinic acid, one of four short-chain fatty acids (SCFAs) detected in fermented media by nuclear magnetic resonance (NMR) analysis, effectively inhibits the growth of P. acnes in vitro and in vivo. Both intralesional injection and topical application of succinic acid to P. acnes-induced lesions markedly suppress the P. acnes-induced inflammation in mice. We demonstrate for the first time that bacterial members in the skin microbiome can undergo fermentation to rein in the overgrowth of P. acnes. The concept of bacterial interference between P. acnes and S. epidermidis via fermentation can be applied to develop probiotics against acne vulgaris and other skin diseases. In addition, it will open up an entirely new area of study for the biological function of the skin microbiome in promoting human health.     

Anti‐inflammatory function of Withangulatin A by targeted inhibiting COX‐2 expression via MAPK and NF‐κB pathways

Withangulatin A (WA), an active component isolated from Physalis angulata L., has been reported to possess anti‐tumor and trypanocidal activities in model systems via multiple biochemical mechanisms. The aim of this study is to investigate its anti‐inflammatory potential and the possible underlying mechanisms. In the current study, WA significantly suppressed mice T lymphocytes proliferation stimulated with LPS in a dose‐ and time‐dependent manner and inhibited pro‐inflammation cytokines (IL‐2, IFN‐γ, and IL‐6) dramatically. Moreover, WA targeted inhibited COX‐2 expression mediated by MAPKs and NF‐κB nuclear translocation pathways in mice T lymphocytes, and this result was further confirmed by the COX‐1/2 luciferase reporter assay. Intriguingly, administration of WA inhibited the extent of mice ear swelling and decreased pro‐inflammatory cytokines production in mice blood serum. Based on these evidences, WA influences the mice T lymphocytes function through targeted inhibiting COX‐2 expression via MAPKs and NF‐κB nuclear translocation signaling pathways, and this would make WA a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 109: 532–541, 2010. © 2009 Wiley‐Liss, Inc.     

Proteomic identification of secreted proteins of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>

Background  

The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups.     

Does inflammatory acne result from imbalance in the keratinocyte innate immune response?

Acne is a multifactorial chronic disease affecting around 80% of teenage population. The pathogenesis of acne involves inflammatory reactions and colonization by the Propionibacterium acnes (P. acnes) strain. P. acnes stimulates the keratinocytes involved in the innate immune response, the intensity of which could be influenced either by bacterial intrinsic factors or by endogenous factors of the host.     

Sampling and detection of skin Propionibacterium acnes: Current status

A connection between acne vulgaris and Propionibacterium acnes has long been suggested. Over the years, several human skin microbiota sampling methods have been evolved and applied, e.g. swab, scrape, extraction techniques including cyanoacrylate gel sampling as well as punch biopsy. Collected samples have been processed following various methodologies ranging from culture studies to probe labelling and molecular analysis. Direct visualization techniques have recently shown the existence of anatomically distinct skin P. acnes populations: epidermal and follicular. P. acnes biofilms appear to be a common phenomenon. Current sampling approaches target different skin populations of P. acnes and the presence of microbial biofilms can influence the retrieval of P. acnes. The anatomical considerations must be taken into account while interpreting microbiological data.     

Lancemaside A inhibits lipopolysaccharide‐induced inflammation by targeting LPS/TLR4 complex

In our previous study, lancemaside A isolated from Codonopsis lanceolata (family Campanulaceae) ameliorated colitis in mice. In this study, the anti‐inflammatory effects of lancemaside A was investigated in lipopolysaccharide (LPS)‐stimulated mice and their peritoneal macrophage cells. Lancemaside A suppressed the production of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in vitro and in vivo. Lancemaside A also down‐regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), as well as the inflammatory mediators, nitric oxide (NO), and PGE₂. Lancemaside A also inhibited the expression of IL‐1 receptor‐associated kinase‐4 (IRAK‐4), the phosphorylation of IKK‐β and IκB‐α, the nuclear translocation of NF‐κB and the activation of mitogen‐activated protein kinases in LPS‐stimulated peritoneal macrophages. Furthermore, lancemaisde A inhibited the interaction between LPS and TLR4, as well as IRAK‐4 expression in peritoneal macrophages. Based on these findings, lancemaside A expressed anti‐inflammatory effects by regulating both the binding of LPS to TLR4 on macrophages. J. Cell. Biochem. 111: 865–871, 2010. © 2010 Wiley‐Liss, Inc.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Physicochemical Properties and Anti-Propionibacterium acnes Activity of Film-Forming Solutions Containing Alpha-Mangostin-Rich Extract

The objective of this study was to study the effect of formulation compositions on physicochemical properties and anti-Propionibacterium acnes activity of film-forming solutions containing alpha-mangostin-rich extract (AM). Film-forming solution bases and film-forming solutions containing AM were prepared by using Eudragit RL PO or Klucel LF or combinations of them as film-forming polymers. Rheological properties, pH values of the solutions, and mechanical properties of the dry films were investigated. An optimized formulation was selected and evaluated for the film surface, in vitro AM release, an anti-P. acnes activity, and potential for being a skin irritant. It was found that mechanical properties of the dry films were affected by total polymer contents, ratios of Klucel LF/Eudragit RL PO, AM, and contents of triethyl citrate. The film-forming solutions containing AM had pH values around 7.0. Their flow curves exhibited Newtonian flow behaviors. The optimized formulation provided films possessing smooth and nonporous surfaces. These films showed greater anti-P. acnes activity than their base films without toxicity to skin fibroblasts. Furthermore, AM released from the film matrix obeyed Higuchi's equation. In conclusion, the film-forming solutions containing AM had potential for treatment of acne vulgaris caused by P. acnes. However, further in vivo study is necessary to determine their efficacy and safety for using in patients suffering from acne vulgaris.