Rhodomyrtone

Antibacterial mechanism of rhodomyrtone involves the disruption of nucleoid segregation checkpoint in Streptococcus suis.[Pubmed:32514868]

AMB Express. 2020 Jun 8;10(1):110.

Rhodomyrtone has been recently demonstrated to possess a novel antibiotic mechanism of action against Gram-positive bacteria which involved the multiple targets, resulting in the interference of several bacterial biological processes including the cell division. The present study aims to closely look at the downstream effect of Rhodomyrtone treatment on nucleoid segregation in Streptococcus suis, an important zoonotic pathogen. The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) values of Rhodomyrtone against the recombinant S. suis ParB-GFP, a nucleoid segregation reporter strain, were 0.5 and 1 microg/ml, respectively, which were equivalent to the potency of vancomycin. Using the fluorescence live-cell imaging, we demonstrated that Rhodomyrtone at 2x MIC caused incomplete nucleoid segregation and septum misplacement, leading to the generation of anucleated cells. FtsZ immune-staining of Rhodomyrtone-treated S. suis for 30 min revealed that the large amount of FtsZ was trapped in the region of high fluidity membrane and appeared to be able to polymerize to form a complete Z-ring. However, the Z-ring was shifted away from the midcell. Transmission electron microscopy further confirmed the disruption of nucleoid segregation and septum misplacement at 120 min following the Rhodomyrtone treatment. Asymmetric septum formation resulted in either generation of minicells without nucleoid, septum formed over incomplete segregated nucleoid (guillotine effect), or formation of multi-constriction of Z-ring within a single cell. This finding spotlights on antibacterial mechanism of Rhodomyrtone involves the early stage in bacterial cell division process.

Evaluation of a Rhodomyrtus tomentosa ethanolic extract for its therapeutic potential on Staphylococcus aureus infections using in vitro and in vivo models of mastitis.[Pubmed:31221210]

Vet Res. 2019 Jun 20;50(1):49.

An ethanolic extract from Rhodomyrtus tomentosa leaves (RTL) was studied as a natural alternative to control Staphylococcus aureus, which is an important pathogen responsible for bovine mastitis. The minimal inhibitory concentrations (MICs) of the RTL extract and of Rhodomyrtone, a pure compound isolated from the plant, were determined by a microdilution method. Rhodomyrtone and the RTL extract exhibited antibacterial activity against S. aureus, including its persistent phenotype (SCV: small-colony variant) and a biofilm hyperproducer strain, with MICs of 0.25-0.5 and 8-16 microg/mL, respectively. Time-kill kinetics showed a strong bactericidal activity for both the RTL extract- and Rhodomyrtone-treated bacteria at 2 x MIC as early as 4 h post-exposure. An additive effect of the extract at 0.5 x MIC was observed in a combination with oxytetracycline or pirlimycin against S. aureus by showing a 64- to 128-fold reduction in antibiotic MICs. Moreover, the RTL extract significantly decreased the number of intracellular SCVs inside bovine mammary epithelial cells. However, the extract or its combination with pirlimycin only slightly improved the activity of pirlimycin against the bacterial colonization of mouse mammary glands. In vitro MICs determined in the presence of casein indicated that the limited activity of the RTL extract in the murine model of mastitis could be linked to neutralization of active components by milk proteins. While the RTL extract showed interesting antibacterial properties in vitro, to be considered as an alternative to antibiotics in dairy farms, formulation studies are needed to cope with the observed reduction of activity in vivo.

Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus.[Pubmed:31191485]

Front Microbiol. 2019 May 28;10:1157.

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (Rom(R)) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the Rom(R) clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the Rom(R) clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the Rom(R) clone compared to its parental strain HG001. If farE is deleted in the Rom(R) clone, or, if native farR is expressed in the Rom(R) strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the Rom(R) clone, that FarR is an important regulator, and that the point mutation in farR (Rom(R) clone) makes the clone hyper-virulent.

Antimicrobial activity of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk.[Pubmed:30600706]

Nat Prod Res. 2019 Jan 2:1-6.

Rhodomyrtone was isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk grown in Vietnam using chromatographic methods. Its chemical structure was confirmed by means of spectroscopic data analysis. The pH drop measurement, enzyme activity assays and fluorescence stain were used to examine Rhodomyrtone anticaries activity. It was found that Rhodomyrtone suppressed acid production by Streptococcus mutans, a major cariogenic agent in human by inhibiting enzyme activities responsible for acid production and tolerance, including membrane bound enzymes F-ATPase and phosphotransferase system (PTS), as well as glycolysis enzymes glyceraldehyphosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) in cytoplasm with the IC50 values of 24 muM, 19 muM, 23 muM and 28 muM, respectively. Moreover, 50 muM Rhodomyrtone reduced biofilm biomass formed by S. mutans up to 59% (p < 0.05). Fluorescent images indicated that cells on the biofilms were significantly killed. Thus, Rhodomyrtone is a new and potential anticaries agent against S. mutans.

Rhodomyrtone decreases Staphylococcus aureus SigB activity during exponentially growing phase and inhibits haemolytic activity within membrane vesicles.[Pubmed:30583020]

Microb Pathog. 2019 Mar;128:112-118.

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of Rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of Rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25-8mug/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with Rhodomyrtone for 3h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P<0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of Rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625mug/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5xMIC Rhodomyrtone (P<0.05). Decreased haemolytic activity was detected within Rhodomyrtone-treated membrane vesicles. The results indicated that Rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

Antidepressant Effects of Rhodomyrtone in Mice with Chronic Unpredictable Mild Stress-Induced Depression.[Pubmed:30407505]

Int J Neuropsychopharmacol. 2019 Feb 1;22(2):157-164.

Background: Rhodomyrtone is one of the main active compounds derived from Rhodomyrtus tomentosa, which belongs to the Myrtaceae family. In the current study, we investigated the properties of Rhodomyrtone as a potential drug candidate for the treatment of stress-caused depression. Methods: We assessed the function of Rhodomyrtone in chronic unpredictable mild stress, a well-validated depression model in mice. Depression-like behavior tests, including a sucrose performance test, social interaction test, and forced swimming test, were used to validate the antidepressant effects of Rhodomyrtone. The Morris water maze was used to evaluate the mice's learning and memory ability. Spine density, glycogen synthase kinase-3beta, brain-derived neurotrophic factor, postsynaptic density protein 95, and apoptosis-associated protein were detected to reveal the underlying mechanism. Results: Rhodomyrtone was found to prevent source consumption decrease, decreased social behaviors, and increase immobility in the forced swimming test, suggesting a protective effect of Rhodomyrtone against depression-like behaviors. Additionally, Rhodomyrtone prevented the impairment of spatial memory in mice exposed to chronic unpredictable mild stress. Rhodomyrtone administration also reversed dendritic spine density defects in chronic unpredictable mild stress. Furthermore, Rhodomyrtone inhibited the increase of glycogen synthase kinase-3beta activity and reversed the decrease of brain-derived neurotrophic factor and postsynaptic density protein 95 in chronic unpredictable mild stress mice. Elevated expression of apoptosis-associated protein Bax and cleaved-caspase 3 was also reversed by Rhodomyrtone treatment. Conclusions: These results suggested that the antidepressant effect of Rhodomyrtone involves the regulation of neurogenesis, neuronal survival, and synaptic plasticity in the hippocampus.

The small molecule rhodomyrtone suppresses TNF-alpha and IL-17A-induced keratinocyte inflammatory responses: A potential new therapeutic for psoriasis.[Pubmed:30321197]

PLoS One. 2018 Oct 15;13(10):e0205340.

Psoriasis is a common skin disease pathogenically driven by TNF and IL-17A-induced epidermal hyperproliferation and inflammatory responses. The ongoing need for new therapeutic agents for psoriasis has highlighted medicinal plants as sources of phytochemicals useful for treating psoriatic disease. Rhodomyrtone, a bioactive phytochemical from Rhodomyrtus tomentosa, has well-established anti-proliferative activities. This study assessed the potential of Rhodomyrtone for curtailing TNF/IL-17A-driven inflammation. Stimulating human skin organ cultures with TNF+IL-17A to model the skin inflammation in psoriasis, we found that Rhodomyrtone significantly decreased inflammatory gene expression and the expression and secretion of inflammatory proteins, assessed by qRT-PCR, immunohistochemistry and ELISA assays respectively. RNA-seq analysis of monolayer primary keratinocytes treated with IL-17A/TNF showed that Rhodomyrtone inhibited 724/1587 transcripts >2-fold altered by IL-17A/TNF (p<0.01), a number of which were confirmed at the mRNA and protein level. Suggesting that Rhodomyrtone acts by modulating MAP kinase and NF-kappaB signaling pathways, Rhodomyrtone inhibited TNF-induced ERK, JNK, p38, and NF-kappaBp65 phosphorylation. Finally, assessing the in vivo anti-inflammatory potential of Rhodomyrtone, we examined its effects on imiquimod-induced skin inflammation in mice, finding Rhodomyrtone reversed imiquimod-induced skin hyperplasia and epidermal thickening (p< 0.001). Taken together, these results suggest that Rhodomyrtone may be useful in preventing or slowing the progression of inflammatory skin disease.

Antibacterial activity of rhodomyrtone on Clostridium difficile vegetative cells and spores in vitro.[Pubmed:30145248]

Int J Antimicrob Agents. 2018 Nov;52(5):724-729.

The increasing incidence and severity of diarrhoea and colitis caused by Clostridium difficile, together with a high rate of relapse following treatment with currently recommended antimicrobials, calls for novel interventions for C. difficile infection (CDI). Rhodomyrtone, a bioactive compound derived from the leaves of the rose myrtle (Rhodomyrtus tomentosa) has demonstrated antibacterial activity against several Gram-positive bacteria. This study compared the in vitro antimicrobial activity of Rhodomyrtone on C. difficile with that of vancomycin, a recommended agent for the treatment of CDI. Determination of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Rhodomyrtone and vancomycin for ten C. difficile isolates showed that the MICs of Rhodomyrtone for C. difficile vegetative cells (0.625-2.5 mg/L) were comparable with that of vancomycin (1.25 mg/L), but the MBCs of Rhodomyrtone (1.25-5 mg/L) were significantly lower than those for vancomycin (5 mg/L to >40 mg/L; P < 0.001). Time-kill assays showed rapid bactericidal activity for Rhodomyrtone, with >/=99% killing within 4 h. Rhodomyrtone was also four-fold more potent than vancomycin in inhibiting C. difficile spore outgrowth. Transmission electron microscopy of Rhodomyrtone-treated C. difficile revealed cell lysis and evidence of defective cell division and spore formation. These studies indicate that Rhodomyrtone should be further investigated as a potential treatment for CDI.

Apoptosis and antimigration induction in human skin cancer cells by rhodomyrtone.[Pubmed:29904398]

Exp Ther Med. 2018 Jun;15(6):5035-5040.

Rhodomyrtone is a bioactive compound extracted from Rhodomyrtus tomentosa leaves. It has been used as a traditional herb medicine for many years. Rhodomyrtone exhibits antibacterial activity, anti-inflammatory and antioxidant activities. However, the anticancer activity of Rhodomyrtone has not been previously reported. The present study investigated the anticancer effect of rhomyrtone on human epidermoid carcinoma A431 cells. The cytotoxic and antiproliferative effects of Rhodomyrtone on A431 cells were investigated by an MTT assay. Cell morphological alterations and apoptotic cells were observed with Hoechst 33342 staining following Rhodomyrtone treatment. Flow cytometry and western blotting were performed to detect cell cycle and apoptosis induction. The results demonstrated that Rhodomyrtone inhibited proliferation of A431 cells in a dose-dependent manner with IC(50) value of 8.04+/-0.11 microg/ml. The results also indicated that Rhodomyrtone increased chromatin condensation, nuclear fragmentation and apoptotic bodies in treated A431 cells in a time-dependent manner. Apoptosis was also induced through the activation of caspase-7 and poly (ADP-Ribose) polymerase cleavage. Flow cytometry analysis revealed that Rhodomyrtone induced cell cycle arrest at the G1 phase. Notably, the non-toxic concentration of Rhodomyrtone markedly inhibited A431 cell migration in a dose- and time-dependent manner. These finding suggested that Rhodomyrtone may be used as an anticancer agent for human skin cancer.

Virtual Screening for the Development of New Effective Compounds Against Staphylococcus aureus.[Pubmed:29589530]

Curr Med Chem. 2018;25(42):5975-5985.

Staphylococcus aureus is a notorious pathogenic bacterium causing a wide range of diseases from soft-tissue contamination, to more serious and deep-seated infections. This species is highlighted by its ability to express several kinds of virulence factors and to acquire genes related to drug resistance. Target this number of factors to design any drug is not an easy task. In this review, we discuss the importance of computational methods to impulse the development of new drugs against S. aureus. The application of docking methods to screen large libraries of natural or synthetic compounds and to provide insights into action mechanisms is demonstrated. Particularly, the studies that validated in silico results with biochemical and microbiological assays are highlighted. We also comment on the computer-aided design of new molecules using some known inhibitors. The confirmation of in silico results with biochemical and microbiological assays allowed the identification of lead molecules that could be used for drug design such as Rhodomyrtone, quinuclidine, berberine (and their derivative compounds). The fast development of the computational methods is essential to improve our ability to discover new drugs, as well as to expand understanding about drug-target interactions.

The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity.[Pubmed:29451901]

PLoS Pathog. 2018 Feb 16;14(2):e1006876.

The acylphloroglucinol Rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clinical applications, very little is known about the mechanism of action of Rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resolution, and electron microscopy we demonstrate that Rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, Rhodomyrtone did not seem to be a typical membrane-inserting molecule. In fact, molecular dynamics simulations showed that instead of inserting into the bilayer, Rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein-trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action.

Rhodomyrtone (Rom) is a membrane-active compound.[Pubmed:29317198]

Biochim Biophys Acta Biomembr. 2018 May;1860(5):1114-1124.

Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is Rhodomyrtone (Rom) isolated from Rhodomyrtus tomentosa leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in Bacillus subtilis indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Staphylococcus aureus, Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.

Immunomodulatory effects of Rhodomyrtus tomentosa leaf extract and its derivative compound, rhodomyrtone, on head kidney macrophages of rainbow trout (Oncorhynchus mykiss).[Pubmed:29238889]

Fish Physiol Biochem. 2018 Apr;44(2):543-555.

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, Rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10 mug mL(-1) of R. tomentosa and 1 mug mL(-1) of Rhodomyrtone for 4 and 24 h. R. tomentosa and Rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1beta, il8, and tnfalpha), anti-inflammatory cytokines (il10 and tgfbeta), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1beta, il8, tnfalpha, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of Rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1beta, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and Rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.

Early Effects of Rhodomyrtone on Membrane Integrity in Methicillin-Resistant Staphylococcus aureus.[Pubmed:29215320]

Microb Drug Resist. 2018 Sep;24(7):882-889.

Strong evidence of high potency of Rhodomyrtone as a promising antibacterial agent against pathogenic gram-positive bacteria has been clearly demonstrated in our previous work. The aim of this study was to provide insight into early action of Rhodomyrtone, an acylphloroglucinol, on membrane damage in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA). Early effects of Rhodomyrtone on the bacterial membrane integrity were detected in a time-course study. Flow cytometry revealed a reduction in green fluorescent emission and increase in uptake of propidium iodide in Rhodomyrtone-treated bacterial cells in a concentration- and time-dependent manner. Disruption of cytoplasmic membrane was further monitored by measuring cellular adenosine triphosphate (ATP) and potassium ion (K(+)). Leakage of both ATP and K(+) and significant decrease in intracellular ATP in MRSA were observed following treatment. Pronounced changes in the bacterial ultrastructure and morphology were confirmed by transmission electron microscopy and scanning electron microscopy. Bacterial cell disruption, holes in cell surface, and bulge formations were noted in Rhodomyrtone-treated cells. In this study, we provided relevant data to clarify that Rhodomyrtone is a bacterial cell membrane-damaging agent. A possible early effect of this novel compound involves bacterial membrane disruption.

Integrated proteomic and metabolomic analysis reveals that rhodomyrtone reduces the capsule in Streptococcus pneumoniae.[Pubmed:28578394]

Sci Rep. 2017 Jun 2;7(1):2715.

The emergence of antibiotic-resistant pathogenic bacteria is a healthcare problem worldwide. We evaluated the antimicrobial activity of Rhodomyrtone, an acylphloroglucinol present in Rhodomyrtus tomentosa leaves, against the human Gram-positive pathogen Streptococcus pneumoniae. The compound exhibited pronounced anti-pneumococcal activity against a broad collection of clinical isolates. We studied the effects at the molecular level by integrated proteomic and metabolomic analysis. The results revealed alterations in enzymes and metabolites involved in several metabolic pathways including amino acid biosynthesis, nucleic acid biosynthesis, glucid, and lipid metabolism. Notably, the levels of two enzymes (glycosyltransferase and UTP-glucose-1-phosphate uridylyltransferase) and three metabolites (UDP-glucose, UDP-glucuronic acid and UDP-N-acetyl-D-galactosamine) participating in the synthesis of the pneumococcal capsule clearly diminished in the bacterial cells exposed to Rhodomyrtone. Rhodomyrtone-treated pneumococci significantly possessed less amount of capsule, as measured by a colorimetric assay and visualized by electron microscopy. These findings reveal the utility of combining proteomic and metabolomic analyses to provide insight into phenotypic features of S. pneumoniae treated with this potential novel antibiotic. This can lead to an alternative antibiotic for the treatment of S. pneumoniae infections, because of the growing concern regarding antimicrobial resistance.