Phloretin Exerts Anti-Tuberculosis Activity and Suppresses Lung Inflammation.

An increase in the prevalence of the drug-resistant Mycobacteria tuberculosis necessitates developing new types of anti-tuberculosis drugs. Here, we found that phloretin, a naturally-occurring flavonoid, has anti-mycobacterial effects on H37Rv, multi-drug-, and extensively drug-resistant clinical isolates, with minimum inhibitory concentrations of 182 and 364 µM, respectively. Since Mycobacteria cause lung inflammation that contributes to tuberculosis pathogenesis, anti-inflammatory effects of phloretin in interferon-γ-stimulated MRC-5 human lung fibroblasts and lipopolysaccharide (LPS)-stimulated dendritic cells were investigated. The release of interleukin (IL)-1ß, IL-12, and tumor necrosis factor (TNF)-α was inhibited by phloretin. The mRNA levels of IL-1ß, IL-6, IL-12, TNF-α, and matrix metalloproteinase-1, as well as p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, were suppressed. A mouse in vivo study of LPS-stimulated lung inflammation showed that phloretin effectively suppressed the levels of TNF-α, IL-1ß, and IL-6 in lung tissue with low cytotoxicity. Phloretin was found to bind M. tuberculosis ß-ketoacyl acyl carrier protein synthase III (mtKASIII) with high affinity (7.221 × 107 M-1); a binding model showed hydrogen bonding of A-ring 2'-hydroxy and B-ring 4-hydroxy groups of phloretin with Asn261 and Cys122 of mtKASIII, implying that mtKASIII can be a potential target protein. Therefore, phloretin can be a useful dietary natural product with anti-tuberculosis benefits.

Antituberculosis Activity of a Naturally Occurring Flavonoid, Isorhamnetin.

Isorhamnetin (1) is a naturally occurring flavonoid having anticancer and anti-inflammatory properties. The present study demonstrated that 1 had antimycobacterial effects on Mycobacterium tuberculosis H37Rv, multi-drug- and extensively drug-resistant clinical isolates with minimum inhibitory concentrations of 158 and 316 µM, respectively. Mycobacteria mainly affect the lungs, causing an intense local inflammatory response that is critical to the pathogenesis of tuberculosis. We investigated the effects of 1 on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells. Isorhamnetin suppressed the release of tumor necrosis factor (TNF)-α and interleukin (IL)-12. A nontoxic dose of 1 reduced mRNA expression of TNF-α, IL-1ß, IL-6, IL-12, and matrix metalloproteinase-1 in IFN-γ-stimulated cells. Isorhamnetin inhibited IFN-γ-mediated stimulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase and showed high-affinity binding to these kinases (binding constants: 4.46 × 10(6) M(-1) and 7.6 × 10(6) M(-1), respectively). The 4'-hydroxy group and the 3'-methoxy group of the B-ring and the 5-hydroxy group of the A-ring of 1 play key roles in these binding interactions. A mouse in vivo study of lipopolysaccharide-induced lung inflammation revealed that a nontoxic dose of 1 reduced the levels of IL-1ß, IL-6, IL-12, and INF-γ in lung tissue. These data provide the first evidence that 1 could be developed as a potent antituberculosis drug.

Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1.

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC)  = 1.6 µM) to Pis-1 (average MIC  = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.

Cytotoxic activity of 3,6-dihydroxyflavone in human cervical cancer cells and its therapeutic effect on c-Jun N-terminal kinase inhibition.

Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF) is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR) with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 µM and 9.8 µM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR) 4/CD14, p38 mitogen-activated protein kinase (MAPK), Jun-N terminal kinase (JNK), extracellular-signaling regulated kinase (ERK), and cyclooxygenase (COX)-2 pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM) value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM) in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M-1) and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

Anti-inflammatory activity of rhamnetin and a model of its binding to c-Jun NH2-terminal kinase 1 and p38 MAPK.

Rhamnetin (1), a commonly occurring plant O-methylated flavonoid, possesses antioxidant properties. To address the potential therapeutic efficacy of 1, its anti-inflammatory activity and mode of action in mouse macrophage-derived RAW264.7 cells stimulated with lipopolysaccharide (LPS) or interferon (IFN)-γ were investigated. Rhamnetin (1) suppressed mouse tumor necrosis factor (mTNF)-α, mouse macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine production in LPS-stimulated macrophages. A nontoxic dose of 1 suppressed nitric oxide production. It was found that the anti-inflammatory effects of 1 are mediated by actions on the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and cyclooxygenase (COX)-2 pathways in LPS- or IFN-γ-stimulated RAW264.7 cells. It was determined that 1 binds to human JNK1 (9.7 × 10(8) M(-1)) and p38 MAPK (2.31 × 10(7) M(-1)) with good affinity. The binding model showed interactions with the 3'- and 4'-hydroxy groups of the B-ring and the 5-hydroxy group of the A-ring of 1. Further, 1 exerted an anti-inflammatory effect, reducing the levels of pro-inflammatory cytokines and mediators.

Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway.

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 10(5) M(-1). Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK.

Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea.

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide capreomycin antibiotics in Korea.

Aminoglycosides are key drugs for the treatment of multidrug-resistant tuberculosis. A total of 97 extensively drug-resistant (XDR) and 29 pan-susceptible Mycobacterium tuberculosis isolates from Korean tuberculosis patients were analyzed to characterize mutations within the rrs, rpsL, gidB, eis and tlyA genes. Thirty (56.6 %) of the 53 streptomycin (STR)-resistant strains had a rpsL mutation and eight strains (15.1 %) had a rrs (514 or 908 site) mutation, whereas 11 (20.8 %) of the 53 STR-resistant strains had a gidB mutation without rpsL or either rrs mutation. Most of the gidB mutations conferred low-level STR resistance, and 22 of these mutations were novel. Mutation at position 1401 in rrs lead to resistance to kanamycin (80/95 = 84.2 %; KAN), amikacin (80/87 = 92.0 %; AMK), and capreomycin (74/86 = 86.0 %; CAP). In this study, 13.7 % (13/95) of KAN-resistant strains showed eis mutations, including 4 kinds of novel mutations. Isolates with eis structural gene mutations were cross-resistant to STR, KAN, CAP, and AMK. Here, 5.8 % (5/86) of the CAP-resistant strains harbored a tlyA mutation that included 3 different novel point mutations. Detection of the A1401G mutation appeared to be 100 % specific for the detection of resistance to KAN and AMK. These data establish the presence of phenotypic XDR strains using molecular profiling and are helpful to understanding of aminoglycoside resistance at the molecular level.

Correlation of the phenotypic ethambutol susceptibility of Mycobacterium tuberculosis with embB gene mutations in Korea.

The phenotypic resistance to ethambutol (EMB) in Mycobacterium tuberculosis with embB gene mutations is still unclear. This study was designed to better understand EMB resistance due to embB gene mutation. Sequencing analysis of the embB gene was performed for 124 EMB-susceptible and 93 EMB-resistant M. tuberculosis strains isolated from South Korea. The MIC was determined for EMB-susceptible M. tuberculosis strains with the embB mutation and wild-type on Löwenstein-Jenson (LJ) solid medium in duplicate. Two (2.8 %) of 72 pan-susceptible, two (9.1 %) of 22 any-drug-resistant but EMB-susceptible, nine (30.0 %) of 30 multidrug-resistant (MDR) but EMB-susceptible and 84 (90.3 %) of 93 EMB-resistant M. tuberculosis strains possessed embB mutations at various codons including 306, 319, 354, 360, 399, 405, 406, 459 and 497. Strains with embB mutations at codons 306, 354, 399, 405 and 497 had highly pronounced EMB resistance, while strains with mutations at codons 319 and 406 mutations were moderately resistant and those with an embB459 mutation were EMB-susceptible at the critical concentration (2.0 µg ml(-1)) on LJ solid medium. However, the mean MIC for strains with embB mutations (1.42 µg ml(-1)) was higher than that for strains without the embB mutation (1.0 µg ml(-1)) in EMB-susceptible M. tuberculosis isolates (P = 0.0052). Three novel embB mutations at codons 399, 405 and 459 were identified in this study. These results support the hypothesis that embB mutation except for a few specific mutation types may be the main cause of EMB resistance.

Improvement of clavulanic acid production in Streptomyces clavuligerus by genetic manipulation of structural biosynthesis genes.

To enhance clavulanic acid production, four structural clavulanic acid biosynthesis genes, carboxyethylarginine synthase (ceas2), ß-lactam synthetase (bls2), clavaminate synthase (cas2) and proclavaminate amidinohydrolase (pah2), were amplified from Streptomyces clavuligerus genomic DNA. They were cloned in the pSET152 integration and pIBR25 expression vectors containing the strong ermE* promoter to generate pHN18 and pHN19, respectively, and both plasmids were introduced into S. clavuligerus by protoplast transformation. Clavulanic acid production was increased by 8.7-fold (to ~310 mg/l) in integrative pHN18 transformants and by 5.1-fold in pHN19 transformants compared to controls. Transcriptional analyses showed that the expression levels of ceas2, bls2, cas2 and pah2 were markedly increased in both transformants as compared with wild-type. The elevation of the ceas2, bls2, cas2 and pah2 transcripts was consistent with the enhanced production of clavulanic acid.

Superoxide dismutase (SOD) genes in Streptomyces peucetius: effects of SODs on secondary metabolites production.

Two superoxide dismutase (SOD) genes; sod1 and sod2, from Streptomyces peucetius ATCC 27952 show high similarity to other known SODs from Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680. These sod1 and sod2 were cloned into pIBR25 expression vector under a strong ermE* promoter to enhance secondary metabolites from Streptomyces strains. The recombinant expression plasmids; pIBR25SD1 and pIBR25SD2, were constructed to overexpress sod1 and sod2 respectively to enhance production of doxorubicin (DXR) in S. peucetius, clavulanic acid (CA) in Streptomyces clavuligerus NRRL 3585 and actinorhodin (ACT) and undecylprodigiosin (Red) in Streptomyces lividans TK24. Biomass variation, antibiotics production and transcriptional analysis of regulatory genes in recombinant strains have been studied to understand the effect of sod1 and sod2. The cell growth analysis shows that life span of all recombinant strains was found to be elevated as compared to wild type cells. In S. peucetius, overexpression of sod1 and sod2 was not effective in DXR production but in case of S. clavuligerus, CA production was increased by 2.5 and 1.5 times in sod1 and sod2 overexpression, respectively while in case of S. lividans, ACT production was increased by 1.4 and 1.6 times and Red production by 1.5 and 1.2 times upon sod1 and sod2 overexpressions, respectively as compared to the corresponding wild type strains.

Role of σ-factor (orf21) in clavulanic acid production in Streptomyces clavuligerus NRRL3585.

A putative sigma factor gene, orf21, was disrupted or overexpressed in the wild-type clavulanic acid (CA) producer Streptomyces clavuligerus NRRL3585 and characterized. An orf21 mutant (Streptomyces clavuligerus HN14) of S. clavuligerus was obtained by insertional inactivation via double-crossover. Although there was little reduction of sporulation in the mutant, the growth pattern was similar between mutant and wild-type. The production was reduced by 10-15% in S. clavuligerus HN14 compared to that in wild-type. Overexpression of orf21 in wild-type cells caused hyperproduction of spores on solid medium and increased clavulanic acid production by 1.43-fold. The overexpression of orf21 in wild-type S. clavuligerus stimulated the expression of the early clavulanic acid genes, ceas2 and cas2, and the regulatory gene, ccaR, as demonstrated by RT-PCR. The elevation of the ceas2, cas2 and ccaR transcripts was consistent with the enhanced production of clavulanic acid.

Enhancement of clavulanic acid production by expressing regulatory genes in gap gene deletion mutant of Streptomyces clavuligerus NRRL3585.

Streptomyces clavuligerus NRRL3585 produces a clinically important ss-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into deleted mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared to the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared to the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

Functional characterization of kanB by complementing in engineered Streptomyces fradiae Deltaneo6::tsr.

A putative aminotransferase gene, kanB, lies in the biosynthetic gene cluster of Streptomyces kanamyceticus ATCC 12853 and has 66% identity with neo6 in neomycin biosynthesis. Streptomyces fradiae Deltaneo6::tsr was generated by disrupting neo6 in the neomycin producer Streptomyces fradiae. Neomycin production was completely abolished in the disruptant mutant but was restored through self-complementation of neo6. S. fradiae HN4 was generated through complementation with kanB in Streptomyces fradiae neo6::tsr. Based on metabolite analysis by ESI/MS and LC/MS, neomycin production was restored in Streptomyces fradiae HN4. Thus, like neo6, kanB also functions as a 2-deoxy-scyllo-inosose aminotransferase that has dual functions in the formation of 2-deoxy-scyllo-inosose (DOS).

A two-component regulatory system involved in clavulanic acid production.

A pair of genes encoding the bacterial two-component regulatory system, orf22 and orf23, was found next to the clavulanic acid gene cluster of Streptomyces clavuligerus NRRL3585. Orf23 was deleted for the construction of S. clavuligerus/Deltaorf23. Although growth and morphological analyses showed no differences between S. clavuligerus/Deltaorf23 and wild-type, the production of clavulanic acid in S. clavuligerus/Deltaorf23 was found to be decreased. In addition, the co-overexpression of orf22/orf23 in wild-type resulted in an enhanced 1.49-fold production of clavulanic acid, and the complementation of orf22/orf23 in S. clavuligerus/Deltaorf23 restored clavulanic acid production about 80% as normal levels. These results demonstrate that the orf22/orf23 two-component regulatory system participates as a positive regulator of the biosynthesis of clavulanic acid and increased levels of orf22/orf23 can contribute to enhanced production of clavulanic acid in S. clavuligerus.