Bacterial resistance modifying agents from Lycopus europaeus

As part of an ongoing project to identify plant natural products which modulate bacterial multidrug resistance (MDR), bioassay-guided isolation of an extract of Lycopus europaeus yielded two new isopimarane diterpenes, namely methyl-1alpha-acetoxy-7alpha 14alpha-dihydroxy-8,15-isopimaradien-18-oate (1) and methyl-1alpha,14alpha-diacetoxy-7alpha-hydroxy-8,15-isopimaradien-18-oate (2). The structures were established by spectroscopic methods. These compounds and several known diterpenes were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing the Tet(K), Msr(A), and Nor(A) multidrug resistance efflux mechanisms. At 512 microg/ml none of the compounds displayed any antibacterial activity but individually in combination with tetracycline and erythromycin, a two-fold potentiation of the activities of these antibiotics was observed against two strains of S. aureus that were highly resistant to these agents due to the presence of the multidrug efflux mechanisms Tet(K) (tetracycline resistance) and Msr(A) (macrolide resistance).     

Complex anthocyanins from Ipomoea congesta

Six acylated anthocyanins have been isolated from the flowers of Ipomoea congesta R. Brown. One has been previously described as an acylated peonidin derivative. Three others are isomers, derived from peonidin-3-(caffeylsophoroside)-5-glucoside. The fifth was characterised as peonidin-3-(p-coumarylcaffeylsophoroside)-5-glucoside and the last as peonidin-3-(coumarylsophoroside)-5-glucoside. It is noteworthy that the anthocyanins found in this species have the same glycosidic pattern, 3-sophoroside-5-glucoside, as those reported for the cyanidin derivatives in Ipomoea cairica flowers. Acylated anthocyanin occurrence in Tubiflorae order is of chemotaxonomical value.     

Batatins III-VI, glycolipid ester-type dimers from Ipomoea batatas

Batatins III-VI (1-4), glycolipid ester-type dimers, were isolated from the tuberous roots of sweet potato (Ipomoea batatas) using recycle high performance liquid chromatography. Their structures were characterized by means of several high-resolution NMR and mass spectrometry techniques. These compounds are the first examples of ester-type dimers which consist of two units of the heterotetrasaccharide operculinic acid C. Each unit was esterified by a different amount and type of acid residues: (2S)-methylbutanoic, cinnamic, decanoic (capric) and dodecanoic (lauric) acids. Batatins III-VI (1-4) are an example of the presence of a large number of resin glycoside congeners in each morning glory species caused by partial acylation of their constitutive saccharide cores.     

Novel acylated lipo-oligosaccharides from the tubers of Ipomoea batatas

Nine new lipo-oligosaccharides, batatosides H-P, were isolated from the tubers of Ipomoea batatas (Convolvulaceae). Spectral and chemical methods allowed to characterize them as tetra- or penta-saccharides that form a macrolactone with the aglycone, (11S)-hydroxyhexadecanoic acid (jalapinolic acid), the absolute configuration of which was established by Mosher’s method. Batatosides L and O showed a weak inhibitory effect on the growth of Hep-2 cells, while the others proved to be inactive.     

Crystal structure of FtsA from Staphylococcus aureus

The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12–S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.     

Antibacterial and resistance modifying activity of Rosmarinus officinalis

As part of a project to characterise plant-derived natural products that modulate bacterial multidrug resistance (MDR), bioassay-guided fractionation of a chloroform extract of the aerial parts of Rosmarinus officinalis led to the characterisation of the known abietane diterpenes carnosic acid (1), carnosol (2) and 12-methoxy-trans-carnosic acid. Additionally, a new diterpene, the cis A/B ring junction isomer of 12-methoxy-trans-carnosic acid, 12-methoxy-cis-carnosic acid (5), was isolated. The major components were assessed for their antibacterial activities against strains of Staphylococcus aureus possessing efflux mechanisms of resistance. Minimum inhibitory concentrations ranged from 16 to 64 microg/ml. Incorporation of 1 and 2 into the growth medium at 10 microg/ml caused a 32- and 16-fold potentiation of the activity of erythromycin against an erythromycin effluxing strain, respectively. Compound 1 was evaluated against a strain of S. aureus possessing the NorA multidrug efflux pump and was shown to inhibit ethidium bromide efflux with an IC50 of 50 microM, but this activity is likely to be related to the inhibition of a pump(s) other than NorA. The antibacterial and efflux inhibitory activities of these natural products make them interesting potential targets for synthesis.     

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His₆-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted l-Ala, l-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for l-Ala. S. aureus MurE was very specific for l-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and l-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (l-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and l-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis.     

Dibenzofuran and pyranone metabolites from Hypericum revolutum ssp. revolutum and Hypericum choisianum

In a project to isolate and characterise anti-staphylococcal compounds from members of the genus Hypericum, a dibenzofuran and a pyranone were isolated from the dichloromethane and hexane extracts of Hypericum revolutum ssp. revolutum Vahl (Guttiferae) and Hypericum choisianum Wall. ex. N. Robson (Guttiferae), respectively. The structures of these compounds were elucidated by 1- and 2D-NMR spectroscopy and mass spectrometry as 3-hydroxy-1,4,7-trimethoxydibenzofuran (1) and 4-(3-O-3″)-3″-methylbutenyl-6-phenyl-pyran-2-one (2). The metabolites were evaluated against a panel of multidrug-resistant strains of Staphylococcus aureus. Compound 1 exhibited a minimum inhibitory concentration (MIC) of 256 μg/ml, whereas compound 2 was inactive at a concentration of 512 μg/ml.     

SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA

SsrA RNA (small stable RNA A), also known as tmRNA and 10Sa RNA, functions both as tRNA and mRNA through its unique structure. The carotenoid pigment is the eponymous feature of human pathogen Staphylococcus aureus. Here, we found that the pigment of the mutant strain with ssrA deletion was increased. Furthermore, it was demonstrated that ssrA could act as an antisense RNA aside from its well-known biological function, and crtMN, encoding two essential enzymes for the pigment synthesis, was identified as target of ssrA. Further investigation showed ssrA could specifically base pair with the RBS (ribosomal binding site) region of the crtMN mRNA. Our results revealed a new mechanism by which ssrA regulated the biosynthesis of pigment in S. aureus.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Multidrug resistance reversal agent from Jatropha elliptica

As part of an ongoing project to identify plant natural products as resistance-modifying agents, bioassay-guided fractionation of an extract of Jatropha elliptica (Pohl) Muell Arg. led to the isolation of a penta-substituted pyridine, namely 2,6-dimethyl-4-phenyl-pyridine-3,5-dicarboxylic acid diethyl ester (8). The structure was established by spectroscopic methods. This known compound was assayed for in vitro antibacterial and resistance-modifying activities against strains of Staphylococcus aureus possessing the MsrA and NorA resistance efflux mechanisms. Antibiotic efflux studies indicated that (8) acts as an inhibitor of the NorA efflux pump and restores the level of intracellular drug concentration.     

Glucogalactan: A polysaccharide isolated from the cell-wall of Verticillium Lecanii

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D ¹H and ¹³C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E₃S_IV fraction) was established:     

Carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, from yellow sweet potato "Benimasari", Ipomoea batatas Lam

A series of carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, named ipomoeaxanthins A (1), B (2), C1 (3) and C2 (4) were isolated from the flesh of yellow sweet potato "Benimasari", Ipomoea batatas Lam. Their structures were determined to be (5R,6S,3'R)-5,6-dihydro-beta,beta-carotene-5,6,3'-triol (1), (5R,6S,5'R,6'S)-5,6,5',6'-tetrahydro-beta,beta-carotene-5,6,5'6'-tetrol (2), (5R,6S,5'R,8'R)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (3), and (5R,6S,5'R,8'S)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (4) by UV-Vis, NMR, MS and CD data.     

Biofunctionalization of cellulosic fibres with l-cysteine: Assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae

The main purpose of this work is to obtain a cotton-based textile material functionalized with l-cysteine (l-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of l-cys to cotton fibres was assessed through different functionalization strategies—surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard ( JISL, 2008). To determine the mechanism of action of l-cys on the selected strains, flow cytometry was used.     

A rapid microtiter plate assay for measuring the effect of compounds on Staphylococcus aureus membrane potential

We developed a homogenous microtiter based assay using the cationic dye 3, 3′-Diethyloxacarbocyanine iodide, DiOC₂(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.     

A protocol for the investigation of the intracellular Staphylococcus aureus metabolome

Systems biology studies assume the acquisition of reliable and reproducible data sets. Metabolomics, in particular, requires comprehensive evaluated workflows to enable the analysis of hundreds of different compounds. Therefore, a protocol to elucidate the metabolome of the gram-positive pathogen, Staphylococcus aureus COL strain, grown in a chemically defined medium is introduced here. Different standard operating procedures in the field of metabolome experiments were tested for common pitfalls. These included suitable and fast sampling processes, efficient metabolite extraction, quenching effectiveness (energy charge), and estimation of leakage and recovery of metabolites. Moreover, a cell disruption protocol for S. aureus was developed and optimized for metabolome analyses, for the express purpose of obtaining reproducible data. We used complementary methods (e.g., gas chromatography and/or liquid chromatography coupled with mass spectrometry) to detect the highly chemically diverse groups of metabolites for a global insight into the intracellular metabolism of S. aureus.     

Cryopreservation of sweetpotato (Ipomoea batatas) and its pathogen eradication by cryotherapy

Sweetpotato (Ipomoea batatas) ranks as the seventh most important staple crop in the world and the fifth in developing countries after rice, wheat, maize and cassava. Sweetpotato is mainly grown in developing countries, which account for more than 95% of total production of the whole world. Genetic resources, including cultivated varieties and wild species, are a prerequisite for novel sweetpotato breeding in both conventional and genetic engineering programs. Various cryopreservation protocols have been developed for shoot tips and embryogenic tissues. The former explants are preferred for long-term conservation of sweetpotato genetic resources, while the latter are valuable for sweetpotato genetic improvement. This review provides update comprehensive information on cryopreservation of sweetpotato shoot tips and embryogenic tissues.Plant pathogens such as viruses and phytoplasma severely hamper high yield and high quality production of sweetpotato. Thus, usage of pathogen-free planting materials is pivotal for sustainable sweetpotato production. Cryotherapy of shoot tips can efficiently eradicate sweetpotato pathogens such as viruses and phytoplasma. The mechanism behind pathogen eradication by cryotherapy of shoot tips has been elucidated. Pathogen eradication by cryotherapy provides an alternative, efficient strategy for production of pathogen-free plants. In addition, cryopreserved tissues may also be considered to be safer for exchange of germplasm between countries and regions.     

Structural investigation of a heteropolysaccharide isolated from the green fruits of Capsicum annuum

A water-soluble polysaccharide isolated from the hot water extract of the green fruits of Capsicum annuum was found to consist of 3-O-acyl-l-rhamnose, d-methyl galacturonate, 6-O-methyl-d-galactose in a molar proportion of nearly 1:2:1. Structural investigation of the polysaccharide was carried out using total hydrolysis, methylation analysis, periodate oxidation followed by GLC-MS, and NMR experiments. On the basis of the above-mentioned experiments it is concluded that the following repeating unit is present in the polysaccharide.     

Seco-tabersonine alkaloids from Tabernaemontana corymbosa

Two seco-tabersonine alkaloids, jerantiphyllines A and B, in addition to a tabersonine hydroxyindolenine, jerantinine H, and a recently reported vincamine alkaloid 7, were isolated from the leaf extract of the Malayan Tabernaemontana corymbosa and the structures were established using NMR and MS analysis. Biomimetic conversion of jerantinines A and E to their respective vincamine and 16-epivincamine derivatives were also carried out.