[Show abstract][Hide abstract] ABSTRACT: This report describes the whole-genome sequence of an alkalitolerant microcystin-degrading bacterium, Sphingopyxis sp. strain C-1, isolated from a lake in China
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas aeruginosa shows multidrug resistance, which is mainly attributable to its expression of xenobiotic efflux pumps. However, it is unclear
how silent pumps are expressed in clinical isolates. Here, we sequenced the complete genome of P. aeruginosa strain 8380, which was isolated from a human gut.
[Show abstract][Hide abstract] ABSTRACT: The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound - ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate - was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.
[Show abstract][Hide abstract] ABSTRACT: Ultraviolet-A (UV-A) can damage microbes by generating reactive oxygen species (ROS), singlet oxygen, superoxides, hydrogen peroxide and hydroxyl radicals. These species readily react with lipids, proteins, DNA and other constituents of cells, leading to oxidative deterioration and the eventual death of the microbe. However, the oxidative ability of these reactive species also harms the viability of mammalian cells such as fibroblasts and keratinocytes, as they cause both acute and chronic damage, photo-aging, and photo-carcinogenesis. This study describes a UV-A treatment that does not affect the viability or growth of human neonate dermal fibroblasts, as determined by examining the post-irradiation cell density after the addition of polyphenols as antioxidants. The results demonstrate the possible wide applicability of UV-A sterilization. The potency of polyphenols for attenuating UV-A-induced ROS generation in cells was tested using (+)-catechin hydrate, (-)- epigallocatechin gallate hydrate, morin hydrate, quercetin hydrate and resveratrol. The lowest concentration of polyphenols required to reduce ROS by 50% in cells upon exposure to a dose of 15 J cm(-2) was determined and defined as its IC50. Pre-treatment with morin hydrate at its IC50 allowed cells irradiated with 5.0 J cm(-2) UV-A to recover to the level of the specific growth rate of cells incubated without UV-A irradiation. However, the growth rate of cells exposed to 15 J cm(-2) UV-A irradiation was scarcely influenced by co-incubation with morin hydrate; this dose of UV-A also suppressed cell growthcompletely in the absence of morin hydrate, although co-incubation resulted in no decrease in cell viability. This study demonstrates the potential of polyphenols for protecting both the viability of cells and their ability to proliferate from damage caused by UV-A-irradiation.
[Show abstract][Hide abstract] ABSTRACT: The signal molecule, 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C12-HSL concentrations (>200 μM) on mammalian cellular functions because ~600 μM 3-oxo-C12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C12-HSL concentration (30 μM) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30 min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C12-HSL.
[Show abstract][Hide abstract] ABSTRACT: N-Acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.
[Show abstract][Hide abstract] ABSTRACT: We investigated to determine whether a variety of acyl-homoserine lactones (AHLs) influences epithelial cell proliferation and mucosal permeability. 3-Oxo-C12-homoserine lactone (HSL) and 3-oxo-C14-HSL significantly suppressed IEC-6 cell proliferation. A significant increase in mucosal permeability was observed in isolated rat colon tissue exposed to C12-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL. These data indicate that AHLs suppress epithelial proliferation and disrupt barrier function in intestinal mucosa.
[Show abstract][Hide abstract] ABSTRACT: In this study, we describe a rational design strategy to develop a latent ratiometric fluorescent pH probe from self-assembled seminaphthorhodafluor (SNARF) derivatives in aqueous conditions. Analysis of the characteristics of SNARF derivatives with protected phenolic groups allowed classification of the threshold between the assembled and monomeric states according to the Hansch-Fujita hydrophobic parameters for a substituent inserted as a SNARF-OH phenolic-protecting group. The esterase-activated latent ratiometric fluorescent pH probe SNARF-OBn(OAc) was characterized in the application of tuneable small-molecule aggregation and disaggregation. Before esterase treatment, SNARF-OBn(OAc) formed self-assembled clusters, emitting no fluorescence. The significant fluorescence was observed after esterase treatment because the self-assembled clusters of SNARF-OBn(OAc) diffuse as SNARF-OH monomers, demonstrating the intracellular application of SNARF-OBn(OAc) as a latent ratiometric fluorescent pH probe for floating cells. Furthermore, to directly visualize the state of self-assembled SNARF derivatives for investigating the mechanism of their cellular uptake, SNARF-Dan was rationally designed and applied for cellular imaging. Through the cellular application of SNARF-Dan, it was suggested that the self-assembled SNARF derivatives were introduced into the cell via macropinocytosis.
[Show abstract][Hide abstract] ABSTRACT: Five ethyl (5-alkyl-2-amino-1,3-thiazol-4-yl) acetates (designated compounds 4a-e) incorporating octyl, decyl, dodecyl, tetradecyl, and hexadecyl alkyl chains, respectively, were prepared by reacting 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters (3a-e) with thiourea in dried acetonitrile. Compounds 3a-e were synthesized by reacting alkylated ethyl acetoacetates with bromine. The newly synthesized compounds were characterized by mass spectrometry, NMR, and elemental analysis. Compounds 4a-c demonstrated good in vitro antiamoebic activity against Acanthamoeba polyphaga exposed to 10 mg L(-1) for 6 h at 28 °C. Compound 4b showed the highest antiamoebic activity among the tested compounds, comparable to that of chlorhexidine dihydrochloride (CHX), decreasing the number of viable cells to below the detection limit of 1 cell mL(-1). The activity of compounds 4a and 4c was similar to that of the commercial antifungal agent fluconazole (Flu). The cytotoxic and hemolytic activity of the compounds was assayed against human neonate dermal fibroblasts and sheep erythrocytes, respectively. Compounds 4a-c were less cytotoxic than Flu and CHX. Our results suggest that compound 4b, which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent.
[Show abstract][Hide abstract] ABSTRACT: This study examined the utility of synergistic disinfection employing a gemini-quaternary ammonium salt (a gemini-QUAT, namely 3,3'-(2,7-dioxaoctane)bis(1-decylpyridinium bromide)), as an organic biocide in combination with irradiation by an ultraviolet-A (UV-A) light-emitting diode (LED) with a peak wavelength of 365nm. The combined system represents a novel disinfection method utilizing facilitated in situ oxidation depending on overproduction of reactive oxygen species (ROS) triggered by the initial action of the gemini-QUAT on the bacterial membrane. We demonstrate that this combination decreased the viability of pathogenic bacteria in a significant and rapid manner, and depended on doses of the gemini-QUAT and the fluence: the viability of Escherichia coli was reduced by greater than 5.0-logs by the combination procedure, but the decrease in viability was only 2.3-logs for exposure to UV at the same fluence dose in the absence of the gemini-QUAT. Adding catalase as a radical scavenger decreased bacterial inactivation by the combined disinfection procedure. Flow cytometric analysis indicated superoxide and hydrogen peroxide overproduction within cells treated with the combined disinfection procedure. The excessive superoxide, detected only in the combined system, appeared to be generated by the action of the gemini-QUAT at the bacterial membrane, leading to excessive and rapid generation of ROS in the system. Our data strongly suggested that this ROS promoted bacterial membrane peroxidation during initial treatment by the combination method, resulting in increased oxidative modification of DNA. These oxidative reactions may play an important role in the efficacy of this disinfection procedure.
Journal of photochemistry and photobiology. B, Biology 12/2013; 130C:226-233. DOI:10.1016/j.jphotobiol.2013.11.027 · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies.
Journal of Bioscience and Bioengineering 12/2013; 117(5). DOI:10.1016/j.jbiosc.2013.10.022 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus spp. bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl-CoA transferase (tlsA), fructose-bisphosphate aldolase (fda) and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in the Rhodococcus species.
[Show abstract][Hide abstract] ABSTRACT: 5a-h, a series of (5-substituted-2-methyl-1,3-thiazole-4-yl) acetic acids as heterocyclic acetic acid derivatives, was designed and synthesized from ethyl acetoacetate. The synthesized compounds were screened for their antimicrobial activities against bacterial and fungal strains, and their characteristics were investigated by assays under various temperature and pH conditions. Cytotoxicity was evaluated with the use of sheep erythrocytes and human neonate dermal fibroblasts. Similarly, agents such as lauric acid 6 and parabens 7a-b, which are used as preservative agents for commercial cosmetics and detergents, were assayed for comparison. Although the structure of 5a is simple, comprising a thiazole attached with an octyl group and acetic acid moiety, the compound showed stronger and broader antibacterial and antifungal activities among the 5 series against the tested microbes other than gram-negative bacteria. Interestingly, 5a overcame the weak antifungal activity of parabens 7a-b. Also, the cytotoxicity of 5a was less than that of parabens 7a-b, especially to human dermal fibroblasts. These results suggest that thiazolyl-acetic acid 5a is a potentially effective biocide, and that it could be used as a preservative agent in commercially sold cosmetics and detergents, facilitated by the hydrophilic and charge properties of its carboxylic acid moiety.
[Show abstract][Hide abstract] ABSTRACT: The NfxC-type mutant of Pseudomonas aeruginosa produces the MexEF-OprN efflux pump and down-regulates expression of the quorum-sensing-dependent efflux pump MexAB-OprM and production of virulence factors in the presence of an active transcriptional regulator, MexT. Consequently, these cells are resistant to chloramphenicol and hypersusceptible to β-lactam antibiotics. An upper negative regulator, MexS, has been assumed to inactivate MexT in wild-type strains, hence shutting down production of the MexEF-OprN pump. This observation was, however, reported in only one clinical strain and not confirmed in well-characterized laboratory strains. Moreover, it is not known whether MexS is involved in the quorum-sensing-dependent regulation of virulence factor production. To assess these issues, a plasmid carrying wild-type mexS was introduced into three NfxC-type mutants from laboratory strains, which carry an impaired mexS and unimpaired mexT. Unexpectedly, all the transformants produced an increased amount of MexEF-OprN proteins. Three clinical NfxC strains were similarly transformed and although MexEF-OprN was undetectable in two of these strains, one produced an increased amount of these proteins, similar to the laboratory strains. These results were interpreted to mean that P. aeruginosa takes two separate routes in MexT-mediated regulation of mexEF-oprN expression: the MexS-bypassed pathway and MexS-mediated pathway. On the other hand, the transformants of both the laboratory and clinically derived NfxC-type cells produced increased amounts of MexAB-OprM and virulence factors, suggesting that production of these proteins occurs via the MexS-mediated pathway.
Microbiology and Immunology 04/2013; 57(4):263-72. DOI:10.1111/1348-0421.12032 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The NfxC-type mutant of Pseudomonas aeruginosa produces the MexEF-OprN efflux pump and down-regulates the expression of the quorum-sensing-dependent efflux pump MexAB-OprM and production of virulence factors in the presence of an active transcriptional regulator, MexT. Consequently, the cells are resistant to chloramphenicol and hypersusceptible to ß-lactam antibiotics. An upper negative regulator, MexS, was assumed to inactivate MexT in wild-type strains, hence shutting down production of the MexEF-OprN pump. This observation was, however, reported in only one clinical strain and not established in well-characterized laboratory strains. Moreover, it is not known whether MexS is involved in the quorum-sensing-dependent regulation of virulence factor production. To assess these issues, we introduced a plasmid carrying the wild-type mexS into three NfxC-type mutants from laboratory strains, which carry an impaired mexS and unimpaired mexT. Unexpectedly, all the transformants produced an elevated level of the MexEF-OprN proteins. Three clinical NfxC strains were similarly transformed and although two strains showed an undetectable level of MexEF-OprN, one strain produced an elevated level of the proteins similar to the laboratory strains. These results were interpreted to mean that P. aeruginosa takes two separate routes in the MexT-mediated regulation of mexEF-oprN expression; the MexS-bypassed pathway and the MexS-mediated pathway. On the other hand, the transformants of both the laboratory and clinically derived NfxC-type cells produced elevated levels of MexAB-OprM and the virulence factors suggesting that production of these proteins takes the MexS-mediated pathway.
Microbiology and Immunology 02/2013; DOI:10.1111/j.1348-0421.12032 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mlr gene cluster consisting of mlrA, mlrB, mlrC, and mlrD is involved in the degradation of the cyanobacterial toxin microcystin. However, it is unclear which degradation intermediates are metabolized by MlrB and MlrC. To address these questions, we constructed recombinant Escherichia coli to overproduce MlrB and MlrC from Sphingopyxis sp. C-1, and determined which intermediates were degraded in cell-free extracts. The cell-free extract containing MlrB degraded linearized microcystin-LR, giving rise to a tetrapeptide. The cell-free extract of MlrC degraded linearized microcystin-LR and also degraded the tetrapeptide to the amino acid Adda. These results indicate that linearized microcystin-LR is degraded by both MlrB and MlrC, and tetrapeptide is degraded by specifically by MlrC in Sphingopyxis sp. C-1.
Journal of Bioscience and Bioengineering 08/2012; 114(6). DOI:10.1016/j.jbiosc.2012.07.004 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously found that the gemini quaternary salt (gemini-QUAT) containing two pyridinium residues per molecule, 3,3'- (2,7-dioxaoctane) bis (1-decylpyridinium bromide) (3DOBP-4,10) , exerted fungicidal activity against Saccharomyces cerevisiae and caused respiration inhibition and the cytoplasmic leakage of ATP, magnesium, and potassium ions. Here, we investigated how the gemini-QUAT, 3DOBP-4,10, exerts more powerful antimicrobial activity than the mono-QUAT N-cetylpyridinium chloride (CPC) and examined the association between reactive oxygen species (ROS) and the antimicrobial mechanism. Antifungal assays showed that the activity of 3DOBP-4,10 against two yeasts, S. cerevisiae and Candida albicans, was significantly elevated under aerobic conditions, and largely reduced under anaerobic conditions (nitrogen atmosphere) . Adding radical scavengers such as superoxide dismutase, catalase and potassium iodide (KI) also decreased the fungicidal activity of 3DOBP-4,10 but negligibly affected that of CPC. We measured survival under static conditions and found that the rapid fungicidal profile of 3DOBP-4,10 was lost, whereas that of CPC was slightly affected in the presence of KI. Our results suggest that 3DOBP-4,10 exerts powerful antimicrobial activity by penetrating the cell wall and membrane, which then allows oxygen to enter the cells, where it participates in the generation of intracellular ROS. The activity could thus be attributable to a synergic antimicrobial combination of the disruption of organelle membranes by the QUAT and oxidative stress imposed by ROS.
[Show abstract][Hide abstract] ABSTRACT: The potential of winter biofilm for microcystin-LR (MCLR) biodegradation was comparatively evaluated under various nutrient conditions. Results indicated that MCLR was completely biodegraded by Day 7 without nutrient addition. MCLR-biodegradation was inhibited in the presence of phosphate or glucose addition, with complete MCLR removal observed by Day 10. MCLR was totally biodegraded by Day 7 with dual nutrients comprising glucose and nitrate, suggesting that additional nitrate alleviated the inhibitory effect of glucose alone on the biodegradation. Simultaneously, MCLR-degrading gene (mlrA) abundance were detected to increase with increasing amount of MCLR being degraded under the respective conditions, implying that MCLR-biodegradation depended on the population of indigenous MCLR-degrading bacteria (MCLRDB), which was related to the population of non-degrading bacteria in the biofilm. MCLRDB was found to primarily use MCLR for proliferation rather than other nutrients. This is the first report verifying MCLR as a primary substrate for bacteria under various nutrient conditions.