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Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus and Micrococcus luteus

Novo, David J.; Perlmutter, Nancy G.; Hunt, Richard H.; Shapiro, Howard M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2000 EN
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780.83875%
Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability...

Studies of the Novel Ketolide ABT-773: Transport, Binding to Ribosomes, and Inhibition of Protein Synthesis in Streptococcus pneumoniae

Capobianco, John O.; Cao, ZhenSheng; Shortridge, Virginia D.; Ma, ZhenKun; Flamm, Robert K.; Zhong, Ping
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2000 EN
Relevância na Pesquisa
780.847%
Macrolide resistance in Streptococcus pneumoniae has been associated with two main mechanisms: target modification by Erm methyltransferases and efflux by macrolide pumps. The ketolide ABT-773, which has a 3-keto group and no l-cladinose sugar, represents a new class of drugs with in vitro activity against a variety of resistant bacteria. Several approaches were undertaken to understand how ABT-773 was able to defeat resistance mechanisms. We demonstrated tighter ribosome binding of ABT-773 than erythromycin. We also showed that ABT-773 (i) accumulated in macrolide-sensitive S. pneumoniae at a higher rate than erythromycin, (ii) was able to bind with methylated ribosomes, though at lower affinities than with wild-type ribosomes, and (iii) accumulated in S. pneumoniae strains with the efflux-resistant phenotype.

Selective Targeting of Topoisomerase IV and DNA Gyrase in Staphylococcus aureus: Different Patterns of Quinolone- Induced Inhibition of DNA Synthesis

Fournier, Bénédicte; Zhao, Xilin; Lu, Tao; Drlica, Karl; Hooper, David C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2000 EN
Relevância na Pesquisa
782.1385%
The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

Inhibitory Activities of Lansoprazole against Respiration in Helicobacter pylori

Nagata, Kumiko; Sone, Nobuhito; Tamura, Toshihide
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2001 EN
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781.69086%
Lansoprazole and its derivative AG-1789 dose-dependently inhibited cellular respiration by an endogenous substrate and decreased the ATP level in Helicobacter pylori cells. The inhibitory action of lansoprazole and AG-1789 against respiration was specific to substrates such as pyruvate and α-ketoglutarate and similar to the inhibitory action of rotenone, which is an inhibitor for the mitochondrial respiratory chain. Growth inhibition by lansoprazole and AG-1789 as well as by rotenone was augmented at high oxygen concentrations under atmospheric conditions. Since the 50% inhibitory concentrations of these compounds for the respiration were close to their MICs for H. pylori growth, the growth inhibition might be due to respiratory inhibition by these compounds.

Inhibitory Action of a Truncated Derivative of the Amphibian Skin Peptide Dermaseptin s3 on Saccharomyces cerevisiae

Coote, Peter J.; Holyoak, Caroline D.; Bracey, Dani; Ferdinando, Dudley P.; Pearce, James A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1998 EN
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782.2308%
The inhibitory activity of a truncated derivative of the natural amphibian skin peptide dermaseptin s3-(1-16)-NH2 [DS s3 (1-16)] against Saccharomyces cerevisiae was studied. Significant growth inhibition was observed after exposure to 3.45 μg of the peptide per ml at pH 6.0 and 7.0, with complete growth inhibition occurring at 8.63 μg of peptide per ml for all pH values tested. Using confocal scanning laser microscopy, we have shown that DS s3 (1-16) disrupted the yeast cell membrane resulting in the gross permeabilization of the cell to the nuclear stain ethidium bromide. However, the principal inhibitory action of the peptide was not due to disruption of intracellular pH homeostasis. Instead, growth inhibition by the peptide correlated with the efflux of important cellular constituents such as ADP, ATP, RNA, and DNA into the surrounding medium. The combination of DS s3 (1-16) with mild heating temperatures as low as 35°C significantly enhanced the inhibitory effect of the peptide (8.63 μg/ml), and at 45°C greater than 99% of the population was killed in 10 min. In summary, a derivative of a natural antimicrobial peptide has potential, either alone or in combination with mild heating, to prevent the growth of or kill spoilage yeast.

Nitric Oxide Generated from Isoniazid Activation by KatG: Source of Nitric Oxide and Activity against Mycobacterium tuberculosis

Timmins, Graham S.; Master, Sharon; Rusnak, Frank; Deretic, Vojo
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2004 EN
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781.9661%
Isonicotinic acid hydrazide (INH) is a frontline antituberculosis agent. Once taken up by Mycobacterium tuberculosis, INH requires activation by the catalase-peroxidase KatG, converting INH from its prodrug form into a range of bactericidal reactive species. Here we used 15N-labeled INH together with electron paramagnetic resonance spin trapping techniques to demonstrate that nitric oxide (NȮ) is generated from oxidation at the hydrazide nitrogens during the activation of INH by M. tuberculosis KatG. We also observed that a specific scavenger of NȮ provided protection against the antimycobacterial activity of INH in bacterial culture. No significant increases in mycobacterial protein nitration were detected, suggesting that NȮ and not peroxynitrite, a nitrating metabolite of NO·, is involved in antimycobacterial action. In conclusion, INH-derived NO· has biological activity, which directly contributes to the antimycobacterial action of INH.

Biphasic Effects of Geranylgeraniol, Teprenone, and Phytol on the Growth of Staphylococcus aureus

Inoue, Yoshihiro; Hada, Toshiko; Shiraishi, Akiko; Hirose, Kazuma; Hamashima, Hajime; Kobayashi, Shigeki
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2005 EN
Relevância na Pesquisa
781.7331%
We examined the antibacterial activities against Staphylococcus aureus of three diterpenes, namely, geranylgeraniol, teprenone, and phytol, by using a broth dilution with shaking method to identify the effects of diterpenes with long aliphatic carbon chains. We also performed time-kill assays and measured the leakage of K+ ions from bacterial cells in response to these diterpenes. The diterpenes used inhibited the growth of S. aureus at concentrations of 0.15 μg/ml, as determined by damage to the cell membranes, and had clear bactericidal activity. However, the inhibitory effects of the diterpenes decreased when the concentration of each was raised above a certain level. The diterpenes tested in this study appeared to have both growth-inhibitory and growth-accelerating effects, and the net effect of each depended on its concentration.

Search for Cyclodextrin-Based Inhibitors of Anthrax Toxins: Synthesis, Structural Features, and Relative Activities▿

Karginov, Vladimir A.; Nestorovich, Ekaterina M.; Yohannes, Adiamseged; Robinson, Tanisha M.; Fahmi, Nour Eddine; Schmidtmann, Frank; Hecht, Sidney M.; Bezrukov, Sergey M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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782.21195%
Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of β-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new β-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of β-cyclodextrin with alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and channel blocking were found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to identify a new class of drugs for anthrax treatment...

Transcriptional Profiling Reveals that Daptomycin Induces the Staphylococcus aureus Cell Wall Stress Stimulon and Genes Responsive to Membrane Depolarization▿ †

Muthaiyan, Arunachalam; Silverman, Jared A.; Jayaswal, Radheshyam K.; Wilkinson, Brian J.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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782.2475%
Daptomycin is a lipopeptide antibiotic that has recently been approved for treatment of gram-positive bacterial infections. The mode of action of daptomycin is not yet entirely clear. To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out. The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal, Curr. Med. Chem. Anti-Infect. Agents 4:259-276, 2005) were significantly induced by daptomycin and by the cell wall-active antibiotics vancomycin and oxacillin. Comparison of the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon in the mutant. Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined. Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon. However...

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei▿

Lanteri, Charlotte A.; Tidwell, Richard R.; Meshnick, Steven R.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
782.1907%
Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the species Trypanosoma brucei gambiense and T. b. rhodesiense. An oral prodrug, DB289, is a promising new therapy undergoing phase III clinical trials for early-stage HAT. DB289 is metabolically converted to the active trypanocidal diamidine DB75 [2,5-bis(4-amidinophenyl)furan]. We previously determined that DB75 inhibits yeast mitochondrial function (C. A. Lanteri, B. L. Trumpower, R. R. Tidwell, and S. R. Meshnick, Antimicrob. Agent Chemother. 48:3968-3974, 2004). The purpose of this study was to investigate if DB75 targets the mitochondrion of T. b. brucei bloodstream forms. DB75 rapidly accumulates within the mitochondria of living trypanosomes, as indicated by the fluorescent colocalization of DB75 with a mitochondrion-specific dye. Fluorescence-activated cell sorting analysis of rhodamine 123-stained living trypanosomes shows that DB75 and other trypanocidal diamidines (pentamidine and diminazene) collapse the mitochondrial membrane potential. DB75 inhibits ATP hydrolysis within T. brucei mitochondria and appears to inhibit the oligomycin-sensitive F1F0-ATPase and perhaps other ATPases. DB75 is most likely not an inhibitor of electron transport within trypanosome mitochondria...

Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials▿

Creek, Darren J.; Charman, William N.; Chiu, Francis C. K.; Prankerd, Richard J.; Dong, Yuxiang; Vennerstrom, Jonathan L.; Charman, Susan A.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
782.0677%
The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study.

Effect of Subinhibitory Concentrations of Antibiotics on Intrachromosomal Homologous Recombination in Escherichia coli▿

López, Elena; Blázquez, Jesús
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
780.8756%
Subinhibitory concentrations of some antibiotics, such as fluoroquinolones, have been reported to stimulate mutation and, consequently, bacterial adaptation to different stresses, including antibiotic pressure. In Escherichia coli, this stimulation is mediated by alternative DNA polymerases induced via the SOS response. Sublethal concentrations of the fluoroquinolone ciprofloxacin have been shown to stimulate recombination between divergent sequences in E. coli. However, the effect of ciprofloxacin on recombination between homologous sequences and its SOS dependence have not been studied. Moreover, the possible effects of other antibiotics on homologous recombination remain untested. The aim of this work was to study the effects of sublethal concentrations of ciprofloxacin and 10 additional antibiotics, including different molecular families with different molecular targets, on the rate of homologous recombination of DNA in E. coli. The antibiotics tested were ciprofloxacin, ampicillin, ceftazidime, imipenem, chloramphenicol, tetracycline, gentamicin, rifampin (rifampicin), trimethoprim, fosfomycin, and colistin. Our results indicate that only ciprofloxacin consistently stimulates the intrachromosomal recombinogenic capability of homologous sequences in E. coli. The ciprofloxacin-based stimulation occurs at concentrations and times that apparently do not dramatically compromise the viability of the whole population...

Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia▿

Wakabayashi, Hiroyuki; Yamauchi, Koji; Kobayashi, Tetsuo; Yaeshima, Tomoko; Iwatsuki, Keiji; Yoshie, Hiromasa
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
780.8645%
Lactoferrin (LF) is an iron-binding antimicrobial protein present in saliva and gingival crevicular fluids, and it is possibly associated with host defense against oral pathogens, including periodontopathic bacteria. In the present study, we evaluated the in vitro effects of LF-related agents on the growth and biofilm formation of two periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, which reside as biofilms in the subgingival plaque. The planktonic growth of P. gingivalis and P. intermedia was suppressed for up to 5 h by incubation with ≥130 μg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and ≥6 μg/ml of bLF-derived antimicrobial peptide lactoferricin B (LFcin B); but those effects were weak after 8 h. The biofilm formation of P. gingivalis and P. intermedia over 24 h was effectively inhibited by lower concentrations (≥8 μg/ml) of various iron-bound forms (the apo, native, and holo forms) of bLF and hLF but not LFcin B. A preformed biofilm of P. gingivalis and P. intermedia was also reduced by incubation with various iron-bound bLFs, hLF, and LFcin B for 5 h. In an examination of the effectiveness of native bLF when it was used in combination with four antibiotics...

Staphylococcus aureus TargetArray: Comprehensive Differential Essential Gene Expression as a Mechanistic Tool To Profile Antibacterials▿ ¶

Xu, H. Howard; Trawick, John D.; Haselbeck, Robert J.; Forsyth, R. Allyn; Yamamoto, Robert T.; Archer, Rich; Patterson, Joe; Allen, Molly; Froelich, Jamie M.; Taylor, Ian; Nakaji, Danny; Maile, Randy; G. C., Kedar; Pilcher, Marshall; Brown-Driver, Vickie;
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
782.21195%
The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.

Structure-Activity Studies and Therapeutic Potential of Host Defense Peptides of Human Thrombin▿

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2011 EN
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781.7936%
Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally...

Virulence-Suppressing Effects of Linezolid on Methicillin-Resistant Staphylococcus aureus: Possible Contribution to Early Defervescence

Yoshizawa, Sadako; Tateda, Kazuhiro; Saga, Tomoo; Ishii, Yoshikazu; Yamaguchi, Keizo
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2012 EN
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781.90305%
In the present study, immunomodulatory effects of linezolid (LZD) on methicillin-resistance Staphylococcus aureus (MRSA) infections were evaluated. We have retrospectively reviewed treatment effects of LZD on 52 patients with severe MRSA infections. Sixty-four percent of the febrile patients demonstrated significant defervescence within 3 days, despite the presence of positive culture results. We speculated that this finding might be due to early anti-inflammatory effects of LZD, and to investigate this further we initiated in vivo experiments using mice MRSA pneumonia models. Mice were treated with either LZD or vancomycin (VCM) immediately after intranasal administration of MRSA. Bacterial numbers and levels of inflammatory cytokines in the lungs were determined. Although the bacterial burden in the lungs was not apparently different between the two groups, LZD but not VCM treatment significantly reduced induction of inflammatory cytokines in the lungs (P < 0.05). To evaluate whether this anti-inflammatory response was due to suppression of virulence factor expression, filter-sterilized supernatants of MRSA incubated in broth overnight with sub-MICs of LZD were subcutaneously administered to mice. To clarify whether LZD possesses direct host-modulating activity...

Effects of 14-Alpha-Lipoyl Andrographolide on Quorum Sensing in Pseudomonas aeruginosa

Ma, Li; Liu, Xiangyang; Liang, Haihua; Che, Yizhou; Chen, Caixia; Dai, Huanqin; Yu, Ke; Liu, Mei; Ma, Luyan; Yang, Ching-Hong; Song, Fuhang; Wang, Yuqiang; Zhang, Lixin
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2012 EN
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781.69484%
In Pseudomonas aeruginosa, the quorum-sensing (QS) system is closely related to biofilm formation. We previously demonstrated that 14-alpha-lipoyl andrographolide (AL-1) has synergistic effects on antibiofilm and antivirulence factors (pyocyanin and exopolysaccharide) of P. aeruginosa when combined with conventional antibiotics, while it has little inhibitory effect on its growth. However, its molecular mechanism remains elusive. Here we investigated the effect of AL-1 on QS systems, especially the Las and Rhl systems. This investigation showed that AL-1 can inhibit LasR–3-oxo-C12-homoserine lactone (HSL) interactions and repress the transcriptional level of QS-regulated genes. Reverse transcription (RT)-PCR data showed that AL-1 significantly reduced the expression levels of lasR, lasI, rhlR, and rhlI in a dose-dependent manner. AL-1 not only decreased the expression level of Psl, which is positively regulated by the Las system, but also increased the level of secretion of ExoS, which is negatively regulated by the Rhl system, indicating that AL-1 has multiple effects on both the Las and Rhl systems. It is no wonder that AL-1 showed synergistic effects with other antimicrobial agents in the treatment of P. aeruginosa infections.

The Broad-Spectrum Antiviral Compound ST-669 Restricts Chlamydial Inclusion Development and Bacterial Growth and Localizes to Host Cell Lipid Droplets within Treated Cells

Sandoz, Kelsi M.; Valiant, William G.; Eriksen, Steven G.; Hruby, Dennis E.; Allen, Robert D.; Rockey, Daniel D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2014 EN
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782.1665%
Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions...

Relationship between Chloroquine Toxicity and Iron Acquisition in Saccharomyces cerevisiae

Emerson, Lyndal R.; Nau, Martin E.; Martin, Rodger K.; Kyle, Dennis E.; Vahey, Maryanne; Wirth, Dyann F.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2002 EN
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782.1907%
Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing. 55FeCl3 accumulation was inhibited in the presence of chloroquine...

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans ▿ †

Vediyappan, Govindsamy; Rossignol, Tristan; d'Enfert, Christophe
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Candida albicans can form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. The molecular mechanisms involved in the antifungal resistance of biofilms remain poorly understood. We have used transcript profiling to explore the early transcriptional responses of mature C. albicans biofilms exposed to various antifungal agents. Mature C. albicans biofilms grown under continuous flow were exposed for as long as 2 h to concentrations of fluconazole (FLU), amphotericin B (AMB), and caspofungin (CAS) that, while lethal for planktonic cells, were not lethal for biofilms. Interestingly, FLU-exposed biofilms showed no significant changes in gene expression over the course of the experiment. In AMB-exposed biofilms, 2.7% of the genes showed altered expression, while in CAS-exposed biofilms, 13.0% of the genes had their expression modified. In particular, exposure to CAS resulted in the upregulation of hypha-specific genes known to play a role in biofilm formation, such as ALS3 and HWP1. There was little overlap between AMB- or CAS-responsive genes in biofilms and those that have been identified as AMB, FLU, or CAS responsive in C. albicans planktonic cultures. These results suggested that the resistance of C. albicans biofilms to azoles or polyenes was due not to the activation of specific mechanisms in response to exposure to these antifungals but rather to the intrinsic properties of the mature biofilms. In this regard...