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1.
J Ind Microbiol Biotechnol ; 49(4)2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35595506

RESUMO

Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40-72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 µg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.


Assuntos
Bacillus , Desinfetantes , Desinfetantes/farmacologia , Muramidase , Hipoclorito de Sódio/farmacologia , Esporos Bacterianos
2.
Ann Microbiol ; 71(1)2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34744534

RESUMO

PURPOSE: There has been an interest in the microbial azo dye degradation as an optional method for the treatment of azo dye-containing wastes. Tattoo ink is an extremely unique azo dye-rich environment, which have never been explored in terms of microorganisms capable of degrading azo dyes. Previously, we isolated 81 phylogenetically diverse bacteria, belonging to 18 genera and 52 species, contaminated in tattoo inks. In this study, we investigated if these bacteria, which can survive in the azo dye-rich environment, have an ability to degrade azo dyes. METHODS: We conducted a two-step azo dye degradation (or decolorization) assay. In step 1, a high-throughput degradability assay was done for 79 bacterial isolates using Methyl Red and Orange II. In step 2, a further degradation assay was done for 10 selected bacteria with a representative of 11 azo dyes, including 3 commercial tattoo ink azo dyes. Degradation of azo dyes were calculated from measuring optical absorbance of soluble dyes at specific wavelengths. RESULTS: The initial high-throughput azo dye assay (step 1) showed that 79 isolates had a complete or partial degradation of azo dyes; > 90% of Methyl Red and Orange II were degraded within 24 h, by 74 and 20 isolates, respectively. A further evaluation of azo dye degradability for 10 selected isolates in step 2 showed that the isolates, belonging to Bacillus, Brevibacillus, Paenibacillus, and Pseudomonas, exhibited an excellent decolorization ability for a wide range of azo dyes. CONCLUSIONS: This study showed that phylogenetically diverse bacteria, isolated from azo dye-rich tattoo inks, is able to degrade a diverse range of azo dyes, including 3 azo dyes used in commercial tattoo inks. Some of the strains would be good candidates for future studies to provide a systematic understanding of azo dye degradation mechanisms.

3.
Anaerobe ; 52: 29-42, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29852249

RESUMO

The use of smokeless tobacco products (STPs) can cause many serious health problems. The oral microbiota plays important roles in oral and systemic health, and the disruption in the oral microbial population is linked to periodontal disease and other health problems. To assess the impact of smokeless tobacco on oral microbiota in vivo, high-throughput sequencing was used to examine the oral microbiota present in Syrian Golden hamster cheek pouches. Sixteen hamsters were divided into four groups and treated with the STP Grizzly snuff (0, 2.5, 25, or 250 mg) twice daily for 4 weeks. After 0, 1, 2, 3, and 4 weeks of treatment, bacterial genomic DNA was extracted from oral swabs sampled from the cheek pouches of the hamsters. The oral bacterial communities present in different hamster groups were characterized by sequencing the hypervariable regions V1-V2 and V4 of 16S rRNA using the Illumina MiSeq platform. Fifteen phyla, 27 classes, 59 orders, 123 families, and 250 genera were identified from 4,962,673 sequence reads from the cheek pouch samples. The bacterial diversity and taxonomic abundances for the different treatment groups were compared to the non-treated hamsters. Bacterial diversity was significantly decreased after 4 weeks of exposure to 2.5 mg, and significantly increased by exposure to 250 mg STP. Treatment with 250 mg STP significantly increased Firmicutes, transiently increased Cyanobacteria and TM7, and decreased Bacteroidetes and Fusobacteria compared to the control group. At the genus level, 4 weeks of administration of 250 mg STP significantly increased Granulicatella, Streptococcus, Oribacterium, Anaerococcus, Acidaminococcus, Actinomyces, Eubacterium, Negativicoccus, and Staphylococcus, and decreased Bacteroides, Buleidia, Dialister, and Leptotrichia, and transiently decreased Arcanobacterium compared to the control group. For the first time, an animal model was used for evaluating the effects of STP on oral microbiota by metagenomic sequencing. Our results provide a view of the shift of the oral microbiota in response to STP exposure in Syrian Golden hamster. Our findings indicate that the use of smokeless tobacco significantly disrupts the oral microbiota.


Assuntos
Bactérias/isolamento & purificação , Carcinogênese/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Neoplasias Bucais/etiologia , Neoplasias Bucais/microbiologia , Boca/microbiologia , Tabaco sem Fumaça/efeitos adversos , Animais , Bactérias/classificação , Bactérias/genética , Cricetinae , DNA Bacteriano/genética , Modelos Animais de Doenças , Humanos , Masculino , Mesocricetus , Filogenia , RNA Ribossômico 16S/genética
4.
Biochem Biophys Res Commun ; 482(2): 296-300, 2017 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-27856243

RESUMO

SecA is an essential component in the bacterial Sec-dependent protein translocation process. We previously showed that in addition to the ubiquitous, high-affinity SecYEG-SecDF·YajC protein translocation channel, there is a low-affinity SecA-only channel that elicits ion channel activity and promotes protein translocation. The SecA-only channels are less efficient, and like Prl suppressors, lack signal peptide specificity; they function in the absence of signal peptides. The presence of SecYEG-SecDF·YajC alters the sensitivity of ATPase inhibitor Rose Bengal. In this study, we found that the suppressor membranes are much more resistant to inhibition by Rose Bengal. Similar results have been found for a SecA-specific inhibitor. Moreover, biphasic responses of inhibition of ion current and protein translocation activities were observed for many PrlA/SecY and PrlG/SecE suppressor membranes, with a low IC50 value similar to that of the SecA-only channels and a very high IC50. However, the suppressor strains are as sensitive to the inhibitor as the parental strain, suggesting that SecA-only channels have some essential physiological function(s) in the cells that are inhibited by the specific SecA inhibitor.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transporte Proteico/fisiologia , Rosa Bengala/administração & dosagem , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/metabolismo , Escherichia coli/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas SecA
5.
J Ind Microbiol Biotechnol ; 44(10): 1471-1481, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28786013

RESUMO

Dyes containing one or more azo linkages are widely applied in cosmetics, tattooing, food and drinks, pharmaceuticals, printing inks, plastics, leather, as well as paper industries. Previously we reported that bacteria living on human skin have the ability to reduce some azo dyes to aromatic amines, which raises potential safety concerns regarding human dermal exposure to azo dyes such as those in tattoo ink and cosmetic colorant formulations. To comprehensively investigate azo dye-induced toxicity by skin bacteria activation, it is very critical to understand the mechanism of metabolism of the azo dyes at the systems biology level. In this study, an LC/MS-based metabolomics approach was employed to globally investigate metabolism of azo dyes by Staphylococcus aureus as well as their effects on the metabolome of the bacterium. Growth of S. aureus in the presence of Sudan III or Orange II was not affected during the incubation period. Metabolomics results showed that Sudan III was metabolized to 4-(phenyldiazenyl) aniline (48%), 1-[(4-aminophenyl) diazenyl]-2-naphthol (4%) and eicosenoic acid Sudan III (0.9%). These findings indicated that the azo bond close to naphthalene group of Sudan III was preferentially cleaved compared with the other azo bond. The metabolite from Orange II was identified as 4-aminobenzene sulfonic acid (35%). A much higher amount of Orange II (~90×) was detected in the cell pellets from the active viable cells compared with those from boiled cells incubated with the same concentration of Orange II. This finding suggests that Orange II was primarily transported into the S. aureus cells for metabolism, instead of the theory that the azo dye metabolism occurs extracellularly. In addition, the metabolomics results showed that Sudan III affected energy pathways of the S. aureus cells, while Orange II had less noticeable effects on the cells. In summary, this study provided novel information regarding azo dye metabolism by the skin bacterium, the effects of azo dyes on the bacterial cells and the important role on the toxicity and/or inactivation of these compounds due to microbial metabolism.


Assuntos
Compostos Azo/metabolismo , Compostos Azo/farmacologia , Metaboloma/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Benzenossulfonatos/metabolismo , Benzenossulfonatos/farmacologia , Cor , Naftóis/química , Naftóis/metabolismo , Ácidos Sulfanílicos/metabolismo , Espectrometria de Massas em Tandem
6.
Anaerobe ; 42: 152-161, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27756619

RESUMO

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 µg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.


Assuntos
Misturas Complexas/farmacologia , Microbiota/efeitos dos fármacos , Boca/microbiologia , Nitrosaminas/farmacologia , Tabaco sem Fumaça/análise , Meios de Cultura/química , Eubacterium/efeitos dos fármacos , Eubacterium/isolamento & purificação , Eubacterium/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Viabilidade Microbiana/efeitos dos fármacos , Microbiota/fisiologia , Peptostreptococcus/efeitos dos fármacos , Peptostreptococcus/isolamento & purificação , Peptostreptococcus/fisiologia , Especificidade da Espécie , Streptococcus anginosus/efeitos dos fármacos , Streptococcus anginosus/isolamento & purificação , Streptococcus anginosus/fisiologia , Streptococcus constellatus/efeitos dos fármacos , Streptococcus constellatus/isolamento & purificação , Streptococcus constellatus/fisiologia , Veillonella/efeitos dos fármacos , Veillonella/isolamento & purificação , Veillonella/fisiologia
7.
Anal Biochem ; 480: 58-66, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25862083

RESUMO

Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP-Mg(2+). Such activity was enhanced by reconstituted SecYEG-SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca(2+)-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Conexinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oócitos/metabolismo , Proteolipídeos/metabolismo , Análise de Célula Única , Animais , Linhagem Celular , Conexina 26 , Insetos , Camundongos , Canais de Translocação SEC , Proteínas SecA , Xenopus laevis/metabolismo
8.
Bioorg Med Chem ; 23(1): 105-17, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25498235

RESUMO

Protein translocation is essential for bacterial survival and the most important translocation mechanism is the secretion (Sec) pathway in which SecA is a central core driving force. Thus targeting SecA is a promising strategy for developing novel antibacterial therapeutics. Herein, we report the syntheses and evaluation of a series of nearly 60 4-oxo-5-cyano thiouracil derivatives based upon our previously reported core pyrimidine structure. Introduction of polar group such as -N3 and linker groups such as -CH2-O- enhanced the potency several fold. Apart from being potential antibacterial agents, these inhibitors can be indispensable tools for biologists to probe the mechanism of protein translocation via the SecA machinery in bacteria.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tiouracila/síntese química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-Atividade , Tiouracila/química
9.
Bioorg Med Chem ; 23(21): 7061-8, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26432604

RESUMO

Due to the emergence and rapid spread of drug resistance in bacteria, there is an urgent need for the development of novel antimicrobials. SecA, a key component of the general bacterial secretion system required for viability and virulence, is an attractive antimicrobial target. Earlier we reported that systematical dissection of a SecA inhibitor, Rose Bengal (RB), led to the development of novel small molecule SecA inhibitors active against Escherichia coli and Bacillus subtilis. In this study, two potent RB analogs were further evaluated for activities against methicillin-resistant Staphylococcus aureus (MRSA) strains and for their mechanism of actions. These analogs showed inhibition on the ATPase activities of S. aureus SecA1 (SaSecA1) and SecA2 (SaSecA2), and inhibition of SaSecA1-dependent protein-conducting channel. Moreover, these inhibitors reduce the secretion of three toxins from S. aureus and exert potent bacteriostatic effects against three MRSA strains. Our best inhibitor SCA-50 showed potent concentration-dependent bactericidal activity against MRSA Mu50 strain and very importantly, 2-60 fold more potent inhibitory effect on MRSA Mu50 than all the commonly used antibiotics including vancomycin, which is considered the last resort option in treating MRSA-related infections. Protein pull down experiments further confirmed SaSecA1 as a target. Deletion or overexpression of NorA and MepA efflux pumps had minimal effect on the antimicrobial activities against S. aureus, indicating that the effects of SecA inhibitors were not affected by the presence of these efflux pumps. Our studies show that these small molecule analogs target SecA functions, have potent antimicrobial activities, reduce the secretion of toxins, and have the ability to overcome the effect efflux pumps, which are responsible for multi-drug resistance. Thus, targeting SecA is an attractive antimicrobial strategy against MRSA.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Anti-Infecciosos/química , Proteínas de Bactérias/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/enzimologia , Rosa Bengala/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Luz , Proteínas de Membrana Transportadoras/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oxirredução , Estrutura Terciária de Proteína , Rosa Bengala/farmacologia , Canais de Translocação SEC , Proteínas SecA , Staphylococcus aureus/efeitos dos fármacos
10.
Biochem Biophys Res Commun ; 454(2): 308-12, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25450394

RESUMO

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Rosa Bengala/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC , Proteínas SecA , Xenopus
11.
FEMS Microbiol Lett ; 365(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30007321

RESUMO

Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Canais de Translocação SEC/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecA
12.
Biochem Biophys Rep ; 12: 240-244, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29214224

RESUMO

We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from Enterococcus faecalis, which cleaves the azo bonds (R-NË­N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutational spots in AzoA has not been obtained in terms of substrate binding and azoreductase activity. In this study, we further quantified, visualized, and described the pleiotropic and/or epistatic behavior of six single or double mutations at the positions, Arg-21 and Asn-121, as a further research endeavor for beneficial fine-tuning of AzoA. Based on this network-based mutational analysis, we showed that pleiotropy and epistasis are common, sensitive, and complex mutational behaviors, depending mainly on the structural and functional responsibility and the physicochemical properties of the residue(s) in AzoA.

13.
Toxicol In Vitro ; 36: 133-141, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27480511

RESUMO

The association between exposure to smokeless tobacco products (STP) and oral diseases is partially due to the physiological and pathological changes in the composition of the oral microbiome and its metabolic profile. However, it is not clear how STPs affect the physiology and ecology of oral microbiota. A UPLC/QTof-MS-based metabolomics study was employed to analyze metabolic alterations in oral bacterium, Capnocytophaga sputigena as a result of smokeless tobacco exposure and to assess the capability of the bacterium to metabolize nicotine. Pathway analysis of the metabolome profiles indicated that smokeless tobacco extracts caused oxidative stress in the bacterium. The metabolomics data also showed that the arginine-nitric oxide pathway was perturbed by the smokeless tobacco treatment. Results also showed that LC/MS was useful in identifying STP constituents and additives, including caffeine and many flavoring compounds. No significant changes in levels of nicotine and its major metabolites were found when C. sputigena was cultured in a nutrient rich medium, although hydroxylnicotine and cotinine N-oxide were detected in the bacterial metabolites suggesting that nicotine metabolism might be present as a minor degradation pathway in the bacterium. Study results provide new insights regarding the physiological and toxicological effects of smokeless tobacco on oral bacterium C. sputigena and associated oral health as well as measuring the ability of the oral bacterium to metabolize nicotine.


Assuntos
Capnocytophaga/efeitos dos fármacos , Tabaco sem Fumaça/toxicidade , Capnocytophaga/metabolismo , Metabolômica , Nicotina/metabolismo , Estresse Oxidativo/efeitos dos fármacos
14.
ChemMedChem ; 11(1): 43-56, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26607404

RESUMO

SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure-activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation activity at low- to sub-micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug-affinity-responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pirimidinas/farmacologia , Triazóis/farmacologia , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Proteínas de Membrana Transportadoras/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/química , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-Atividade , Triazóis/química
15.
ChemMedChem ; 11(22): 2511-2521, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27753464

RESUMO

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram-negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Canais de Translocação SEC/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Canais de Translocação SEC/metabolismo , Proteínas SecA , Relação Estrutura-Atividade
16.
Future Med Chem ; 7(8): 989-1007, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26062397

RESUMO

There is a consensus in the medical profession of the pressing need for novel antimicrobial agents due to issues related to drug resistance. In practice, solutions to this problem to a large degree lie with the identification of new and vital targets in bacteria and subsequently designing their inhibitors. We consider SecA a very promising antimicrobial target. In this review, we compile and analyze information available on SecA to show that inhibition of SecA has a multitude of consequences. Furthermore, we discuss issues critical to the design and evaluation of SecA inhibitors.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Adenosina Trifosfatases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Estrutura Molecular , Canais de Translocação SEC , Proteínas SecA
17.
ChemMedChem ; 8(8): 1384-93, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23794293

RESUMO

SecA, a key component of bacterial Sec-dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure-activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E. coli and B. subtilis.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Anti-Infecciosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Rosa Bengala/química , Adenosina Trifosfatases/metabolismo , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Rosa Bengala/metabolismo , Rosa Bengala/farmacologia , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-Atividade
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