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1.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
2.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologia
4.
PLoS One ; 16(2): e0246755, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33566816

RESUMO

Campylobacter jejuni and Campylobacter coli are globally recognized as a major cause of bacterial foodborne gastroenteritis. A cross-sectional study was conducted from October 2015 to May 2016 in Mekelle city to isolate, identify, and estimate the prevalence of C. jejuni and C. coli in raw meat samples and to determine their antibiotic susceptibility pattern. A total of 384 raw meat samples were randomly collected from bovine (n = 210), goat (n = 108), and chicken (n = 66), and isolation and identification of Campylobacter spp. were performed using standard bacteriological techniques and PCR. Antibiotic susceptibility test was performed using disc diffusion method. Of the total 384 raw meat samples, 64 (16.67%) were found positive for Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (43.93%) followed by bovine meat (11.90%) and goat meat (9.25%). The most prevalent Campylobacter spp. isolated from meat samples was C. jejuni (81.25%). The overall prevalence of Campylobacter in restaurants, butcher shops, and abattoir was 43.93%, 18.30%, and 9.30%, respectively. 96.8%, 81.25%, 75%, and 71% of the Campylobacter spp. isolates were sensitive to norfloxacin, erythromycin, chloramphenicol, and sulphamethoxazole-trimethoprim, respectively. However, 96.9%, 85.9%, and 50% of the isolates were resistant to ampicillin, amoxicillin, and streptomycin, respectively. Strains that developed multi-drug resistant were 68.7%. The result of this study revealed the occurrence of Campylobacter in bovine, goat, and chicken meats. Hence, there is a chance of acquiring infection via consumption of raw or undercooked meat. Thus, implementation of hygienic practices from a slaughterhouse to the retailers, proper handling and cooking of foods of meat are very important in preventing Campylobacter infection.


Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter coli/efeitos dos fármacos , Campylobacter jejuni/efeitos dos fármacos , Galinhas/microbiologia , Farmacorresistência Bacteriana Múltipla , Cabras/microbiologia , Carne/microbiologia , Amoxicilina/farmacologia , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Campylobacter coli/isolamento & purificação , Campylobacter jejuni/isolamento & purificação , Bovinos , Cloranfenicol/farmacologia , Estudos Transversais , Eritromicina/farmacologia , Etiópia , Testes de Sensibilidade Microbiana , Norfloxacino/farmacologia , Prevalência , Estreptomicina/farmacologia , Sulfametoxazol/farmacologia , Trimetoprima/farmacologia
5.
Eur Rev Med Pharmacol Sci ; 25(2): 605-608, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33577013

RESUMO

OBJECTIVE: Nocardia kroppenstedtii was isolated from the spinal vertebral abscess of a 78-year-old patient presenting with mid-thoracic pain and bilateral lower limb weakness and numbness. The patient was on long-term immunosuppressive therapy with steroids for underlying autoimmune hemolytic anemia. Investigations showed a T5 pathological fracture and vertebra plana with the erosion of the superior and inferior endplates. There was evidence of paraspinal collection from the T4-T6 vertebrae with an extension into the spinal canal. Analysis of Nocardia 16S rRNA (99.9%, 1395/1396 nt) and secA1 gene (99.5%, 429/431 nt) fragments showed the highest sequence similarity with Nocardia kroppenstedtii type strain (DQ157924), and next with Nocardia farcinica (Z36936). The patient was treated with intravenous carbapenem and oral trimethoprim-sulfamethoxazole for four weeks, followed by another six months of oral trimethoprim-sulfamethoxazole. Despite the improvement of neurological deficits, the patient required assistive devices to ambulate at discharge. This study reports the first isolation of N. kroppenstedtii from the spinal vertebral abscess of a patient from Asia. Infections caused by N. kroppenstedtii may be underdiagnosed as the bacterium can be misidentified as N. farcinica in the absence of molecular tests in the clinical laboratory.


Assuntos
Abscesso Epidural/microbiologia , Nocardiose/microbiologia , Nocardia/isolamento & purificação , Administração Oral , Idoso , Anemia Hemolítica Autoimune/tratamento farmacológico , Anemia Hemolítica Autoimune/microbiologia , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Abscesso Epidural/tratamento farmacológico , Feminino , Humanos , Imunossupressores/uso terapêutico , Nocardia/efeitos dos fármacos , Nocardiose/tratamento farmacológico , Esteroides/uso terapêutico , Sulfametoxazol/administração & dosagem , Sulfametoxazol/farmacologia , Trimetoprima/administração & dosagem , Trimetoprima/farmacologia
6.
Microb Genom ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33459584

RESUMO

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.


Assuntos
Burkholderia gladioli/classificação , Fibrose Cística/microbiologia , Doenças das Plantas/microbiologia , Sequenciamento Completo do Genoma/métodos , Vias Biossintéticas , Ácido Bongcréquico/metabolismo , Burkholderia gladioli/genética , Burkholderia gladioli/patogenicidade , Burkholderia gladioli/fisiologia , Microbiologia de Alimentos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Filogenia , Trimetoprima/farmacologia
7.
Can J Microbiol ; 67(8): 599-612, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33481681

RESUMO

Bacterial resistance has become one of the most serious public health problems, globally, and drug repurposing is being investigated to speed up the identification of effective drugs. The aim of this study was to investigate the repurposing of escitalopram oxalate and clonazepam drugs individually, and in combination with the antibiotics ciprofloxacin and sulfamethoxazole-trimethoprim, to treat multidrug-resistant (MDR) microorganisms and to evaluate the potential chemical nuclease activity. The minimum inhibitory concentration, minimum bactericidal concentration, fractional inhibitory concentration index, and tolerance level were determined for each microorganism tested. In vitro antibacterial activity was evaluated against 47 multidrug-resistant clinical isolates and 11 standard bacterial strains from the American Type Culture Collection. Escitalopram oxalate was mainly active against Gram-positive bacteria, and clonazepam was active against both Gram-positive and Gram-negative bacteria. When associated with the two antibiotics mentioned, they had a significant synergistic effect. Clonazepam cleaved plasmid DNA, and the mechanisms involved were oxidative and hydrolytic. These results indicate the potential for repurposing these non-antibiotic drugs to treat bacterial infections. However, further studies on the mechanism of action of these drugs should be performed to ensure their safe use.


Assuntos
Ciprofloxacina , Bactérias Gram-Negativas , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Citalopram/farmacologia , Clonazepam/farmacologia , DNA , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Positivas , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Sulfametoxazol/farmacologia , Trimetoprima/farmacologia
8.
PLoS Biol ; 18(9): e3000856, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941420

RESUMO

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy.


Assuntos
Antibacterianos/administração & dosagem , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana/métodos , Algoritmos , Andinocilina/administração & dosagem , Andinocilina/farmacologia , Antibacterianos/farmacologia , Quimioterapia Combinada/métodos , Quimioterapia Combinada/normas , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Testes de Sensibilidade Microbiana/instrumentação , Nitrofurantoína/administração & dosagem , Nitrofurantoína/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Reprodutibilidade dos Testes , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Trimetoprima/administração & dosagem , Trimetoprima/farmacologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
9.
Int J Food Microbiol ; 335: 108857, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32947144

RESUMO

Cronobacter species (Cronobacter spp.) are important foodborne pathogens that can infect and cause serious life-threatening diseases in infants and immunocompromised elderly. This study aimed to acquire data on Cronobacter spp. contamination of aquatic products in China from 2011 to 2016. In total, 800 aquatic products were tested, and the overall contamination rate for Cronobacter spp. was 3.9% (31/800). The average contamination level of the positive samples was 2.05 MPN/g. Four species and nine serotypes were identified among 33 isolates, of which the C. sakazakii serogroup O1 (n = 9) was the primary serotype. The majority of Cronobacter spp. strains harbored highest resistance against cephalothin (84.8%), followed by tetracycline (6.1%), trimethoprim/sulfameth-oxazole (3.0%) and chloramphenicol (3.0%). Two isolates were resistant to three antibiotics. In total, 26 sequence types and 33 CRISPR types (including 6 new STs and 26 new CTs) were identified, which indicates the extremely high diversity of Cronobacter spp. in aquatic products. Pathogenic C. sakazakii ST4, ST1, and C. malonaticus ST7 were also observed. Overall, this large-scale study revealed the relatively low prevalence and high genetic diversity of Cronobacter spp. in aquatic products in China, and the findings provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp. in aquatic products during production processes.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cronobacter/classificação , Cronobacter/isolamento & purificação , Farmacorresistência Bacteriana , Alimentos Marinhos/microbiologia , Antibacterianos/farmacologia , Cefalotina/farmacologia , China , Cloranfenicol/farmacologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Variação Genética , Tipagem de Sequências Multilocus , Prevalência , Sorotipagem , Tetraciclina/farmacologia , Trimetoprima/farmacologia
11.
N Z Med J ; 133(1519): 62-69, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32777796

RESUMO

AIM: To assess whether trimethoprim remains an appropriate empiric treatment for uncomplicated cystitis in women 15-55 years old. METHODS: General practitioners in Auckland, Nelson-Marlborough, Otago and Southland were invited to participate in this audit of current practice. Participating general practitioners were asked to submit urine to the laboratory for microscopy and culture from any woman aged 15-55 years presenting with uncomplicated cystitis. Urine samples submitted as part of the audit were identified by a "copy to" code. Data on laboratory results were extracted from the laboratory information system. RESULTS: Data were collected from June 2016 to August 2018. Four hundred and eighty-one samples were submitted, of which 340 (70.7%) met the inclusion criteria of the audit. A urinary pathogen was identified in 181 (53.2%) specimens, of which 148 (81.8%) were E. coli, 13 (7.2%) other coliforms and 20 (11.0%) Staphylococcus saprophyticus. Of the E. coli isolates, 109 of 148 (73.6%, 95% CI 66.6-80.7) were susceptible to trimethoprim, 144 of 144 (100%, 95% CI 100-100) to nitrofurantoin and 143 of 148 (96.6%, 95% CI 93.7-99.5) to cefalexin. Of the urinary pathogens, 139 of 185 (75.1%, 95% CI 68.9-81.4) were susceptible to trimethoprim, 164 of 177 tested (92.7%, 95% CI 88.8-96.5) to nitrofurantoin and 166 of 178 tested (93.3%, 95% CI 89.6-96.9) to cefalexin. Overall, a uropathogen resistant to trimethoprim was detected in 13.5%, to nitrofurantoin in 3.8%, and to cefalexin in 3.5% of samples tested. CONCLUSION: Similar rates of resistance to trimethoprim were seen in women 15-55 years old presenting with cystitis compared with unselected samples submitted from the general community. Given the high rates of resistance, trimethoprim is no longer appropriate as an empiric treatment option for cystitis in this group. Nitrofurantoin or cefalexin are appropriate alternative empiric treatment options. Given the current recommendation that a urine sample should not be submitted to the laboratory from women with uncomplicated cystitis, ongoing audits will be required to ensure that empiric treatment recommendations remain appropriate.


Assuntos
Antibacterianos/uso terapêutico , Cistite , Farmacorresistência Bacteriana/efeitos dos fármacos , Prescrição Inadequada/estatística & dados numéricos , Trimetoprima/uso terapêutico , Adolescente , Adulto , Antibacterianos/farmacologia , Cistite/tratamento farmacológico , Cistite/microbiologia , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Feminino , Clínicos Gerais , Humanos , Auditoria Médica , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Nova Zelândia , Trimetoprima/farmacologia , Adulto Jovem
12.
Int J Antimicrob Agents ; 56(4): 106131, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763373

RESUMO

BACKGROUND: High antimicrobial use (AMU) and antimicrobial resistance (AMR) in veal calves remain a source of concern. As part of the EFFORT project, the association between AMU and the abundance of faecal antimicrobial resistance genes (ARGs) in veal calves in three European countries was determined. METHODS: In 2015, faecal samples of veal calves close to slaughter were collected from farms located in France, Germany and the Netherlands (20 farms in France, 20 farms in the Netherlands and 21 farms in Germany; 25 calves per farm). Standardized questionnaires were used to record AMU and farm characteristics. In total, 405 faecal samples were selected for DNA extraction and quantitative polymerase chain reaction to quantify the abundance (16S normalized concentration) of four ARGs [aph(3')-III, ermB, sul2 and tetW] encoding for resistance to frequently used antimicrobials in veal calves. Multiple linear mixed models with random effects for country and farm were used to relate ARGs to AMU and farm characteristics. RESULTS: A significant positive association was found between the use of trimethoprim/sulfonamides and the concentration of sul2 in faeces from veal calves. A higher weight of calves on arrival at the farm was negatively associated with aph(3')-III and ermB. Lower concentrations of aph(3')-III were found at farms with non-commercial animals present. Furthermore, farms using only water for the cleaning of stables had a significantly lower abundance of faecal ermB and tetW compared with other farms. CONCLUSION: A positive association was found between the use of trimethoprim/sulfonamides and the abundance of sul2 in faeces in veal calves. Additionally, other relevant risk factors associated with ARGs in veal calves were identified, such as weight on arrival at the farm and cleaning practices.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Sulfonamidas/farmacologia , Trimetoprima/farmacologia , Animais , Antibacterianos/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Bovinos , Doenças dos Bovinos/microbiologia , Combinação de Medicamentos , Fezes/microbiologia , França , Alemanha , Canamicina Quinase/genética , Metiltransferases/genética , Países Baixos , Uso Excessivo de Medicamentos Prescritos , Reação em Cadeia da Polimerase em Tempo Real , Inquéritos e Questionários
13.
Cell Chem Biol ; 27(5): 622-634.e6, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330442

RESUMO

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.


Assuntos
Antibacterianos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antibacterianos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Feminino , Antagonistas do Ácido Fólico/química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/química , Triazinas/química , Triazinas/farmacologia , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia
14.
ACS Chem Biol ; 15(4): 837-843, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32182034

RESUMO

Inducing protein translocation to the plasma membrane (PM) is an important approach for manipulating diverse signaling molecules/pathways in living cells. We previously devised a new chemogenetic system, in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation turned out to be transient and spontaneously reversed within 1 h, limiting its application. Here, we first demonstrated that the spontaneous reverse translocation was caused by cellular degradation of mgcTMP, presumably by proteases. To address this problem, we newly developed a proteolysis-resistant SL, mDcTMP. This mDcTMP now allows sustained PM localization of eDHFR-fusion proteins (over several hours to a day), and it was applicable to inducing prolonged signal activation and cell differentiation. mDcTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.


Assuntos
Cisteína/análogos & derivados , Cisteína/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cisteína/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Complexo de Golgi/metabolismo , Humanos , Ligantes , Lipoilação , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Transdução de Sinais/fisiologia , Estereoisomerismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/metabolismo
15.
mBio ; 11(2)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184236

RESUMO

Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs' mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs.IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.


Assuntos
Farmacorresistência Bacteriana , Leite Humano/química , Oligossacarídeos/química , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/metabolismo , Trimetoprima/farmacologia , Antibacterianos/farmacologia , Humanos , Redes e Vias Metabólicas , Metabolômica , Espectrometria de Massas em Tandem
16.
Sci Rep ; 10(1): 5315, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210253

RESUMO

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).


Assuntos
Antibacterianos/metabolismo , Bdellovibrio bacteriovorus/crescimento & desenvolvimento , Bdellovibrio bacteriovorus/metabolismo , Antibiose/genética , Bdellovibrio/genética , Bdellovibrio/crescimento & desenvolvimento , Bdellovibrio bacteriovorus/genética , Farmacorresistência Bacteriana/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Trimetoprima/farmacologia , Resistência a Trimetoprima/genética
17.
ACS Infect Dis ; 6(5): 1154-1168, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32212725

RESUMO

The Burkholderia cepacia complex is a group of closely related bacterial species with large genomes that infect immunocompromised individuals and those living with cystic fibrosis. Some of these species are found more frequently and cause more severe disease than others, yet metabolomic differences between these have not been described. Furthermore, our understanding of how these species respond to antibiotics is limited. We investigated the metabolomics differences between three most prevalent Burkholderia spp. associated with cystic fibrosis: B. cenocepacia, B. multivorans, and B. dolosa in the presence and absence of the antibiotic trimethoprim. Using a combination of supervised and unsupervised metabolomics data visualization and analysis tools, we describe the overall differences between strains of the same species and between species. Specifically, we report, for the first time, the role of the pyomelanin pathway in the metabolism of trimethoprim. We also report differences in the detection of known secondary metabolites such as fragin, ornibactin, and N-acylhomoserine lactones and their analogs in closely related strains. Furthermore, we highlight the potential for the discovery of new secondary metabolites in clinical strains of Burkholderia spp. The metabolomics differences described in this study highlight the personalized nature of closely related Burkholderia strains.


Assuntos
Antibacterianos/farmacologia , Burkholderia/efeitos dos fármacos , Fibrose Cística/microbiologia , Metaboloma/efeitos dos fármacos , Trimetoprima/farmacologia , Burkholderia/metabolismo , Infecções por Burkholderia , Humanos
18.
J Inorg Biochem ; 205: 111002, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32007697

RESUMO

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania, which has very limited treatment options and affects poor and underdeveloped populations. The current treatment is plagued by many complications, such as high toxicity, high cost and resistance to parasites; therefore, novel therapeutic agents are urgently needed. Herein, the synthesis, characterization and in vitro leishmanicidal potential of new complexes with the general formula [RuCl3(TMP)(dppb)] (1), [PtCl(TMP)(PPh3)2]PF6 (2) and [Cu(CH3COO)2(TMP)2]·DMF (3) (dppb = 1,4-bis(diphenylphosphino)butane, PPH3 = triphenylphosphine and TMP = trimethoprim) were evaluated. The complexes were characterized by infrared, UV-vis, cyclic voltammetry, molar conductance measurements, elemental analysis and NMR experiments. Also, the geometry of (2) and (3) were determined by single crystal X-ray diffraction. Despite being less potent against promastigote L. amazonensis proliferation than amphotericin B reference drug (IC50 = 0.09 ± 0.02 µM), complex (2) (IC50 = 3.6 ± 1.5 µM) was several times less cytotoxic (CC50 = 17.8 µM, SI = 4.9) in comparison with amphotericin B (CC50 = 3.3 µM, SI = 36.6) and gentian violet control (CC50 = 0.8 µM). Additionally, complex (2) inhibited J774 macrophage infection and amastigote number by macrophages (IC50 = 6.6 and SI = 2.7). Outstandingly, complex (2) was shown to be a promising candidate for a new leishmanicidal therapeutic agent, considering its biological power combined with low toxicity.


Assuntos
Antiprotozoários , Complexos de Coordenação , Cobre , Leishmania/crescimento & desenvolvimento , Leishmaniose/tratamento farmacológico , Platina , Rubídio , Trimetoprima , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Cristalografia por Raios X , Leishmaniose/metabolismo , Leishmaniose/patologia , Camundongos , Estrutura Molecular , Platina/química , Platina/farmacologia , Rubídio/química , Rubídio/farmacologia , Trimetoprima/química , Trimetoprima/farmacologia
19.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098820

RESUMO

Low doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism.IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.


Assuntos
Antibacterianos/farmacologia , Burkholderia/efeitos dos fármacos , Burkholderia/metabolismo , Metabolismo Secundário/efeitos dos fármacos , Proteínas de Bactérias , Produtos Biológicos/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Burkholderia/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homosserina/metabolismo , Lactonas/química , Lactonas/metabolismo , Família Multigênica , Metabolismo Secundário/genética , Trimetoprima/farmacologia , Fatores de Virulência/metabolismo
20.
J Antibiot (Tokyo) ; 73(1): 5-27, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31578455

RESUMO

The development of new mechanisms of resistance among pathogens, the occurrence and transmission of genes responsible for antibiotic insensitivity, as well as cancer diseases have been a serious clinical problem around the world for over 50 years. Therefore, intense searching of new leading structures and active substances, which may be used as new drugs, especially against strain resistant to all available therapeutics, is very important. Dihydrofolate reductase (DHFR) has attracted a lot of attention as a molecular target for bacterial resistance over several decades, resulting in a number of useful agents. Trimethoprim (TMP), (2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine) is the well-known dihydrofolate reductase inhibitor and one of the standard antibiotics used in urinary tract infections (UTIs). This review highlights advances in design, synthesis, and biological evaluations in structural modifications of TMP as DHFR inhibitors. In addition, this report presents the differences in the active site of human and pathogen DHFR. Moreover, an excellent review of DHFR inhibition and their relevance to antimicrobial and parasitic chemotherapy was presented.


Assuntos
Desenvolvimento de Medicamentos/métodos , Antagonistas do Ácido Fólico/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologia , Antibacterianos , Desenho de Fármacos , Descoberta de Drogas , Antagonistas do Ácido Fólico/uso terapêutico , Humanos , Trimetoprima/uso terapêutico
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