RESUMO
We used classical mutagens in Gram-negative Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQO > ZEB > 5AZ > 2AP > 5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network.
Assuntos
Antibacterianos/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Mutagênicos/farmacologia , 2-Aminopurina/farmacologia , 4-Nitroquinolina-1-Óxido/farmacologia , Azacitidina/farmacologia , Bromodesoxiuridina/farmacologia , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Citidina/análogos & derivados , Citidina/farmacologia , Eritromicina/farmacologia , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Tetraciclina/farmacologia , Trimetoprima/farmacologia , Vancomicina/farmacologiaRESUMO
Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 µg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755).
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Nitrofurantoína/farmacologia , Trimetoprima/farmacologia , Vancomicina/farmacologia , Proteínas de Transporte/genética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Peptidilprolil Isomerase/genéticaRESUMO
We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Nucleosídeo Desaminases/metabolismo , Vancomicina/farmacologia , Adenosina/farmacologia , Citidina/farmacologia , Citidina Desaminase , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Guanosina/farmacologia , Nucleosídeo Desaminases/genéticaRESUMO
The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.
Assuntos
Bacillus anthracis/efeitos dos fármacos , Cafeína/farmacologia , Ciprofloxacina/farmacologia , Dano ao DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Canamicina/farmacologia , Bacillus anthracis/genética , Interações MedicamentosasRESUMO
Temperature variation-through time and across climatic gradients-affects individuals, populations, and communities. Yet how the thermal response of biological systems is altered by environmental stressors is poorly understood. Here, we quantify two key features-optimal temperature and temperature breadth-to investigate how temperature responses vary in the presence of antibiotics. We use high-throughput screening to measure growth of Escherichia coli under single and pairwise combinations of 12 antibiotics across seven temperatures that range from 22°C to 46°C. We find that antibiotic stress often results in considerable changes in the optimal temperature for growth and a narrower temperature breadth. The direction of the optimal temperature shifts can be explained by the similarities between antibiotic-induced and temperature-induced damage to the physiology of the bacterium. We also find that the effects of pairs of stressors in the temperature response can often be explained by just one antibiotic out of the pair. Our study has implications for a general understanding of how ecological systems adapt and evolve to environmental changes. IMPORTANCE The growth of living organisms varies with temperature. This dependence is described by a temperature response curve that is described by an optimal temperature where growth is maximized and a temperature range (termed breadth) across which the organism can grow. Because an organism's temperature response evolves or acclimates to its environment, it is often assumed to change over only evolutionary or developmental timescales. Counter to this, we show here that antibiotics can quickly (over hours) change the optimal growth temperature and temperature breadth for the bacterium Escherichia coli. Moreover, our results suggest a shared-damage hypothesis: when an antibiotic damages similar cellular components as hot (or cold) temperatures do, this shared damage will combine and compound to more greatly reduce growth when that antibiotic is administered at hot (or cold) temperatures. This hypothesis could potentially also explain how temperature responses are modified by stressors other than antibiotics.
RESUMO
Environmental factors like temperature, pressure, and pH partly shaped the evolution of life. As life progressed, new stressors (e.g., poisons and antibiotics) arose as part of an arms race among organisms. Here we ask if cells co-opted existing mechanisms to respond to new stressors, or whether new responses evolved de novo. We use a network-clustering approach based purely on phenotypic growth measurements and interactions among the effects of stressors on population growth. We apply this method to two types of stressors-temperature and antibiotics-to discover the extent to which their cellular responses overlap in Escherichia coli. Our clustering reveals that responses to low and high temperatures are clearly separated, and each is grouped with responses to antibiotics that have similar effects to cold or heat, respectively. As further support, we use a library of transcriptional fluorescent reporters to confirm heat-shock and cold-shock genes are induced by antibiotics. We also show strains evolved at high temperatures are more sensitive to antibiotics that mimic the effects of cold. Taken together, our results strongly suggest that temperature stress responses have been co-opted to deal with antibiotic stress.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Temperatura , Evolução Biológica , Escherichia coli/fisiologia , Biblioteca Gênica , Estresse FisiológicoRESUMO
Interactions and emergent processes are essential for research on complex systems involving many components. Most studies focus solely on pairwise interactions and ignore higher-order interactions among three or more components. To gain deeper insights into higher-order interactions and complex environments, we study antibiotic combinations applied to pathogenic Escherichia coli and obtain unprecedented amounts of detailed data (251 two-drug combinations, 1512 three-drug combinations, 5670 four-drug combinations, and 13608 five-drug combinations). Directly opposite to previous assumptions and reports, we find higher-order interactions increase in frequency with the number of drugs in the bacteria's environment. Specifically, as more drugs are added, we observe an elevated frequency of net synergy (effect greater than expected based on independent individual effects) and also increased instances of emergent antagonism (effect less than expected based on lower-order interaction effects). These findings have implications for the potential efficacy of drug combinations and are crucial for better navigating problems associated with the combinatorial complexity of multi-component systems.
RESUMO
HIV incidence is a primary metric for epidemic surveillance and prevention efficacy assessment. HIV incidence assay performance is evaluated via false recency rate (FRR) and mean duration of recent infection (MDRI). We conducted a meta-analysis of 438 incident and 305 chronic specimens' HIV envelope genes from a diverse global cohort. The genome similarity index (GSI) accurately characterized infection stage across diverse host and viral factors. All except one chronic specimen had GSIs below 0.67, yielding a FRR of 0.33 [0-0.98] %. We modeled the incidence assay biomarker dynamics with a logistic link function assuming individual variabilities in a Beta distribution. The GSI probability density function peaked close to 1 in early infection and 0 around two years post infection, yielding MDRI of 420 [361, 467] days. We tested the assay by newly sequencing 744 envelope genes from 59 specimens of 21 subjects who followed from HIV negative status. Both standardized residuals and Anderson-Darling tests showed that the test dataset was statistically consistent with the model biomarker dynamics. This is the first reported incidence assay meeting the optimal FRR and MDRI performance standards. Signatures of HIV gene diversification can allow precise cross-sectional surveillance with a desirable temporal range of incidence detection.
Assuntos
Genômica/métodos , Infecções por HIV/epidemiologia , HIV-1/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Algoritmos , Estudos Transversais , Feminino , Infecções por HIV/virologia , Humanos , Incidência , Análise de Sequência de RNARESUMO
Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.