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1.
Antimicrob Agents Chemother ; : e0045224, 2024 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-38940570

RESUMEN

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

2.
Artículo en Inglés | MEDLINE | ID: mdl-30885897

RESUMEN

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical blaCMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from a human and from poultry meat. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and green fluorescent protein [GFP]), allowing fluorescence-activated cell sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6, and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 102 CFU/ml. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that the plasmids were predominantly acquired by Enterobacteriaceae species, such as E. coli and Hafnia alvei Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Small amounts of the exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment.


Asunto(s)
Enterobacteriaceae/genética , Escherichia coli/genética , Heces/microbiología , Plásmidos/genética , Humanos , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Lactamasas/genética
3.
Molecules ; 24(6)2019 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-30901860

RESUMEN

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure⁻activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2⁻4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2⁻16 µg/mL). Time⁻kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Proteína Básica de Mielina/química , Fragmentos de Péptidos/química , Relación Estructura-Actividad , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular
4.
Artículo en Inglés | MEDLINE | ID: mdl-27956425

RESUMEN

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ß-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.


Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Resistencia betalactámica/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Conjugación Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/deficiencia , Proteínas de Unión a las Penicilinas/genética , Peptidoglicano Glicosiltransferasa/deficiencia , Peptidoglicano Glicosiltransferasa/genética , Plásmidos/química , Plásmidos/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/deficiencia , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , Resistencia betalactámica/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
5.
Antimicrob Agents Chemother ; 60(10): 5995-6002, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27458225

RESUMEN

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.


Asunto(s)
Pared Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Péptidos/farmacología , Peptidomiméticos/farmacología , beta-Galactosidasa/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Pared Celular/química , Pared Celular/metabolismo , Compuestos Cromogénicos/química , Compuestos Cromogénicos/metabolismo , Eritromicina/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácido Fusídico/farmacología , Expresión Génica , Hidrólisis , Pruebas de Sensibilidad Microbiana , Mutación , Nitrofenilgalactósidos/química , Nitrofenilgalactósidos/metabolismo , Novobiocina/farmacología , Biblioteca de Péptidos , Péptidos/química , Peptidomiméticos/química , Permeabilidad/efectos de los fármacos , Rifampin/farmacología , beta-Galactosidasa/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
6.
BMC Vet Res ; 12(1): 126, 2016 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-27342694

RESUMEN

BACKGROUND: New therapeutic strategies are needed to face the rapid spread of multidrug-resistant staphylococci in veterinary medicine. The objective of this study was to identify synergies between antimicrobial and non-antimicrobial drugs commonly used in companion animals as a possible strategy to restore antimicrobial susceptibility in methicillin-resistant Staphylococcus pseudintermedius (MRSP). RESULTS: A total of 216 antimicrobial/non-antimicrobial drug combinations were screened by disk diffusion using a clinical MRSP sequence type (ST) 71 strain resistant to all six antimicrobials tested (ampicillin, ciprofloxacin, clindamycin, doxycycline, oxacillin and trimethoprim/sulfamethoxazole). The most promising drug combination (doxycycline-carprofen) was further assessed by checkerboard testing extended to four additional MRSP strains belonging to ST71 or ST68, and by growth inhibition experiments. Seven non-antimicrobial drugs (bromhexine, acepromazine, amitriptyline, clomipramine, carprofen, fluoxetine and ketoconazole) displayed minimum inhibitory concentrations (MICs) ranging between 32 and >4096 mg/L, and enhanced antimicrobial activity of one or more antimicrobials. Secondary screening by checkerboard assay revealed a synergistic antimicrobial effect between carprofen and doxycycline, with the sum of the fractional inhibitory concentration indexes (ΣFICI) ranging between 0.3 and 0.5 depending on drug concentration. Checkerboard testing of multiple MRSP strains revealed a clear association between synergy and carriage of tetK, which is a typical feature of MRSP ST71. An increased growth inhibition was observed when MRSP ST71 cells in exponential phase were exposed to 0.5/32 mg/L of doxycycline/carprofen compared to individual drug exposure. CONCLUSIONS: Carprofen restores in vitro susceptibility to doxycycline in S. pseudintermedius strains carrying tetK such as MRSP ST71. Further research is warranted to elucidate the molecular mechanism behind the identified synergy and its linkage to tetK.


Asunto(s)
Carbazoles/farmacología , Doxiciclina/farmacología , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Antibacterianos/farmacología , Pruebas Antimicrobianas de Difusión por Disco , Sinergismo Farmacológico , Staphylococcus/genética
7.
Nat Microbiol ; 2024 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-39030344

RESUMEN

Genetic interactions identify functional connections between genes and pathways, establishing gene functions or druggable targets. Here we use CRISPRi-TnSeq, CRISPRi-mediated knockdown of essential genes alongside TnSeq-mediated knockout of non-essential genes, to map genome-wide interactions between essential and non-essential genes in Streptococcus pneumoniae. Transposon-mutant libraries constructed in 13 CRISPRi strains enabled screening of ~24,000 gene pairs. This identified 1,334 genetic interactions, including 754 negative and 580 positive interactions. Network analyses show that 17 non-essential genes pleiotropically interact with more than half the essential genes tested. Validation experiments confirmed that a 7-gene subset protects against perturbations. Furthermore, we reveal hidden redundancies that compensate for essential gene loss, relationships between cell wall synthesis, integrity and cell division, and show that CRISPRi-TnSeq identifies synthetic and suppressor-type relationships between both functionally linked and disparate genes and pathways. Importantly, in species where CRISPRi and Tn-Seq are established, CRISPRi-TnSeq should be straightforward to implement.

8.
J Bacteriol ; 195(20): 4580-91, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23913327

RESUMEN

The Escherichia coli TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B12. The cytoplasmic membrane proteins harvest and transmit the proton motive force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as Δ10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the Δ10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All Δ10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the Δ10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Transporte Biológico Activo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/fisiología , Hierro/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Fuerza Protón-Motriz
9.
FEMS Microbes ; 4: xtad013, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37701421

RESUMEN

In a previous in silico study, we identified an essential outer membrane protein (LptD) as an attractive target for development of novel antibiotics. Here, we characterized the effects of LptD depletion on Escherichia coli physiology and morphology. An E. coli CRISPR interference (CRISPRi) strain was constructed to allow control of lptD expression. Induction of the CRISPRi system led to ∼440-fold reduction of gene expression. Dose-dependent growth inhibition was observed, where strong knockdown effectively inhibited initial growth but partial knockdown exhibited maximum overall killing after 24 h. LptD depletion led to morphological changes where cells exhibited long, filamentous cell shapes and cytoplasmic accumulation of lipopolysaccharide (LPS). Transcriptional profiling by RNA-Seq showed that LptD knockdown led to upregulation of carbohydrate metabolism, especially in the colanic acid biosynthesis pathway. This pathway was further overexpressed in the presence of sublethal concentrations of colistin, an antibiotic targeting LPS, indicating a specific transcriptional response to this synergistic envelope damage. Additionally, exposure to colistin during LptD depletion resulted in downregulation of pathways related to motility and chemotaxis, two important virulence traits. Altogether, these results show that LptD depletion (i) affects E. coli survival, (ii) upregulates carbohydrate metabolism, and (iii) synergizes with the antimicrobial activity of colistin.

10.
bioRxiv ; 2023 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-37398100

RESUMEN

Genetic interaction networks can help identify functional connections between genes and pathways, which can be leveraged to establish (new) gene function, drug targets, and fill pathway gaps. Since there is no optimal tool that can map genetic interactions across many different bacterial strains and species, we develop CRISPRi-TnSeq, a genome-wide tool that maps genetic interactions between essential genes and nonessential genes through the knockdown of a targeted essential gene (CRISPRi) and the simultaneous knockout of individual nonessential genes (Tn-Seq). CRISPRi-TnSeq thereby identifies, on a genome-wide scale, synthetic and suppressor-type relationships between essential and nonessential genes, enabling the construction of essential-nonessential genetic interaction networks. To develop and optimize CRISPRi-TnSeq, CRISPRi strains were obtained for 13 essential genes in Streptococcus pneumoniae, involved in different biological processes including metabolism, DNA replication, transcription, cell division and cell envelope synthesis. Transposon-mutant libraries were constructed in each strain enabling screening of ∼24,000 gene-gene pairs, which led to the identification of 1,334 genetic interactions, including 754 negative and 580 positive genetic interactions. Through extensive network analyses and validation experiments we identify a set of 17 pleiotropic genes, of which a subset tentatively functions as genetic capacitors, dampening phenotypic outcomes and protecting against perturbations. Furthermore, we focus on the relationships between cell wall synthesis, integrity and cell division and highlight: 1) how essential gene knockdown can be compensated by rerouting flux through nonessential genes in a pathway; 2) the existence of a delicate balance between Z-ring formation and localization, and septal and peripheral peptidoglycan (PG) synthesis to successfully accomplish cell division; 3) the control of c-di-AMP over intracellular K + and turgor, and thereby modulation of the cell wall synthesis machinery; 4) the dynamic nature of cell wall protein CozEb and its effect on PG synthesis, cell shape morphology and envelope integrity; 5) functional dependency between chromosome decatenation and segregation, and the critical link with cell division, and cell wall synthesis. Overall, we show that CRISPRi-TnSeq uncovers genetic interactions between closely functionally linked genes and pathways, as well as disparate genes and pathways, highlighting pathway dependencies and valuable leads for gene function. Importantly, since both CRISPRi and Tn-Seq are widely used tools, CRISPRi-TnSeq should be relatively easy to implement to construct genetic interaction networks across many different microbial strains and species.

11.
Nat Commun ; 13(1): 3165, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35672367

RESUMEN

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.


Asunto(s)
Antibacterianos , Streptococcus pneumoniae , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Tolerancia a Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana
12.
Clin Transl Med ; 12(5): e790, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35522900

RESUMEN

BACKGROUND: In patients with atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions and is hypothesized to be linked to disease severity and progression. Treatments that reduce S. aureus colonization without significantly affecting the skin commensal microbiota are needed. METHODS AND FINDINGS: In this study, we tested ATx201 (niclosamide), a small molecule, on its efficacy to reduce S. aureus and propensity to evolve resistance in vitro. Various cutaneous formulations were then tested in a superficial skin infection model. Finally, a Phase 2 randomized, double-blind and placebo-controlled trial was performed to investigate the impact of ATx201 OINTMENT 2% on S. aureus colonization and skin microbiome composition in patients with mild-to-severe AD (EudraCT:2016-003501-33). ATx201 has a narrow minimal inhibitory concentration distribution (.125-.5 µg/ml) consistent with its mode of action - targeting the proton motive force effectively stopping cell growth. In murine models, ATx201 can effectively treat superficial skin infections of methicillin-resistant S. aureus. In a Phase 2 trial in patients with mild-to-severe AD (N = 36), twice-daily treatment with ATx201 OINTMENT 2% effectively reduces S. aureus colonization in quantitative colony forming unit (CFU) analysis (primary endpoint: 94.4% active vs. 38.9% vehicle success rate, p = .0016) and increases the Shannon diversity of the skin microbiome at day 7 significantly compared to vehicle. CONCLUSION: These results suggest that ATx201 could become a new treatment modality as a decolonizing agent.


Asunto(s)
Dermatitis Atópica , Staphylococcus aureus Resistente a Meticilina , Microbiota , Infecciones Estafilocócicas , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Humanos , Ratones , Niclosamida/farmacología , Pomadas/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus
13.
J Bacteriol ; 193(20): 5649-57, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21840979

RESUMEN

The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B(12) across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.


Asunto(s)
Citoplasma/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de la Membrana/metabolismo , Mutación , Periplasma/metabolismo , Reactivos de Enlaces Cruzados/química , Citoplasma/química , Citoplasma/genética , Escherichia coli/química , Escherichia coli/metabolismo , Formaldehído/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Periplasma/química , Periplasma/genética , Unión Proteica , Estructura Terciaria de Proteína
14.
Antibiotics (Basel) ; 10(6)2021 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-34070637

RESUMEN

Novel antimicrobials interfering with pathogen-specific targets can minimize the risk of perturbations of the gut microbiota (dysbiosis) during therapy. We employed an in silico approach to identify essential proteins in Escherichia coli that are either absent or have low sequence identity in seven beneficial taxa of the gut microbiota: Faecalibacterium, Prevotella, Ruminococcus, Bacteroides, Lactobacillus, Lachnospiraceae and Bifidobacterium. We identified 36 essential proteins that are present in hyper-virulent E. coli ST131 and have low similarity (bitscore < 50 or identity < 30% and alignment length < 25%) to proteins in mammalian hosts and beneficial taxa. Of these, 35 are also present in Klebsiella pneumoniae. None of the proteins are targets of clinically used antibiotics, and 3D structure is available for 23 of them. Four proteins (LptD, LptE, LolB and BamD) are easily accessible as drug targets due to their location in the outer membrane, especially LptD, which contains extracellular domains. Our results indicate that it may be possible to selectively interfere with essential biological processes in Enterobacteriaceae that are absent or mediated by unrelated proteins in beneficial taxa residing in the gut. The identified targets can be used to discover antimicrobial drugs effective against these opportunistic pathogens with a decreased risk of causing dysbiosis.

15.
Front Microbiol ; 12: 786173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35069485

RESUMEN

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847-treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

16.
BMC Microbiol ; 9: 20, 2009 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-19178705

RESUMEN

BACKGROUND: Protonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated. RESULTS: Induction of heat-shock-like response in E. coli attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores. CONCLUSION: Our experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in E. coli and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.


Asunto(s)
Escherichia coli/efectos de los fármacos , Respuesta al Choque Térmico , Ionóforos/farmacología , Fuerza Protón-Motriz/efectos de los fármacos , 2,4-Dinitrofenol , Fosfatasa Alcalina/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Citosol/química , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Transporte de Proteínas/efectos de los fármacos , Factor sigma/metabolismo
17.
Mol Membr Biol ; 25(5): 411-22, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18651316

RESUMEN

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42 degrees C0 degrees C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.


Asunto(s)
Cloruro de Calcio/química , Escherichia coli , Fluidez de la Membrana , Lípidos de la Membrana , Plásmidos/química , Transformación Genética , Frío , Escherichia coli/ultraestructura , Calor , Microscopía de Fuerza Atómica , Propiedades de Superficie
18.
Sci Rep ; 9(1): 17834, 2019 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-31780689

RESUMEN

We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the proton gradient. We showed that CPF is a proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising proton-motive-force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.


Asunto(s)
Antibacterianos/farmacología , Carbazoles/farmacología , Doxiciclina/farmacología , Farmacorresistencia Bacteriana Múltiple , Protones , Staphylococcus/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Sinergismo Farmacológico , Transporte Iónico , Proteínas de Transporte de Membrana/metabolismo , Resistencia a la Meticilina , NADP/metabolismo , Staphylococcus/efectos de los fármacos , Staphylococcus/genética , Resistencia a la Tetraciclina
19.
Sci Rep ; 9(1): 3679, 2019 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-30842436

RESUMEN

Integumentary infections like pyoderma represent the main reason for antimicrobial prescription in dogs. Staphylococcus pseudintermedius and Pseudomonas aeruginosa are frequently identified in these infections, and both bacteria are challenging to combat due to resistance. To avoid use of important human antibiotics for treatment of animal infections there is a pressing need for novel narrow-spectrum antimicrobial agents in veterinary medicine. Herein, we characterize the in vitro activity of the novel peptide-peptoid hybrid B1 against canine isolates of S. pseudintermedius and P. aeruginosa. B1 showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius and P. aeruginosa isolates as well rapid killing kinetics. B1 was found to disrupt the membrane integrity and affect cell-wall synthesis in methicillin-resistant S. pseudintermedius (MRSP). We generated 28 analogues of B1, showing comparable haemolysis and MICs against MRSP and P. aeruginosa. The most active analogues (23, 26) and B1 were tested against a collection of clinical isolates from canine, of which only B1 showed potent activity. Our best compound 26, displayed activity against P. aeruginosa and S. pseudintermedius, but not the closely related S. aureus. This work shows that design of target-specific veterinary antimicrobial agents is possible, even species within a genus, and deserves further exploration.


Asunto(s)
Antibacterianos/farmacología , Enfermedades de los Perros/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Enfermedades Cutáneas Bacterianas/microbiología , Enfermedades Cutáneas Bacterianas/veterinaria , Staphylococcus/efectos de los fármacos , Animales , Antibacterianos/química , Perros , Pruebas de Sensibilidad Microbiana , Péptidos/química , Péptidos/farmacología , Peptoides/química , Peptoides/farmacología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/veterinaria , Pseudomonas aeruginosa/patogenicidad , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/veterinaria , Staphylococcus/patogenicidad , Relación Estructura-Actividad
20.
Int J Antimicrob Agents ; 53(6): 868-872, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30447380

RESUMEN

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.


Asunto(s)
Antibacterianos/farmacología , Azitromicina/farmacología , Reposicionamiento de Medicamentos , Sinergismo Farmacológico , Bacterias Gramnegativas/efectos de los fármacos , Péptidos/farmacología , Rifampin/farmacología , Acinetobacter baumannii , Supervivencia Celular/efectos de los fármacos , Escherichia coli , Células Eucariotas/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Klebsiella pneumoniae , Péptidos/toxicidad
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