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1.
Nature ; 625(7995): 566-571, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38172634

RESUMEN

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.


Asunto(s)
Antibacterianos , Lipopolisacáridos , Proteínas de Transporte de Membrana , Animales , Humanos , Ratones , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/clasificación , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Desarrollo de Medicamentos
3.
Mol Cell ; 68(6): 1147-1154.e3, 2017 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-29225037

RESUMEN

Physiologic and environmental factors can modulate antibiotic activity and thus pose a significant challenge to antibiotic treatment. The quinolone class of antibiotics, which targets bacterial topoisomerases, fails to kill bacteria that have grown to high density; however, the mechanistic basis for this persistence is unclear. Here, we show that exhaustion of the metabolic inputs that couple carbon catabolism to oxidative phosphorylation is a primary cause of growth phase-dependent persistence to quinolone antibiotics. Supplementation of stationary-phase cultures with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing. Using this approach, we successfully sensitize high-density populations of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis to quinolone antibiotics. Our findings link growth-dependent quinolone persistence to discrete impairments in respiratory metabolism and identify a strategy to kill non-dividing bacteria.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Carbono/metabolismo , Respiración de la Célula/fisiología , Farmacorresistencia Bacteriana , Oxígeno/metabolismo , Quinolonas/farmacología , Bacterias/crecimiento & desarrollo , Infecciones Bacterianas/microbiología , Pruebas de Sensibilidad Microbiana , Fosforilación Oxidativa
4.
J Virol ; 95(3)2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33177204

RESUMEN

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.


Asunto(s)
Cuello del Útero/virología , Infecciones por VIH/transmisión , VIH-1/genética , Leucocitos Mononucleares/virología , Membrana Mucosa/virología , Pene/virología , Proteínas Virales/genética , Femenino , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , ARN Viral/análisis , ARN Viral/genética
5.
Proc Natl Acad Sci U S A ; 112(27): 8173-80, 2015 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-26100898

RESUMEN

Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes--the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Adenosina Trifosfato/biosíntesis , Antibacterianos/clasificación , Bacterias/genética , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Interacciones Farmacológicas , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Metaboloma/efectos de los fármacos , Metabolómica/métodos , Pruebas de Sensibilidad Microbiana , Mutación , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(20): E2100-9, 2014 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-24803433

RESUMEN

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.


Asunto(s)
Antibacterianos/farmacología , Catalasa/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Oxidación-Reducción , Antioxidantes/química , Respiración de la Célula , Reparación del ADN , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Peróxido de Hidrógeno/química , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Oxígeno/metabolismo , Plásmidos/metabolismo , Especies Reactivas de Oxígeno
7.
Nano Lett ; 15(7): 4808-13, 2015 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-26044909

RESUMEN

The increasing incidence of antibiotic-resistant bacterial infections is creating a global public health threat. Because conventional antibiotic drug discovery has failed to keep pace with the rise of resistance, a growing need exists to develop novel antibacterial methodologies. Replication-competent bacteriophages have been utilized in a limited fashion to treat bacterial infections. However, this approach can result in the release of harmful endotoxins, leading to untoward side effects. Here, we engineer bacterial phagemids to express antimicrobial peptides (AMPs) and protein toxins that disrupt intracellular processes, leading to rapid, nonlytic bacterial death. We show that this approach is highly modular, enabling one to readily alter the number and type of AMPs and toxins encoded by the phagemids. Furthermore, we demonstrate the effectiveness of engineered phagemids in an in vivo murine peritonitis infection model. This work shows that targeted, engineered phagemid therapy can serve as a viable, nonantibiotic means to treat bacterial infections, while avoiding the health issues inherent to lytic and replicative bacteriophage use.


Asunto(s)
Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/genética , Bacteriófagos/genética , Ingeniería Genética , Peritonitis/terapia , Plásmidos/uso terapéutico , Toxinas Biológicas/genética , Animales , Escherichia coli/fisiología , Infecciones por Escherichia coli/complicaciones , Terapia Genética , Humanos , Ratones , Ratones Endogámicos C57BL , Peritonitis/genética , Peritonitis/microbiología , Plásmidos/genética
8.
Antimicrob Agents Chemother ; 57(6): 2640-50, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23529732

RESUMEN

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.


Asunto(s)
Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , Quimiocina CCL5/farmacología , Ciclohexanos/farmacología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Triazoles/farmacología , Animales , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/uso terapéutico , Línea Celular , Quimiocina CCL5/metabolismo , Quimiocina CCL5/uso terapéutico , Ciclohexanos/metabolismo , Ciclohexanos/uso terapéutico , Regulación hacia Abajo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Macaca , Maraviroc , Pruebas de Sensibilidad Microbiana/métodos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Triazoles/metabolismo , Triazoles/uso terapéutico , Internalización del Virus/efectos de los fármacos , Replicación Viral
9.
PLoS Pathog ; 7(5): e1002038, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21625572

RESUMEN

The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50) ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (-) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.


Asunto(s)
Fármacos Anti-VIH/farmacología , Duplicado del Terminal Largo de VIH/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Péptidos Cíclicos/farmacología , Transcripción Reversa/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Expresión Génica , Regulación Viral de la Expresión Génica , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/virología , Péptidos Cíclicos/metabolismo , ARN Viral/genética , Activación Transcripcional/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
10.
Cell Chem Biol ; 29(2): 276-286.e4, 2022 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-34990601

RESUMEN

ß-Lactam antibiotics disrupt the assembly of peptidoglycan (PG) within the bacterial cell wall by inhibiting the enzymatic activity of penicillin-binding proteins (PBPs). It was recently shown that ß-lactam treatment initializes a futile cycle of PG synthesis and degradation, highlighting major gaps in our understanding of the lethal effects of PBP inhibition by ß-lactam antibiotics. Here, we assess the downstream metabolic consequences of treatment of Escherichia coli with the ß-lactam mecillinam and show that lethality from PBP2 inhibition is a specific consequence of toxic metabolic shifts induced by energy demand from multiple catabolic and anabolic processes, including accelerated protein synthesis downstream of PG futile cycling. Resource allocation into these processes is coincident with alterations in ATP synthesis and utilization, as well as a broadly dysregulated cellular redox environment. These results indicate that the disruption of normal anabolic-catabolic homeostasis by PBP inhibition is an essential factor for ß-lactam antibiotic lethality.


Asunto(s)
Amdinocilina/farmacología , Antibacterianos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Amdinocilina/química , Antibacterianos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Homeostasis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/metabolismo
11.
J Biol Chem ; 285(38): 29326-35, 2010 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-20624919

RESUMEN

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2-4 (A3FΔ2-4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2-4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.


Asunto(s)
Citosina Desaminasa/metabolismo , VIH-1/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Western Blotting , Línea Celular , Citosina Desaminasa/genética , Humanos , Unión Proteica/genética , Unión Proteica/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética
12.
PLoS Pathog ; 5(4): e1000377, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19360131

RESUMEN

Elite suppressors (ES) are a rare subset of HIV-1-infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals.


Asunto(s)
Productos del Gen env/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Internalización del Virus , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Citometría de Flujo , Humanos , Receptores CCR5/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Viral
13.
PLoS Pathog ; 5(4): e1000365, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19343217

RESUMEN

Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.


Asunto(s)
VIH-1/inmunología , Mutación , Linfocitos T Citotóxicos/inmunología , Replicación Viral , Secuencia de Aminoácidos , Epítopos de Linfocito T/inmunología , Evolución Molecular , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo
14.
BMJ Open ; 11(7): e049232, 2021 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-34226231

RESUMEN

OBJECTIVE: To assess the SARS-CoV-2 transmission in healthcare workers (HCWs) using seroprevalence as a surrogate marker of infection in our tertiary care centre according to exposure. DESIGN: Seroprevalence cross-sectional study. SETTING: Single centre at the end of the first COVID-19 wave in Lausanne, Switzerland. PARTICIPANTS: 1874 of 4074 responders randomly selected (46% response rate), stratified by work category among the 13 474 (13.9%) HCWs. MAIN OUTCOME MEASURES: Evaluation of SARS-CoV-2 serostatus paired with a questionnaire of SARS-CoV-2 acquisition risk factors internal and external to the workplace. RESULTS: The overall SARS-CoV-2 seroprevalence rate among HCWs was 10.0% (95% CI 8.7% to 11.5%). HCWs with daily patient contact did not experience increased rates of seropositivity relative to those without (10.3% vs 9.6%, respectively, p=0.64). HCWs with direct contact with patients with COVID-19 or working in COVID-19 units did not experience increased seropositivity rates relative to their counterparts (10.4% vs 9.8%, p=0.69 and 10.6% vs 9.9%, p=0.69, respectively). However, specific locations of contact with patients irrespective of COVID-19 status-in patient rooms or reception areas-did correlate with increased rates of seropositivity (11.9% vs 7.5%, p=0.019 and 14.3% vs 9.2%, p=0.025, respectively). In contrast, HCWs with a suspected or proven SARS-CoV-2-infected household contact had significantly higher seropositivity rates than those without such contacts (19.0% vs 8.7%, p<0.001 and 42.1% vs 9.4%, p<0.001, respectively). Finally, consistent use of a mask on public transportation correlated with decreased seroprevalence (5.3% for mask users vs 11.2% for intermittent or no mask use, p=0.030). CONCLUSIONS: The overall seroprevalence was 10% without significant differences in seroprevalence between HCWs exposed to patients with COVID-19 and HCWs not exposed. This suggests that, once fully in place, protective measures limited SARS-CoV-2 occupational acquisition within the hospital environment. SARS-CoV-2 seroconversion among HCWs was associated primarily with community risk factors, particularly household transmission.


Asunto(s)
COVID-19 , SARS-CoV-2 , Estudios Transversales , Personal de Salud , Humanos , Estudios Seroepidemiológicos , Suiza/epidemiología , Centros de Atención Terciaria
15.
J Virol ; 83(21): 11016-26, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19692480

RESUMEN

The affinity of human immunodeficiency virus (HIV) envelope for CD4 and CCR5 appears to be associated with aspects of R5 virus (virus using the CCR5 coreceptor) pathogenicity. However, entry efficiency results from complex interactions between the viral envelope glycoprotein and both CD4 and CCR5, which limits attempts to correlate viral pathogenicity with surrogate measures of envelope CD4 and CCR5 affinities. Here, we present a system that provides a quantitative and comprehensive characterization of viral entry efficiency as a direct interdependent function of both CD4 and CCR5 levels. This receptor affinity profiling system also revealed heretofore unappreciated complexities underlying CD4/CCR5 usage. We first developed a dually inducible cell line in which CD4 and CCR5 could be simultaneously and independently regulated within a physiologic range of surface expression. Infection by multiple HIV type 1 (HIV-1) and simian immunodeficiency virus isolates could be examined simultaneously for up to 48 different combinations of CD4/CCR5 expression levels, resulting in a distinct usage pattern for each virus. Thus, each virus generated a unique three-dimensional surface plot in which viral infectivity varied as a function of both CD4 and CCR5 expression. From this functional form, we obtained a sensitivity vector along with corresponding metrics that quantified an isolate's overall efficiency of CD4/CCR5 usage. When applied to viral isolates with well-characterized sensitivities to entry/fusion inhibitors, the vector metrics were able to encapsulate their known biological phenotypes. The application of the vector metrics also indicated that envelopes derived from elite suppressors had overall-reduced entry efficiencies compared to those of envelopes derived from chronically infected viremic progressors. Our affinity-profiling system may help to refine studies of R5 virus tropism and pathogenesis.


Asunto(s)
Antígenos CD4/fisiología , VIH-1/fisiología , Receptores CCR5/fisiología , Virus de la Inmunodeficiencia de los Simios/fisiología , Internalización del Virus , Marcadores de Afinidad , Animales , Antígenos CD4/genética , Línea Celular , Ecdisterona/análogos & derivados , Ecdisterona/metabolismo , Humanos , Conceptos Matemáticos , Minociclina/metabolismo , Receptores CCR5/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
16.
J Virol ; 82(6): 2938-51, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18199654

RESUMEN

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5alpha did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5' of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3'-to-5' siRNA amplification and spreading. In contrast, HIV-1 RNA 3' of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively "naked" cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5alpha, or other host restriction factors.


Asunto(s)
VIH-1/fisiología , ARN Interferente Pequeño/genética , Replicación Viral , Secuencia de Bases , Western Blotting , Células Cultivadas , Cartilla de ADN , ADN Viral/genética , Duplicado del Terminal Largo de VIH , VIH-1/genética , Humanos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética
17.
Cell Metab ; 30(2): 251-259, 2019 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-31279676

RESUMEN

Antibiotics target energy-consuming processes. As such, perturbations to bacterial metabolic homeostasis are significant consequences of treatment. Here, we describe three postulates that collectively define antibiotic efficacy in the context of bacterial metabolism: (1) antibiotics alter the metabolic state of bacteria, which contributes to the resulting death or stasis; (2) the metabolic state of bacteria influences their susceptibility to antibiotics; and (3) antibiotic efficacy can be enhanced by altering the metabolic state of bacteria. Altogether, we aim to emphasize the close relationship between bacterial metabolism and antibiotic efficacy as well as propose areas of exploration to develop novel antibiotics that optimally exploit bacterial metabolic networks.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Animales , Humanos , Pruebas de Sensibilidad Microbiana
18.
Cell Chem Biol ; 24(2): 195-206, 2017 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-28111098

RESUMEN

Metabolically dormant bacteria present a critical challenge to effective antimicrobial therapy because these bacteria are genetically susceptible to antibiotic treatment but phenotypically tolerant. Such tolerance has been attributed to impaired drug uptake, which can be reversed by metabolic stimulation. Here, we evaluate the effects of central carbon metabolite stimulations on aminoglycoside sensitivity in the pathogen Pseudomonas aeruginosa. We identify fumarate as a tobramycin potentiator that activates cellular respiration and generates a proton motive force by stimulating the tricarboxylic acid (TCA) cycle. In contrast, we find that glyoxylate induces phenotypic tolerance by inhibiting cellular respiration with acetyl-coenzyme A diversion through the glyoxylate shunt, despite drug import. Collectively, this work demonstrates that TCA cycle activity is important for both aminoglycoside uptake and downstream lethality and identifies a potential strategy for potentiating aminoglycoside treatment of P. aeruginosa infections.


Asunto(s)
Antibacterianos/farmacología , Carbono/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/química , Biopelículas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/metabolismo
19.
Cell Rep ; 13(5): 968-80, 2015 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-26565910

RESUMEN

Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (?-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 min that then diverged into more distinct profiles at later time points. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products.


Asunto(s)
Ampicilina/farmacología , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Kanamicina/farmacología , Norfloxacino/farmacología , Estrés Oxidativo , Roturas del ADN de Doble Cadena
20.
Cell Host Microbe ; 13(6): 632-42, 2013 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-23768488

RESUMEN

Microbial drug persistence is a widespread phenomenon in which a subpopulation of microorganisms is able to survive antimicrobial treatment without acquiring resistance-conferring genetic changes. Microbial persisters can cause recurrent or intractable infections, and, like resistant mutants, they carry an increasing clinical burden. In contrast to heritable drug resistance, however, the biology of persistence is only beginning to be unraveled. Persisters have traditionally been thought of as metabolically dormant, nondividing cells. As discussed in this review, increasing evidence suggests that persistence is in fact an actively maintained state, triggered and enabled by a network of intracellular stress responses that can accelerate processes of adaptive evolution. Beyond shedding light on the basis of persistence, these findings raise the possibility that persisters behave as an evolutionary reservoir from which resistant organisms can emerge. As persistence and its consequences come into clearer focus, so too does the need for clinically useful persister-eradication strategies.


Asunto(s)
Farmacorresistencia Microbiana , Células Eucariotas/efectos de los fármacos , Células Procariotas/efectos de los fármacos , Estrés Fisiológico
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