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1.
Nat Commun ; 13(1): 6557, 2022 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-36450721

RESUMO

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.


Assuntos
Streptococcus pyogenes , Sulfametoxazol , Sulfametoxazol/farmacologia , Antibacterianos/farmacologia , Especificidade por Substrato , Ácido Fólico
2.
J Microbiol Methods ; 190: 106346, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34637818

RESUMO

Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 µL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.


Assuntos
Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Especificidade de Hospedeiro , Pseudomonas aeruginosa/virologia , Águas Residuárias/virologia , Antibacterianos , DNA Viral , Farmacorresistência Bacteriana Múltipla , Humanos , Terapia por Fagos , Infecções por Pseudomonas/microbiologia , Sensibilidade e Especificidade
3.
Sci Rep ; 8(1): 12538, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135446

RESUMO

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Bacteriófago T7 , Biotinilação , Células CHO , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/toxicidade , Dicroísmo Circular , Cricetulus , Modelos Animais de Doenças , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Distrofia Muscular de Duchenne/tratamento farmacológico , Biblioteca de Peptídeos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
4.
Biomolecules ; 8(3)2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29997382

RESUMO

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 ß-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length ß-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of ß-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split ß-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the ß-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.


Assuntos
Peptídeos Penetradores de Células/química , Citosol/química , beta-Lactamases/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetulus , Sistemas de Liberação de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , beta-Lactamases/química , beta-Lactamases/genética
5.
Sci Rep ; 5: 18329, 2015 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-26671759

RESUMO

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.


Assuntos
Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos/métodos , Endossomos/metabolismo , Proteínas de Fluorescência Verde , Animais , Células CHO , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/farmacologia , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/farmacocinética , Proteínas de Fluorescência Verde/farmacologia , Células HEK293 , Humanos
6.
Antiviral Res ; 71(1): 53-63, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16621037

RESUMO

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.


Assuntos
Antivirais/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Ribavirina/farmacologia , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Citocinas/metabolismo , Efeito Citopatogênico Viral/efeitos dos fármacos , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Ácido Micofenólico/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Ribonucleosídeos/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Organismos Livres de Patógenos Específicos , Células Vero
7.
Antimicrob Agents Chemother ; 49(6): 2378-86, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15917537

RESUMO

Hemorrhagic fever of arenaviral origin is a frequently fatal infectious disease of considerable priority to the biodefense mission. Historically, the treatment of arenaviral infections with alpha interferons has not yielded favorable results. Here we present evidence that interferon alfacon-1, a nonnaturally occurring bioengineered alpha interferon approved for the treatment of chronic hepatitis C, is active against Pichinde and Tacaribe arenaviruses in cell culture. In the hamster model of Pichinde virus (PCV) infection, interferon alfacon-1 treatment significantly protected animals from death, prolonged the survival of those that eventually died, reduced virus titers, and limited liver damage characteristic of PCV-induced disease. Moreover, interferon alfacon-1 also demonstrated therapeutic activity, to a lesser degree, when the initiation of treatment was delayed up to 2 days post-virus challenge. Despite the observed advantages of interferon alfacon-1 therapy, efforts to stimulate the immune system with the known interferon inducer poly(I:C12U) (Ampligen) offered only limited protection against lethal PCV challenge. Taken together, these data suggest that the increased potency of the bio-optimized interferon alfacon-1 molecule may be critical to the observed antiviral effects. These data are the first report demonstrating efficacious treatment of acute arenaviral disease with alpha interferon therapy, and further study is warranted.


Assuntos
Antivirais/uso terapêutico , Infecções por Arenaviridae/prevenção & controle , Interferon Tipo I/uso terapêutico , Vírus Pichinde/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/efeitos dos fármacos , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Interferon Tipo I/administração & dosagem , Interferon Tipo I/farmacologia , Interferon-alfa , Vírus Pichinde/fisiologia , Proteínas Recombinantes
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