RESUMO
Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions-their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide's physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias , Microfluídica , Relação Estrutura-AtividadeRESUMO
Antibiotic resistance is a major challenge for modern medicine, and there is a dire need to refresh the antibiotic development pipeline to treat infections that are resistant to currently available drugs. Peptide-based antimicrobials represent a promising source of novel anti-infectives, but their development is severely impeded due to the lack of suitable techniques to accurately quantify their antimicrobial efficacy. A major problem involves the heterogeneity of cellular phenotypes in response to these peptides, even within a clonal population of bacteria. There is thus a need to develop single-cell resolution assays to quantify drug efficacy for these novel therapeutics. We present here a detailed microfluidics-microscopy protocol for testing the efficacy of peptide-based antimicrobials on hundreds to thousands of individual bacteria in well-defined microenvironments. This enables the study of cell-to-cell differences in drug response within a clonal population. It is a highly versatile tool, which can be used to quantify drug efficacy, including the number of individual survivors at defined drug doses; it even enables the potential exploration of the molecular mechanisms of action of the drug, which are often unknown in the early stages of drug development. We present here protocols for working with Escherichia coli, but organisms of different geometric shapes and sizes may also be tested with suitable modifications of the microfluidic device.
Assuntos
Microfluídica/métodos , Peptídeos/química , Proteínas Citotóxicas Formadoras de Poros/química , Análise de Célula Única/métodos , Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Peptídeos/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologiaRESUMO
We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.
Assuntos
Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Antígenos CD34/metabolismo , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Forma Celular , Citocalasina D/farmacologia , Citoesqueleto , Desenho de Equipamento , Células HL-60/citologia , Células HL-60/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas Analíticas MicrofluídicasRESUMO
Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (