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Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells.

ABSTRACT: Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse imaging to reveal time-dependent statistics of natural killer (NK) killing at the single-cell level. Most kills occurred during serial killing, and a single NK cell killed up to 10 targets over a 6-h assay. The first kill was slower than subsequent kills, especially on poor targets, or when NK signaling pathways were partially inhibited. Spatial analysis showed that sequential kills were usually adjacent. We propose that NK cells integrate signals from the previous and current target, possibly by simultaneous contact. The resulting burst kinetics and spatial coordination may control the activity of NK cells in tissues.

SUBMITTER: Choi PJ

PROVIDER: S-EPMC3631668 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells.

Choi Paul J PJ Mitchison Timothy J TJ

Proceedings of the National Academy of Sciences of the United States of America 20130401 16

Cytotoxic lymphocytes eliminate virus-infected and cancerous cells by immune recognition and killing through the perforin-granzyme pathway. Traditional killing assays measure average target cell lysis at fixed times and high effector:target ratios. Such assays obscure kinetic details that might reveal novel physiology. We engineered target cells to report on granzyme activity, used very low effector:target ratios to observe potential serial killing, and performed low magnification time-lapse ima ...[more]

PMID: 23576740

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Project description:With the spread of multidrug-resistant bacteria, there has been an increasing focus on molecular classes that have not yet yielded an antibiotic. A key capability for assessing and prescribing new antibacterial treatments is to compare the effects antibacterial agents have on bacterial growth at a phenotypic, single-cell level. Here, we combined time-lapse microscopy with microfluidics to investigate the concentration-dependent killing kinetics of stationary-phase Escherichia coli cells. We used antibacterial agents from three different molecular classes, β-lactams and fluoroquinolones, with the known antibiotics ampicillin and ciprofloxacin, respectively, and a new experimental class, protein Ψ-capsids. We found that bacterial cells elongated when treated with ampicillin and ciprofloxacin used at their minimum inhibitory concentration (MIC). This was in contrast to Ψ-capsids, which arrested bacterial elongation within the first two hours of treatment. At concentrations exceeding the MIC, all the antibacterial agents tested arrested bacterial growth within the first 2 h of treatment. Further, our single-cell experiments revealed differences in the modes of action of three different agents. At the MIC, ampicillin and ciprofloxacin caused the lysis of bacterial cells, whereas at higher concentrations, the mode of action shifted toward membrane disruption. The Ψ-capsids killed cells by disrupting their membranes at all concentrations tested. Finally, at increasing concentrations, ampicillin and Ψ-capsids reduced the fraction of the population that survived treatment in a viable but nonculturable state, whereas ciprofloxacin increased this fraction. This study introduces an effective capability to differentiate the killing kinetics of antibacterial agents from different molecular classes and offers a high content analysis of antibacterial mechanisms at the single-cell level. IMPORTANCE Antibiotics act against bacterial pathogens by inhibiting their growth or killing them directly. Different modes of action determine different antibacterial responses, whereas phenotypic differences in bacteria can challenge the efficacy of antibiotics. Therefore, it is important to be able to differentiate the concentration-dependent killing kinetics of antibacterial agents at a single-cell level, in particular for molecular classes which have not yielded an antibiotic before. Here, we measured single-cell responses using microfluidics-enabled imaging, revealing that a novel class of antibacterial agents, protein Ψ-capsids, arrests bacterial elongation at the onset of treatment, whereas elongation continues for cells treated with β-lactam and fluoroquinolone antibiotics. The study advances our current understanding of antibacterial function and offers an effective strategy for the comparative design of new antibacterial therapies, as well as clinical antibiotic susceptibility testing.

Dataset Information

Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells.

Publications

Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells.

Similar Datasets

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