Measuring Thousands of Single-Vesicle Leakage Events Reveals the Mode of Action of Antimicrobial Peptides.

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics.

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.

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.

Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism's phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.

Aerosol-jet-printed, conformable microfluidic force sensors.

Force sensors that are thin, low-cost, flexible, and compatible with commercial microelectronic chips are of great interest for use in biomedical sensing, precision surgery, and robotics. By leveraging a combination of microfluidics and capacitive sensing, we develop a thin, flexible force sensor that is conformable and robust. The sensor consists of a partially filled microfluidic channel made from a deformable material, with the channel overlaying a series of interdigitated electrodes coated with a thin, insulating polymer layer. When a force is applied to the microfluidic channel reservoir, the fluid is displaced along the channel over the electrodes, thus inducing a capacitance change proportional to the applied force. The microfluidic molds themselves are made of low-cost sacrificial materials deposited via aerosol-jet printing, which is also used to print the electrode layer. We envisage a large range of industrial and biomedical applications for this force sensor.

Switching Cytolytic Nanopores into Antimicrobial Fractal Ruptures by a Single Side Chain Mutation.

Disruption of cell membranes is a fundamental host defense response found in virtually all forms of life. The molecular mechanisms vary but generally lead to energetically favored circular nanopores. Here, we report an elaborate fractal rupture pattern induced by a single side-chain mutation in ultrashort (8-11-mers) helical peptides, which otherwise form transmembrane pores. In contrast to known mechanisms, this mode of membrane disruption is restricted to the upper leaflet of the bilayer where it exhibits propagating fronts of peptide-lipid interfaces that are strikingly similar to viscous instabilities in fluid flow. The two distinct disruption modes, pores and fractal patterns, are both strongly antimicrobial, but only the fractal rupture is nonhemolytic. The results offer wide implications for elucidating differential membrane targeting phenomena defined at the nanoscale.

Microfluidic Single-Cell Phenotyping of the Activity of Peptide-Based Antimicrobials.

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.

Single-cell microfluidics facilitates the rapid quantification of antibiotic accumulation in Gram-negative bacteria.

The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to rapidly quantify antibiotic accumulation in hundreds of individual Escherichia coli cells. By serially manipulating the microfluidic environment, we demonstrated that stationary phase Escherichia coli, traditionally more refractory to antibiotics than growing cells, display reduced accumulation of the antibiotic ofloxacin compared to actively growing cells. Our novel microfluidic method facilitates the quantitative comparison of the role of the microenvironment versus that of the absence of key membrane transport pathways in cellular drug accumulation. Unlike traditional techniques, our assay is rapid, studying accumulation as the cells are dosed with the drug. This platform provides a powerful new tool for studying antibiotic accumulation in bacteria, which will be critical for the rational development of the next generation of antibiotics.

Characterization of lipid composition and diffusivity in OLA generated vesicles.

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 µm2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

Aerosol-jet printing facilitates the rapid prototyping of microfluidic devices with versatile geometries and precise channel functionalization.

Microfluidics has emerged as a powerful analytical tool for biology and biomedical research, with uses ranging from single-cell phenotyping to drug discovery and medical diagnostics, and only small sample volumes required for testing. The ability to rapidly prototype new designs is hugely beneficial in a research environment, but the high cost, slow turnaround, and wasteful nature of commonly used fabrication techniques, particularly for complex multi-layer geometries, severely impede the development process. In addition, microfluidic channels in most devices currently play a passive role and are typically used to direct flows. The ability to "functionalize" the channels with different materials in precise spatial locations would be a major advantage for a range of applications. This would involve incorporating functional materials directly within the channels that can partake in, guide or facilitate reactions in precisely controlled microenvironments. Here we demonstrate the use of Aerosol Jet Printing (AJP) to rapidly produce bespoke molds for microfluidic devices with a range of different geometries and precise "in-channel" functionalization. We show that such an advanced microscale additive manufacturing method can be used to rapidly design cost-efficient and customized microfluidic devices, with the ability to add functional coatings at specific locations within the microfluidic channels. We demonstrate the functionalization capabilities of our technique by specifically coating a section of a microfluidic channel with polyvinyl alcohol to render it hydrophilic. This versatile microfluidic device prototyping technique will be a powerful aid for biological and bio-medical research in both academic and industrial contexts.

An Integrated Microfluidic Platform for Quantifying Drug Permeation across Biomimetic Vesicle Membranes.

The low membrane permeability of candidate drug molecules is a major challenge in drug development, and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging because one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological pH and salt concentrations, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin and ciprofloxacin across biomimetic liposomes.

Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes.

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner.

Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones.

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug - we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.

Dependence of norfloxacin diffusion across bilayers on lipid composition.

Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.

Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli.

Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.

The indole pulse: a new perspective on indole signalling in Escherichia coli.

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5-1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.