SPIM-Flow: An Integrated Light Sheet and Microfluidics Platform for Hydrodynamic Studies of Hydra.

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a powerful imaging approach. However, access to and interfacing microscopes with microfluidics have remained challenging. Complex interfacing with microfluidics has limited the SPIM's utility for studying the hydrodynamics of freely moving multicellular organisms. We developed SPIM-Flow, an inexpensive light sheet platform that enables easy integration with microfluidics. We used SPIM-Flow to investigate the hydrodynamics of a freely moving Hydra polyp via particle tracking in millimeter-sized chambers. Initial experiments across multiple animals, feeding on a chip (Artemia franciscana nauplii used as food), and baseline behaviors (tentacle swaying, elongation, and bending) indicated the organisms' health inside the system. Fluidics were used to investigate Hydra's response to flow. The results suggested that the animals responded to an established flow by bending and swaying their tentacles in the flow direction. Finally, using SPIM-Flow in a proof-of-concept experiment, the shear stress required to detach an animal from a surface was demonstrated. Our results demonstrated SPIM-Flow's utility for investigating the hydrodynamics of freely moving animals.

Developing Antimicrobial Synergy With AMPs.

Antimicrobial peptides (AMPs) have been extensively studied due to their vast natural abundance and ability to kill microbes. In an era critically lacking in new antibiotics, manipulating AMPs for therapeutic application is a promising option. However, bacterial pathogens resistant to AMPs remain problematic. To improve AMPs antimicrobial efficacy, their use in conjunction with other antimicrobials has been proposed. How might this work? AMPs kill bacteria by forming pores in bacterial membranes or by inhibiting bacterial macromolecular functions. What remains unknown is the duration for which AMPs keep bacterial pores open, and the extent to which bacteria can recover by repairing these pores. In this mini-review, we discuss various antimicrobial synergies with AMPs. Such synergies might arise if the antimicrobial agents helped to keep bacterial pores open for longer periods of time, prevented pore repair, perturbed bacterial intracellular functions at greater levels, or performed other independent bacterial killing mechanisms. We first discuss combinations of AMPs, and then focus on histones, which have antimicrobial activity and co-localize with AMPs on lipid droplets and in neutrophil extracellular traps (NETs). Recent work has demonstrated that histones can enhance AMP-induced membrane permeation. It is possible that histones, histone fragments, and histone-like peptides could amplify the antimicrobial effects of AMPs, giving rise to antimicrobial synergy. If so, clarifying these mechanisms will thus improve our overall understanding of the antimicrobial processes and potentially contribute to improved drug design.

A novel antibacterial strategy: histone and antimicrobial peptide synergy.

The rate at which antibiotics are discovered and developed has stagnated; meanwhile, antibacterial resistance continually increases and leads to a plethora of untreatable and deadly infections worldwide. Therefore, there is a critical need to develop new antimicrobial strategies to combat this alarming reality. One approach is to understand natural antimicrobial defense mechanisms that higher-level organisms employ in order to kill bacteria, potentially leading to novel antibiotic therapeutic approaches. Mammalian histones have long been reported to have antibiotic activity, with the first observation of their antibacterial properties reported in 1942. However, there have been doubts about whether histones could truly have any such role in the animal, predominantly based on two issues: they are found in the nucleus (so are not in a position to encounter bacteria), and their antibiotic activity in vitro has been relatively weak in physiological conditions. More recent studies have addressed both sets of concerns. Histones are released from cells as part of neutrophil extracellular traps (NETs) and are thus able to encounter extracellular bacteria. Histones are also present intracellularly in the cytoplasm attached to lipid droplets, positioning them to encounter cytosolic bacteria. Our recent work (Doolin et al., 2020, Nat Commun), which is discussed here, shows that histones have synergistic antimicrobial activities when they are paired with antimicrobial peptides (AMPs), which form pores in bacterial membranes and co-localize with histones in NETs. The work demonstrates that histones enhance AMP-mediated pores, impair bacterial membrane recovery, depolarize the bacterial proton gradient, and enter the bacterial cytoplasm, where they restructure the chromosome and inhibit transcription. Here, we examine potential mechanisms that are responsible for these outcomes.

Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization.

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.