The Structure and Function of the Bacterial Osmotically Inducible Protein Y.

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell.

Single-protein Diffusion in the Periplasm of Escherichia coli.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria.

Super-resolving microscopy reveals the localizations and movement dynamics of stressosome proteins in Listeria monocytogenes.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Integrated Microfluidic Preconcentration and Nucleic Amplification System for Detection of Influenza A Virus H1N1 in Saliva.

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (µFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the µFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

Microfluidic approach for the fabrication of cell-laden hollow fibers for endothelial barrier research.

This study reports an advanced approach to effectively generate hollow fibers in a triple-flow polydimethylsiloxane (PDMS) microfluidic device based on the gelation of alginate induced with CaCl2 inside a coaxial flow system. Two PDMS replicas with a semi-cylindrical microchannel were assembled to obtain a complete microchannel with a circular cross-section, which allowed the formation of mild and continuous coaxial flows for the fabrication of hollow fibers without employing complex glass microcapillaries. Mineral oil was introduced into the central flow to serve as an inert space inside the Ca-alginate wall. This was used to maintain the consistent formation of the hollow core of the microfiber and to easily transport fluid through the lumen structure in subsequent applications. The hollow fibers exhibited characteristics such as flexibility while showing robust mechanical strength, high permeability, and biocompatibility, and were used as scaffolds for the attachment and proliferation of human umbilical vein endothelial cells (HUVECs) to mimic a blood vessel. The fully covered HUVEC fibers were further integrated into a neurovascular system and co-cultured with astrocytes forming an on-chip blood brain barrier (BBB) platform. The use of this neurovascular model for drug testing will pave the way for developing or synthesizing a new drug that can cross the BBB in the human brain.

An integrated microfluidic PCR system with immunomagnetic nanoparticles for the detection of bacterial pathogens.

There is growing interest in rapid microbial pre-concentration methods to lower the detection limit of bacterial pathogens of low abundance in samples. Here, we report an integrated microfluidic PCR system that enables bacterial cells of interest in samples to be concentrated prior to PCR. It consists of two major compartments: a preconcentration chamber for the immunomagnetic separation of bacterial cells, and a PCR chamber for the DNA amplification of the concentrated cells. We demonstrate the feasibility of the system for the detection of microbial pathogens by preconcentrating the human pathogen Escherichia coli O157:H7, and also amplifying its DNA. The detection limit of E. coli O157:H7 in the PCR system is 1 × 103 CFU (colony forming unit)/mL. On-chip processing steps, including preconcentration and PCR steps, take less than two hours. Our system can serve as a rapid, specific, and quantitative platform for the detection of microbial pathogens in samples of large volume.