Myosin-X and disease

Myosin-X (Myo10) is a motor protein well known for its role in filopodia formation. New research implicates Myo10 in a number of disease states including cancer metastasis and pathogen infection. This review focuses on these developments with emphasis on the emerging roles of Myo10 in formation of cancer cell protrusions and metastasis. A number of aggressive cancers show high levels of Myo10 expression and knockdown of Myo10 has been shown to dramatically limit cancer cell motility in 2D and 3D systems. Myo10 knockdown also limits spread of intracellular pathogens marburgvirus and Shigella flexneri. Consideration is given to how these properties might arise and potential paths of future research

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile , c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production During Stationary Phase

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability

How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide–copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue

Fast Benchtop Fabrication of Laminar Flow Chambers for Advanced Microscopy Techniques

Background: Fluid handling technology is acquiring an ever more prominent place in laboratory science whether it is in simple buffer exchange systems, perfusion chambers, or advanced microfluidic devices. Many of these applications remain the providence of laboratories at large institutions with a great deal of expertise and specialized equipment. Even with the expansion of these techniques, limitations remain that frequently prevent the coupling of controlled fluid flow with other technologies, such as coupling microfluidics and high-resolution position and force measurements by optical trapping microscopy. Method: Here we present a method for fabrication of multiple-input laminar flow devices that are optically clear [glass] on each face, chemically inert, reusable, inexpensive, and can be fabricated on the benchtop in approximately one hour. Further these devices are designed to allow flow regulation by a simple gravity method thus requiring no specialized equipment to drive flow. Here we use these devices to perform total internal reflection fluorescence microscopy measurements as well as position sensitive optical trapping experiments. Significance: Flow chamber technology needs to be more accessible to the general scientific community. The method presented here is versatile and robust. These devices use standard slides and coverslips making them compatible with nearly all types and models of light microscopes. These devices meet the needs of groups doing advanced optical trapping experiments, but could also be adapted by nearly any lab that has a function for solution flow coupled with microscopy

Fast Benchtop Fabrication of Laminar Flow Chambers for Advanced Microscopy Techniques

Background: Fluid handling technology is acquiring an ever more prominent place in laboratory science whether it is in simple buffer exchange systems, perfusion chambers, or advanced microfluidic devices. Many of these applications remain the providence of laboratories at large institutions with a great deal of expertise and specialized equipment. Even with the expansion of these techniques, limitations remain that frequently prevent the coupling of controlled fluid flow with other technologies, such as coupling microfluidics and high-resolution position and force measurements by optical trapping microscopy.Method: Here we present a method for fabrication of multiple-input laminar flow devices that are optically clear [glass] on each face, chemically inert, reusable, inexpensive, and can be fabricated on the benchtop in approximately one hour. Further these devices are designed to allow flow regulation by a simple gravity method thus requiring no specialized equipment to drive flow. Here we use these devices to perform total internal reflection fluorescence microscopy measurements as well as position sensitive optical trapping experiments.Significance: Flow chamber technology needs to be more accessible to the general scientific community. The method presented here is versatile and robust. These devices use standard slides and coverslips making them compatible with nearly all types and models of light microscopes. These devices meet the needs of groups doing advanced optical trapping experiments, but could also be adapted by nearly any lab that has a function for solution flow coupled with microscopy.</p

Visualizing the Effects of Clostridium difficile Toxins A and B on Mammalian Epithelial Cells

Clostridium difficile is a spore-forming, obligate anaerobe, Gram-positive bacterium that causes Clostridium difficile infection (CDI), responsible for ~30,000 deaths annually. C. difficile secretes Toxin A and Toxin B, closely related proteins which target host small GTPases and have been associated with severe symptoms of CDI. We studied the effects of these toxins on epithelial cell junctions and actin cytoskeletons via live-cell fluorescent microscopy. Epithelial cells form a protective barrier by generating tight, strong junctions on all sides. The actin cytoskeleton allows cells to bind to the substrate and to interact with their neighboring cells. Using real-time, high-resolution fluorescent microscopy we show how C. difficile affects host cell junctions and cytoskeleton. Additionally, we tested the effects of purified toxins at various concentrations on these fluorescently labeled cells to clarify the role of each toxin separately during CDI. Toxins A and B appear to work on different timescales and have different effects on epithelial cell physiology. Cells introduced to Toxin A retracted significantly yet maintained cell junctions relatively well. Cells treated with Toxin B rounded more gradually. Treatment with Toxin A+B caused dramatic rounding and degradation of cells junctions compared to healthy controls. In addition, the tight junctions showed distress slightly sooner than the actin cytoskeleton. Toxin concentration determined how quickly the epithelial cells showed damage, but all concentrations tested were eventually fatal, illustrating how potent these toxins are against mammalian cells. These findings improve our mechanistic understanding of infection bringing us one step closer to effective treatment

Single-Cell Characterization of \u3ci\u3eClostridioidies difficile\u3c/i\u3e Motility Using Anaerobic Live Cell Microscopy

Clostridioidies difficile (C.difficile) is an anaerobic bacterium responsible for CDI (Clostridioidies difficile infection), a common problem in hospitals and for people using antibiotics, due to the bacteria’s resistance to common treatments and ready appetite for sugar byproducts in the intestine. C.difficile has proven to be resistant to multiple antibiotic families, including beta lactams and fluroquinolones. The bacteria have two forms: dormant spores that persist in the environment and spread the infection, and vegetative cells, which proliferate within the host colon and produce virulent toxins. Little is known about the behavior of vegetative cells within hosts, because they are strict anaerobes and killed by the environmental oxygen. It is known that the organism is a motile, flagellated bacterium which swims through liquids and can even traverse the surface of solids through the extension and retraction of pili in vitro. Here, we demonstrate a novel methodology for capturing active imagery of the microbe at the level of individual cells, and show that the epidemic C.difficile strain R20291 has its motility regulated by the presence of different sources of energy that it is cultured in. This provides a superior ability to analyze distribution patterns of bacteria outside the anaerobic chamber, in addition to showing that this distribution is highly regulated by the nutritional substrate available to the microbe. Different concentrations of both arabinose and glucose showed no difference in motility for C.difficile, whereas higher concentrations of the mucus component N-actylneuraminic acid (Neu5Ac) caused significant reductions in movement and direction changes in the bacteria. This suggests that C.difficile responds to substrate nutrient type and concentration through changes in behavior and may actively target gut mucus as a colonization site