Spotlight pubs.acs.org/acschemicalbiology
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MICROBIAL FLAVONOID METABOLITE WARDS OFF FLU EFFECTS
SINGLE-CELL IMAGING TECHNIQUE SLASHES ANTIBIOTIC SUSCEPTIBILITY SCREENING TIMES
From Steed, A. L., et al., Science 2017, 357, 498−502. Reprinted with permission from AAAS, Copyright 2017.
Foods containing flavonoid compounds have long been touted for their proposed antioxidant and cardiovascular benefits. New research indicates that there may be yet another reason to reach for the fruit, wine, chocolate, or black tea: they could reduce the effects of the flu, with the help of friendly gut bacteria. The study was carried out by Thaddeus S. Stappenbeck and co-workers, who wanted to find out how gut microbiota regulate influenza virus pathogenicity. Considering that antibiotic-treated or germ-free mice suffer worse flu outcomes than mice with intact gut microbiomes, and also that many microbial metabolites influence a variety of biological systems including immunity, the research team hypothesized that a specific microbial metabolite might moderate host response to a flu infection. The team screened reporter cells with a panel of 84 known microbial-associated metabolites to find which could trigger signaling pathways controlled by the activity of type 1 interferon (IFN), a cytokine released by cells in response to pathogens in order to elicit immune system responses. One of the most active compounds was desaminotyrosine (DAT), a degradation product of flavonoids that the research team showed to be produced in high levels by the gut bacterium Clostridium orbiscindens. To determine whether microbially synthesized DAT would stimulate type 1 IFN-associated flu defenses in vivo, the research team examined influenza-infected mice whose gut microbiomes had either been knocked down by antibiotics or colonized with specific types of bacteria. Mice colonized with C. orbiscindens fared significantly better than did the negative controls, with higher survival rates presumably stemming from DAT-induced augmentation of type-1 IFN signaling and attenuation of lung damage. Notably, the protective effects of DAT only occurred when mice were exposed to DAT prior to rather than post-flu infection. The authors suggest that given the right microbiome, a flavonoid-rich diet might modulate the immune response to the fluso maybe an apple a day really does keep the doctor away. Heidi A. Dahlmann © 2017 American Chemical Society
Baltekin, O., et al., Proc. Natl. Acad. Sci., U.S.A., 114, 9170−9175. Copyright 2017 National Academy of Sciences, U.S.A.
Initial steps in the development of a rapid antibiotic susceptibility test (AST) have recently been reported by a team of researchers at Uppsala University (Proc. Natl. Acad. Sci., U.S.A. 2017, 114, 9170−9175). Johan Elf and co-workers used their new single-cell imaging-based technique to measure the susceptibility of bacterial strains that commonly cause urinary tract infections to frequently prescribed antibiotics with unprecedented speed. The technique was developed out of the research team’s previous investigation of cell-to-cell variation in the E. coli life cycle (Wallden et al., Cell 2016), which spurred their construction of a microfluidic device in which bacteria can be loaded into narrow wells so that the growth of individual cells can be monitored by microscopy over time. To test the effect of antibiotics on bacterial cell growth, half of the wells get bathed in media with an antibiotic, while the other half get bathed in media without antibiotic; then, the growth rates of the individual cells in both groups are measured as a function of the increase in length of the cells trapped in each well. Susceptible strains of bacteria that had been exposed to an antibiotic grew significantly slower than negative control bacteria, while resistant bacteria that had been treated with an antibiotic typically display a transient growth reduction before leveling off to growth rates similar to those observed for negative control bacteria. These unique growth rate signatures enabled the research team to correctly classify 49 clinical uropathogenic E. coli isolates as either susceptible or resistant to the broad-spectrum antibiotic ciproflaxin in 10 min after loading the sample, impressively faster than is possible using conventional AST’s that require hours or even days to complete. The technology is Published: September 15, 2017 2228
DOI: 10.1021/acschembio.7b00798 ACS Chem. Biol. 2017, 12, 2228−2230
ACS Chemical Biology
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now being developed into a user-friendly diagnostic test that can be used at the point-of-care to guide doctors in prescribing effective antibiotics for each infection and reduce the overprescription of broad-spectrum antibiotics that is contributing to the current scourge of antibiotic resistance. Heidi A. Dahlmann
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NANOMOTORS DRILL THROUGH CELL MEMBRANES
Spotlight
SEEING SIRTUINS AT WORK
Reprinted with permission from Xuan, et al., J. Am. Chem. Soc., DOI: 10.1021/jacs.7b05725. Copyright 2017 American Chemical Society.
Sirtuins are a class of enzymes found in all three domains of life, and they get their name from the founding family member, Sir2 from budding yeast. This enzyme along with most of the seven human family members (SIRTs) are NAD+-dependent protein deacetylases, meaning that they erase acetyl modifications on proteins in an energy-dependent manner. These enzymes have garnered considerable attention and controversy over the past two decades due to their association with lifespan in both yeast and animals. With their activity also linked to cancer, cardiovascular disease, and neurodegenerative disorders, the human surtuins represent a potential pharmaceutical target. Characterizing the enzymatic activity of sirtuins usually involves in vitro assays, but Xuan et al. (J. Am. Chem. Soc. 2017 DOI: 10.1021/jacs.7b05725) recently introduced a clever genetically encoded assay to detect sirtuin deacetylase activity in living cells. The researchers took advantage of the fluorophore maturation pathway for green fluorescent protein (GFP) which depends upon a specific lysine residue. By using a modified GFP gene carrying a UAG codon instead of the lysine codon at this position, a noncanonical amino acid, N-ε-acetyl-Llysine, could be specifically incorporated at the key position using an orthogonal aminoacyl-tRNA synthetase and suppressor tRNA system. With all of these components inserted into E. coli, GFP was only observed if the activity of the endogenous bacterial surtuin, cobB, was intact to remove the blocking acetyl group from the critical lysine. Since a ΔcobB bacterial strain was viable but did not exhibit fluorescence, it became the ideal test environment for the deacetylase activity of human SIRT proteins. The researchers also showed that the system can be ported over to mammalian cells by plasmid transfection, affording an additional control knob on deacetylase activity through specific or general inhibitor drugs. Jason G. Underwood
Reprinted by permission from Macmillan Publishers Ltd.: Nature, Robinson, J. T., et al., 548, 567, copyright 2017.
The movement of molecules into and out of cells is critical for natural biological functions as well as for research or medical purposes. Scientists traditionally rely on intermolecular forces and/or concentration gradients to facilitate the adsorption of cargo molecules to cell surfaces and mediate passive diffusion or active transport of cargo across cell membranes. Now, researchers can also harness mechanical force at the molecular level to puncture cell membranes, improving permeability of chemical species or inducing necrosis (Nature 2017, 548, 567). A research team led by Jacob T. Robinson, Gufeng Wang, Robert Pal, and James M. Tour repurposed molecular motors, which are small molecules that undergo precisely tailored conformational changes in response to external stimuli, to act as drills for opening the lipid bilayers of cell membranes. The basic structure of the molecular drills consisted of a dihydrobenzindene “rotor” connected via a methide bridge to a thioxanthene “stator,” which could be functionalized with various solubilizing, fluorescent, or cell-targeting labels. Irradiating the molecular drill with 355−365 nm ultraviolet (UV) light induces the methide bridge to rapidly undergo double-bond isomerization, which occurs successively to simulate unidirectional rotation of the rotor moiety relative to the stator at a rate of 2−3 million revolutions per second. This rotation propels the drill forward with up to 0.54 nN of force, which corresponds to 2−3 orders of magnitude more pressure than is needed to rupture most bilipid membranes. The research team demonstrated that upon UV light activation, fluorophore-labeled molecular drills could permeate live cells in a passive diffusion- and endocytosis-independent manner, while a homopropargylic alcohol-labeled drill was able to disrupt cell membranes to such an extent that inward ionic currents could be detected by patch-clamp analysis and exogenous dye in the cell media could flow into cells and intercalate into RNA and DNA. Furthermore, molecular drills labeled with specialized peptides could selectively induce necrosis in targeted cancer cells, demonstrating the potential application of the molecular drills in anticancer therapies. Heidi A. Dahlmann
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BRIGHTER DYES IN BETTER YIELDS
Reprinted with permission from Grimm, J. B., et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.7b00247. Copyright 2017 American Chemical Society. 2229
DOI: 10.1021/acschembio.7b00798 ACS Chem. Biol. 2017, 12, 2228−2230
ACS Chemical Biology
Spotlight
The ability to engineer dye molecules with specific absorbance and emission properties, boost brightness, and limit photobleaching is critical for a host of molecular and cellular biology experiments. Many chemical dyes are based on classic fluoresceins or the similar rhodamines, polyaromatic xanthene structures with a set of interesting optical and chemical properties. Such dyes can interconvert between a charged fluorescent form and a lactone form, which is colorless and nonfluorescent, based on pH or polarity, which also makes them useful chemical sensors. Recently, Luke Lavis’s group at Janelia Farm reported the synthesis of fluorosceins and related rhodamines where the xanthene oxygen is replaced with silicon. These dyes absorb at longer wavelengths and with improved brightness, which makes them especially useful for bioimaging. But these initial syntheses were difficult and produced low yields of these compounds. To expand their utility, the group now reports a simpler, general method for synthesizing these compounds in higher yields (ACS. Cent. Sci. 2017, DOI: 10.1021/acscentsci.7b00247). In this new approach, the team centered their strategy around an easily synthesized bisaryl dibromide intermediate that supplied the backbone of the fluorescent dyes. This compound could easily be converted to a diaryl lithium or diaryl Grignard reagent. These electron-rich nucleophiles react twice with an ester or anhydride electrophile to install the pendant aryl group on the dye. This electronically matched reaction resulted in conversion from dibromide to dye in 40 to 60% yield. This series of reactions produced Si-fluoresceins in four fewer steps and in yields 8 times greater than the previous synthetic route. This new strategy also allowed the team to incorporate more fluorine into the dye molecules, a strategy that allows the researchers to lower the pKa and make the dyes fluorescent under a larger range of pH conditions. The researchers then studied the fluorescent properties of various Si-fluoresceins. They used the same synthetic approach to produce a large panel of Si-rhodamines with similar yields and spectral results. This new approach allowed the synthesis of previously inaccessible Si-rhodamines with fully fluorinated pendant aromatic rings. This substitution pushed the spectral properties farther into the red and allows fine-tuning of a lipid membrane stain. These fluorinated fluorophores could also be conjugated by facile reaction with different thiol substrates, resulting in live cell labels for cell nuclei and antibody labels for immunofluorescence with impressive photostability. Overall, these researchers demonstrate a useful synthetic strategy for producing new and improved dyes for bioimaging. Sarah A. Webb
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DOI: 10.1021/acschembio.7b00798 ACS Chem. Biol. 2017, 12, 2228−2230
