Cell-free production of fluorescent proteins for the discovery of novel ribosome-targeting antibiotics.

Various issues including the overuse of antibiotics has led to the development of threatening multidrug-resistant bacterial strains urging development of novel anti-infectives. One quarter of current clinical phase III antibiotic drug candidates address ribosomal protein translation as a target. Here, we describe an effective cell-free in vitro screening system for inhibitors of bacterial ribosome activity with direct fluorescence read-out. Using ribosomal S30 extracts from Escherichia coli, Salmonella enterica, and Pseudomonas putida, the validity of this system is demonstrated by concentration-dependent inhibition of translation by a set of different classes of translation-targeting drugs. The single-compartment cell-free translation reaction is compatible with multi-well formats. Fluorophore formation of green fluorescent protein or monomeric NeonGreen occurs in an hour time frame without the need of adding reagents for secondary enzymatic detection saving handling time, and prohibiting false positives. As label-free readout, the dose response further allows for IC50 determination in the same setup. Together, we show that cell-free production of fluorescent proteins for the discovery of ribosome-targeting antibiotics is feasible and amenable to high-throughput applications.

Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins.

Galectins represent ß-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.

Aquaporins with lactate/lactic acid permeability at physiological pH conditions.

The spectrum of putative and experimentally shown permeants of cellular water and solute channels of the ubiquitous aquaporin family is still increasing. Virtually all AQP substrates, e.g. water, glycerol, urea, hydrogen peroxide, or carbon dioxide, are permanently neutral small molecule compounds. Several reports, however, describe aquaporins that exhibit lactate permeability. Lactate in aqueous solution undergoes a pH-dependent protonation equilibrium with neutral lactic acid, which likely represents the actual substrate form passing the aquaporin channel. Certain aquaporins, however, appear to be better geared for lactate/lactic acid permeability even at low proton availability. Here, we discuss the structural properties of such aquaporins and compare them to the microbial protein family of the formate-nitrite (lactate) transporters that assume the aquaporin fold despite unrelated protein sequences.

Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport.

Microbial formate-nitrite transporter-type proteins (FNT) exhibit dual transport functionality. At neutral pH, electrogenic anion currents are detectable, whereas upon acidification transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of the energy metabolism, such as l-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. Two opposing hypotheses on the site of substrate protonation are plausible. The proton relay mechanism postulates proton transfer from a highly conserved histidine centrally positioned in the transport path. The dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Here, we defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also show that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including l-lactate, a low substrate affinity as is typical for FNTs, and, strikingly, proton motive force-dependent transport as observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules. Furthermore, EhFNT is the sole annotated FNT in the Entamoeba genome suggesting that it could be a putative new drug target with similar utility as that of the malarial PfFNT.