Significance: Extracellular vesicle (EV)-mediated transfer of macromolecules may play a key role in cellular communication and may have utility in directed molecular therapies. In addition, the EV packaged biomolecules in serum may have potential for diagnosing cancer and determining its likelihood of metastasis. EVs are heterogeneous and there are many outstanding questions associated with biogenesis, uptake, and the fate of transferred molecules in recipient cells. In fact, the function, characterization, and even the nomenclature of EVs are being refined. Here we aimed to improve the functional characterization of EVs, and observed that only microvesicles (MVs), but not exosomes, can functionally transfer loaded reporter molecules to recipient cells, largely by delivering plasmid DNA. Our data show that exosomes and MVs are structurally and functionally distinct.

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from Pseudomonas putida ) and YieF (from Escherichia coli )—were examined. Both are dimers and reduce chromate efficiently to Cr(III) ( k _cat / K _m = ∼2 × 10 ⁴ M ⁻¹ · s ⁻¹ ). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.

Summary: Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen‐insensitive nitroreductase of Escherichia coli , is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single‐electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one‐electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni‐ and di‐valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH‐catalysed chromate reduction reaction decreased ROS generation by c . 65, 40, or 20%, respectively, suggesting that these enzymes can pre‐empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.

The nature of the stress experienced by Escherichia coli K-12 exposed to chromate, and mechanisms that may enable cells to withstand this stress, were examined. Cells that had been preadapted by overnight growth in the presence of chromate were less stressed than nonadapted controls. Within 3 h of chromate exposure, the latter ceased growth and exhibited extreme filamentous morphology; by 5 h there was partial recovery with restoration of relatively normal cell morphology. In contrast, preadapted cells were less drastically affected in their morphology and growth. Cellular oxidative stress, as monitored by use of an H ₂ O ₂ -responsive fluorescent dye, was most severe in the nonadapted cells at 3 h postinoculation, lower in the partially recovered cells at 5 h postinoculation, and lower still in the preadapted cells. Chromate exposure depleted cellular levels of reduced glutathione and other free thiols to a greater extent in nonadapted than preadapted cells. In both cell types, the SOS response was activated, and levels of proteins such as SodB and CysK, which can counter oxidative stress, were increased. Some mutants missing antioxidant proteins (SodB, CysK, YieF, or KatE) were more sensitive to chromate. Thus, oxidative stress plays a major role in chromate toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant mechanisms. As bacterial chromate bioremediation is limited by the toxicity of chromate, minimizing oxidative stress during bacterial chromate reduction and bolstering the capacity of these organisms to deal with this stress will improve their effectiveness in chromate bioremediation.

This paper deals with specific targeting of the prodrug/enzyme regimen, CNOB/HChrR6, to treat a serious disease, namely HER2+ human breast cancer with minimal off-target toxicity. HChrR6 is an improved bacterial enzyme that converts CNOB into the cytotoxic drug MCHB. Extracellular vesicles (EV) were used for mRNA-based HchrR6 gene delivery: EVs may cause minimal immune rejection, and mRNA may be superior to DNA for gene delivery. To confine HChrR6 generation and CNOB activation to the cancer, the EVHB chimeric protein was constructed. It contains high-affinity anti-HER2 scFv antibody (ML39) and is capable of latching on to EV surface. Cells transfected with EVHB-encoding plasmid generated EVs displaying this protein (“directed EVs”). Transfection of a separate batch of cells with the new plasmid, XPort/HChrR6, generated EVs containing HChrR6 mRNA; incubation with pure EVHB enabled these to target the HER2 receptor, generating “EXO-DEPT” EVs. EXO-DEPT treatment specifically enabled HER2-overexpressing BT474 cells to convert CNOB into MCHB in actinomycin D–independent manner, showing successful and specific delivery of HChrR6 mRNA. EXO-DEPTs—but not undirected EVs—plus CNOB caused near-complete growth arrest of orthotopic BT474 xenografts in vivo, demonstrating for the first time EV-mediated delivery of functional exogenous mRNA to tumors. EXO-DEPTs may be generated from patients' own dendritic cells to evade immune rejection, and without plasmids and their potentially harmful genetic material, raising the prospect of clinical use of this regimen. This approach can be used to treat any disease overexpressing a specific marker. Mol Cancer Ther; 17(5); 1133–42. ©2018 AACR.

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.