The development of new methods to facilitate direct electron transfer (DET) between enzymes and electrodes is of much interest because of the desire for stable biofuel cells that produce significant amounts of power. In this study, hydroxylated multiwalled carbon nanotubes (MWCNTs) were covalently modified with anthracene groups to help orient the active sites of laccase to allow for DET. The onset of the catalytic oxygen reduction current for these biocathodes occurred near the potential of the T1 active site of laccase, and optimized biocathodes produced background-subtracted current densities up to 140 μA/cm 2 . Potentiostatic and galvanostatic stability measurements of the biocathodes revealed losses of 25% and 30%, respectively, after 24 h of constant operation. Finally, the novel biocathodes were utilized in biofuel cells employing two different anodic enzymes. A compartmentalized cell using a mediated glucose oxidase anode produced an open circuit voltage of 0.819 ( 0.022 V, a maximum power density of 56.8 ((1.8) μW/cm 2 , and a maximum current density of 205.7 ((7.8) μA/cm 2 . A compartment-less cell using a DET fructose dehydrogenase anode produced an open circuit voltage of 0.707 ( 0.005 V, a maximum power density of 34.4 ((2.7) μW/cm 2 , and a maximum current density of 201.7 ((14.4) μA/cm 2 .

Laccase, a blue multicopper oxidoreductase enzyme, is a robust enzyme that catalyzes the reduction of oxygen to water and has been shown previously to perform improved direct electron transfer in a biocathode when mixed with anthracene-modified multi-walled carbon nanotubes. Previous cathode construction used crude laccase enzyme isolated as a brown cell extract powder containing both active and inactive proteins. Purification of this enzyme, yielding a blue solution, resulted in greatly improved enzyme activity and removed insulating protein that competed for docking space in this cathodic system. Cyclic voltammetry of the purified biocathodes showed a background subtracted limiting current density of 1.84 (±0.05) mA/cm 2 in a stationary air-saturated system. Galvanostatic and potentiostatic stability experiments show that the biocathode maintains up to 75% and 80% of the original voltage and current respectively over 24 hours of constant operation. Inclusion of the biocathode in a glucose/O 2 biofuel cell using a mediated glucose oxidase (GOx) anode produced maximum current and power densities of 1.28 (±0.18) mA/cm 2 and 281 (±50) μW/cm 2 at 25 • C and 1.80 (±0.06) mA/cm 2 and 381 (±33) μW/cm 2 at 37 • C, respectively. Enzymatic efficiency of this glucose/O 2 enzymatic fuel cell is among the highest reported for a glucose/O 2 enzymatic fuel cell.Laccase is an oxidoreductase enzyme from a class of multicopper oxidases (MCO) that catalyzes the four-electron reduction of molecular oxygen to water. Laccase has four copper atoms integrated into its two catalytic active sites: a tri-nuclear cluster responsible for the reduction of molecular oxygen, and a mononuclear Cu atom responsible for scavenging electrons from a variety of nonspecific aromatic substrates through one-electron oxidation and radical product formation. 1,2 Laccase is relatively thermostable and has a high turnover rate, making it an ideal target in the field of bioelectrocatalysis. 3,4 Biofuel cells allow for the harnessing of electrical energy that is available from a chemical reaction through the use of bioelectrocatalysts, and oxidoreductase enzymes are common bioelectrocatalysts considered for efficient energy conversion. For this purpose, a large amount of research effort has been put forth developing materials and methods to enhance the electrical connection of catalytic oxidoreductase enzyme active sites to electrode surfaces. 5-8 Two primary methods of electron transfer exist for connecting the enzyme active sites to a conductive electrode surface: mediated electron transfer (MET) and direct electron transfer (DET). MET focuses on using a reversible redox species as a shuttle for electrons from the active site of the enzyme to the electrode surface. This method is suitable for enzymes whose active sites are buried deep inside the insulating protein shell and are not very accessible to pass electrons directly to a conductive surface. Typically, MET employs a polymer matrix to immobilize the enzyme on the electrode surface while the mediator c...

The bacterial pathogen Salmonella uses sophisticated type III secretion systems (T3SS) to translocate and deliver bacterial effector proteins into host cells to establish infection. Monitoring these important virulence determinants in the context of live infections is a key step in defining the dynamic interface between the host and pathogen. Here, we provide a modular labeling platform based on fluorescence complementation with split-GFP that permits facile tagging of new Salmonella effector proteins. We demonstrate enhancement of split-GFP complementation signals by manipulating the promoter or by multimerizing the fluorescent tag and visualize three effector proteins, SseF, SseG and SlrP, that have never before been visualized over time during infection of live cells. Using this platform, we developed a methodology for visualizing effector proteins in primary macrophage cells for the first time and reveal distinct differences in effector defined intracellular niche between primary macrophage and commonly used HeLa and RAW cell lines.

Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc while simultaneously ensuring sufficient zinc to support the immune response.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron availability, increasing exposure to bactericidal copper, and manipulating zinc to restrict the growth of pathogens. Manipulation of zinc at the host-pathogen interface depends on both the pathogen’s identity and the nature of the host cell. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing our results to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response, with 129S6 macrophages transitioning from M1 to M2 polarization over the course of infection and showing signs of recovery. Our results reveal that functional Slc11a1 profoundly affects the transcriptional landscape upon infection. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc while simultaneously ensuring sufficient zinc to support the immune response.Author summaryMetal ions play an important role in influencing how immune cells such as macrophages respond to infection by pathogens. Because metal ions are both essential to survival, as well toxic when present is excessive amounts, the host and the pathogen have evolved diverse strategies to regulate metal acquisition and availability. Here, we show that the metal transporter slc11a1 plays a critical role in defining the host response to Salmonella infection. Infection causes widespread changes in expression of metal regulatory genes to limit the pathogen’s access to iron, increase its exposure to copper, and remodel zinc to ensure increased zinc in the cytosol and limited zinc for the pathogen. Macrophages expressing functional slc11a1 have a different profile of metal regulation and vastly different outcomes compared to immune compromised macrophage, demonstrating significantly different nutritional immune responses in immune competent versus immune compromised macrophages.