Modifications of Epitaxial Graphene on SiC for the Electrochemical Detection and Identification of Heavy Metal Salts in Seawater.

The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.

Multilayer Epitaxial Graphene on Silicon Carbide: A Stable Working Electrode for Seawater Samples Spiked with Environmental Contaminants.

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis.

Machine Learning Techniques for Chemical Identification Using Cyclic Square Wave Voltammetry.

Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.

Acyl carrier protein is a bacterial cytoplasmic target of cationic antimicrobial peptide LL-37.

In addition to membrane disruption, the cathelicidin antimicrobial peptide (AMP) LL-37 translocates through the bacterial inner membrane to target intracellular molecules. The present study aims to identify an alternate mechanism and a cytoplasmic target of LL-37 in Francisella. LL-37 binding proteins from Francisella novicida U112 bacterial lysates were precipitated by using biotinylated LL-37 (B-LL-37) and NeutrAvidin-agarose beads. Bound proteins were identified by LC-MS/MS, validated and characterized by bead pull-down assays and differential scanning fluorimetry (DSF). The cationic AMP (CAMP) LL-37 was able to interact with Francisella cytoplasmic acyl carrier protein (AcpP; FTN1340/FTT1376). Further study confirmed that LL-37 peptide could bind to AcpP and that the sheep cathelicidin SMAP-29 (Sheep Myeloid Antimicrobial Peptide 29) further increased LL-37 binding to AcpP, suggesting a synergistic effect of SMAP-29 on the binding. LL-37 could also bind to both AcpP of Escherichia coli and Bacillus anthracis, implying a mechanism of broad action of LL-37-AcpP binding. Overexpression of the acpP gene in F. novicida led to an increase in LL-37 susceptibility. LL-37 binding to AcpP changed the fatty acid composition profiles. Taken together, we identified a novel cytoplasmic target of LL-37 in Francisella, suggesting a mechanism of action of this peptide beyond membrane permeabilization. Our findings highlight a novel mechanism of antimicrobial activity of this peptide and document a previously unexplored target of α-helical CAMPs.

Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.

In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation.

Analysis of mixed biofilm (Staphylococcus aureus and Pseudomonas aeruginosa) by laser ablation electrospray ionization mass spectrometry.

Pseudomonas aeruginosa and Staphylococcus aureus are ubiquitous pathogens often found together in polymicrobial, biofilm-associated infections. This study is the first to use laser ablation electrospray ionization mass spectrometry (LAESI-MS) to rapidly study bacteria within a mixed biofilm. Fast, direct, non-invasive LAESI-MS analysis of biofilm could significantly accelerate biofilm studies and provide previously unavailable information on both biofilm composition and the effects of antibiofilm treatment. LAESI-MS was applied directly to a polymicrobial biofilm and analyzed with respect to whether P. aeruginosa and S. aureus were co-localized or self-segregated within the mixed biofilm. LAESI-MS was also used to analyze ions following LL-37 antimicrobial peptide treatment of the biofilm. This ambient ionization method holds promise for future biofilm studies. The use of this innovative technique has profound implications for the study of biofilms, as LAESI-MS eliminates the need for lengthy and disruptive sample preparation while permitting rapid analysis of unfixed and wet biofilms.

Multienzymatic Cascades and Nanomaterial Scaffolding-A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products.

Synthetic biology is touted as the next industrial revolution as it promises access to greener biocatalytic syntheses to replace many industrial organic chemistries. Here, it is shown to what synthetic biology can offer in the form of multienzyme cascades for the synthesis of the most basic of new materials-chemicals, including especially designer chemical products and their analogs. Since achieving this is predicated on dramatically expanding the chemical space that enzymes access, such chemistry will probably be undertaken in cell-free or minimalist formats to overcome the inherent toxicity of non-natural substrates to living cells. Laying out relevant aspects that need to be considered in the design of multi-enzymatic cascades for these purposes is begun. Representative multienzymatic cascades are critically reviewed, which have been specifically developed for the synthesis of compounds that have either been made only by traditional organic synthesis along with those cascades utilized for novel compound syntheses. Lastly, an overview of strategies that look toward exploiting bio/nanomaterials for accessing channeling and other nanoscale materials phenomena in vitro to direct novel enzymatic biosynthesis and improve catalytic efficiency is provided. Finally, a perspective on what is needed for this field to develop in the short and long term is presented.

Bacterial Membrane Vesicles for In Vitro Catalysis.

The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems' potential as an enabling tool for biotechnology and biomanufacturing.

Computationally Designed AMPs with Antibacterial and Antibiofilm Activity against MDR Acinetobacter baumannii.

The discovery of new antimicrobials is necessary to combat multidrug-resistant (MDR) bacteria, especially those that infect wounds and form prodigious biofilms, such as Acinetobacter baumannii. Antimicrobial peptides (AMPs) are a promising class of new therapeutics against drug-resistant bacteria, including gram-negatives. Here, we utilized a computational AMP design strategy combining database filtering technology plus positional analysis to design a series of novel peptides, named HRZN, designed to be active against A. baumannii. All of the HRZN peptides we synthesized exhibited antimicrobial activity against three MDR A. baumannii strains with HRZN-15 being the most active (MIC 4 µg/mL). This peptide also inhibited and eradicated biofilm of A. baumannii strain AB5075 at 8 and 16 µg/mL, which is highly effective. HRZN-15 permeabilized and depolarized the membrane of AB5075 rapidly, as demonstrated by the killing kinetics. HRZN 13 and 14 peptides had little to no hemolysis activity against human red blood cells, whereas HRZN-15, -16, and -17 peptides demonstrated more significant hemolytic activity. HRZN-15 also demonstrated toxicity to waxworms. Further modification of HRZN-15 could result in a new peptide with an improved toxicity profile. Overall, we successfully designed a set of new AMPs that demonstrated activity against MDR A. baumannii using a computational approach.

Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles.

All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein "anchors/directors") and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp', SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp'. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp' anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.

Extracellular vesicle production in Gram-positive bacteria.

This is a highlight on the article 'Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage-encoded holin-lysin system' by Yue Liu, Eddy Smid and Tjakko Abee.

Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.

Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid Loligo vulgaris and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 °C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.

Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus.

BACKGROUND: Chronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms. RESULTS: The helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low µg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities. CONCLUSIONS: The NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections.

Isolation and Characterization of Membrane Vesicles from Lactobacillus Species.

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome ( Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules. Graphic abstract: Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows).

PepVAE: Variational Autoencoder Framework for Antimicrobial Peptide Generation and Activity Prediction.

New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here, we report a peptide generation framework, PepVAE, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.

Variational Autoencoder for Generation of Antimicrobial Peptides.

Over millennia, natural evolution has allowed for the emergence of countless biomolecules with highly specific roles within natural systems. As seen with peptides and proteins, often evolution produces molecules with a similar function but with variable amino acid composition and structure but diverging from a common ancestor, which can limit sequence diversity. Using antimicrobial peptides as a model biomolecule, we train a generative deep learning algorithm on a database of known antimicrobial peptides to generate novel peptide sequences with antimicrobial activity. Using a variational autoencoder, we are able to generate a latent space plot that can be surveyed for peptides with known properties and interpolated across a predictive vector between two defined points to identify novel peptides that show dose-responsive antimicrobial activity. These proof-of-concept studies demonstrate the potential for artificial intelligence-directed methods to generate new antimicrobial peptides and motivate their potential application toward peptide and protein design without the need for exhaustive screening of sequence libraries.

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production.

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg2+. In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays.

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

Bacterial bioreactors: Outer membrane vesicles for enzyme encapsulation.

Bacterial membrane vesicles, whether naturally occurring or engineered for enhanced functionality, have significant potential as tools for bioremediation, enzyme catalysis, and the development of therapeutics such as vaccines and adjuvants. In many instances, the vesicles themselves and the naturally occurring proteins are sufficient to lend functionality. Alternatively, additional function can be conveyed to these biological nanoparticles through the directed packaging of peptides and proteins, specifically recombinant enzymes chosen to mediate a specific reaction or facilitate a controlled response. Here we will detail mechanisms for directing the packaging of recombinant proteins and peptides into the nascent membrane vesicles (MVs) of Gram-negative bacteria with a focus on both active and passive packaging using both cellular machinery and engineered molecular systems. Additionally, we detail some of the more common methods for bacterial MVs purification, quantitation, and characterization as these methods are requisite for any subsequent experimentation or processing of MV reagents.