Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins.

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.

Delineation of the Ancestral Tus-Dependent Replication Fork Trap.

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a 'locked' Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for 'back-up' Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies.

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.

Comparison of the analytical sensitivity of COVID-19 rapid antigen tests in Australia and Canada.

Rapid testing has become an indispensable strategy to identify the most infectious individuals and prevent the transmission of SARS-CoV-2 in vulnerable populations. As such, COVID-19 rapid antigen tests (RATs) are being manufactured faster than ever yet lack relevant comparative analyses required to inform on absolute analytical sensitivity and performance, limiting end-user ability to accurately compare brands for decision making. To date, more than 1000 different COVID-19 RATs are commercially available in the world, most of which detect the viral nucleocapsid protein (NP). Here, we examine and compare the analytical sensitivity of 26 RATs that are readily available in Canada and/or Australia using two NP reference materials (RMs) - a fluorescent NP-GFP expressed in bacterial cells and NCAP-1 produced in a mammalian expression system. Both RMs generate highly comparable results within each RAT, indicating minimal bias due to differing expression systems and final buffer compositions. However, we demonstrate orders of magnitude differences in analytical sensitivities among distinct RATs, and find little correlation with the median tissue culture infectious dose (TCID50) assay values reported by manufacturers. In addition, two COVID-19/Influenza A&B combination RATs were evaluated with influenza A NP-GFP. Finally, important logistics considerations are discussed regarding the robustness, ease of international shipping and safe use of these reference proteins. Taken together, our data highlight the need for and practicality of readily available, reliable reference proteins for end-users that will ensure that manufacturers maintain batch-to-batch quality and accuracy of RATs. They will aid international public health and government agencies, as well as health and aged care facilities to reliably benchmark and select the best RATs to curb transmission of future SARS-CoV-2 and influenza outbreaks.

A GFP-tagged nucleoprotein-based aggregation assay for anti-influenza drug discovery and antibody development.

Influenza is a viral pandemic that affects millions of people worldwide. Seasonal variations due to genetic shuffling and antigenic drifts in the influenza viruses have necessitated continual updating of therapeutics. The growing resistance to current influenza drugs has increased demand for new antivirals. The highly conserved nature of NP, a multi-functional viral protein that is serotypically distinct and abundantly expressed during infection, has led to its use in developing universal biotherapeutics and vaccines that could be effective against the virus, irrespective of its strain variations. Compounds causing aggregation of NP have recently been shown to be potent antivirals but require the development of new high-throughput assays capable of screening compounds with similar modes of action. Here, we describe the development of a new bioassay for the Influenza A nucleoprotein (NP). The assay was developed to quantify ligand-induced aggregation of a GFP-tagged NP and was validated with aggregation-inducing compounds such as nucleozin and a NP-specific antibody. The new NP-GFP aggregation assay can be performed with partially purified or mixtures of proteins and is amenable to a high-throughput format. Using this assay, we demonstrate the potential of a new anti-NP polyclonal antibody that we have obtained from chicken. This cost-effective high-yield source of anti-NP IgY has potential for large-scale production and development of therapeutic antibodies. The simplicity, speed and flexibility of this assay make it an invaluable tool for timely development of effective antivirals that can help to control future epidemics.

Real-Time Temperature Sensing Using a Ratiometric Dual Fluorescent Protein Biosensor.

Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development of a simple, inexpensive ratiometric dual fluorescent protein temperature biosensor (DFPTB). A combination of cycle three green fluorescent protein and a monomeric red fluorescent protein enabled the quantification of relative temperature changes and the identification of temperature discrepancies across a wide temperature range of 4-70 °C. The maximal sensitivity of 6.7% °C-1 and precision of 0.1 °C were achieved in a biologically relevant temperature range of 25-42 °C in standard phosphate-buffered saline conditions at a pH of 7.2. Good temperature sensitivity was achieved in a variety of biological buffers and pH ranging from 4.8 to 9.1. The DFPTB can be used in either purified or mixed bacteria-encapsulated formats, paving the way for in vitro and in vivo applications for topologically precise temperature measurements.

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard.

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.

Combining RNA-DNA swapping and quantitative polymerase chain reaction for the detection of influenza A nucleoprotein.

The detection of proteinaceous antigens generally relies on traditional immunoassays and, more recently, on immuno-PCR (polymerase chain reaction) assays and their derivatives, which do not take advantage of the intrinsic function or binding property of a protein. The RNA-binding nucleoprotein has been shown to be an excellent target for the development of various influenza A diagnostics due to its high antigenicity and the presence of large numbers in the virus. It binds nonspecifically to the sugar-phosphate backbone of RNA as well as to single-stranded DNA (ssDNA) in vitro. We decided to take advantage of this property to develop an ssDNA probe for the detection of nucleoprotein by quantitative PCR (qPCR). We found that recombinant influenza A nucleoprotein from avian H5N1 subtype binds strongest to a 74-base-long ssDNA. Two systems, one comprising an antibody-based nucleoprotein capture surface and the other based on direct nucleoprotein adsorption under denaturing conditions, were developed combining the replacement of RNA bound to nucleoprotein by a discrete ssDNA probe and a qPCR for the detection of nucleoprotein in the low picomolar (pM) range.

A polyplex qPCR-based binding assay for protein-DNA interactions.

The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.

A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices.

Protein detection in complex biological fluids and matrices has become a widely diversified field utilizing a number of different technologies. The quantification of target proteins in complex media such as serum remains a challenge for most technologies such as mass spectrometry, ELISA and western blot. Quantitative Immuno-PCR has been heavily used for antigen detection in immunoassays, but minimally so for quantifying affinity-tagged proteins expressed or circulating in complex matrices--despite its high sensitivity and robustness--because it suffers from detrimental background effects arising from its extreme detection power. We report the development of a universal qIPCR-based platform for the reproducible detection of dual affinity-tagged protein analytes in crude complex matrices such as serum and cell culture media or lysates. The system uses a couple of high-affinity antibodies against two affinity tags (GFP and HA) for the detection of dual-tagged proteins. The dual-tagged analyte is immuno-captured by one of its tags, while the second tag is bound by a detection device consisting of a new kind of self-assembled antibody-DNA conjugate. The new qIPCR platform enabled picomolar quantification of dual-tagged sortase in crude serum in 4 h including the PCR step.

Rise of the terminator protein tus: A versatile tool in the biotechnologist's toolbox.

Tus is a protein involved in DNA replication termination that binds specific DNA sequences (Ter) located around the terminus region of the chromosome in Enterobacterales. Tus and Ter form a unique monomeric protein-DNA complex which is one of strongest of its kind. A fascinating aspect of Tus-Ter is its ability to dramatically change conformation into a locked structure upon progression of a replication fork towards the non-permissive face of the complex. Over the last two decades, several new technologies have emerged harnessing the unique and interesting properties of this fascinating DNA-binding protein. This review highlights the important properties of the Tus-Ter complex and their exploitation for the development of diverse and novel ultrasensitive detection devices as well as innovative genomic and proteomic platform technologies. A variety of ex vivo and in vivo bioanalytical applications are discussed, including immuno-PCR diagnostic, bioassay and protein array technologies that are broadly relevant to the fields of cancer biology, microbiology and immunology. A perspective on future research and applications is provided.

A soft Tus-Ter interaction is hiding a fail-safe lock in the replication fork trap of Dickeya paradisiaca.

A variety of replication fork traps have recently been characterised in Enterobacterales, unveiling two different types of architecture. Of these, the degenerate type II fork traps are commonly found in Enterobacteriaceae such as Escherichia coli. The newly characterised type I fork traps are found almost exclusively outside Enterobacteriaceae within Enterobacterales and include several archetypes of possible ancestral architectures. Dickeya paradisiaca harbours a somewhat degenerate type I fork trap with a unique Ter1 adjacent to tus gene on one side of the circular chromosome and three putative Ter2-4 sites on the other side of the fork trap. The two innermost Ter1 and Ter2 sites are only separated by 18 kb, which is the shortest distance between two innermost Ter sites of any chromosomal fork trap identified so far. Of note, the dif site is located between these two sites, coinciding with a sharp GC-skew flip. Here we examined and compared the binding modalities of E. coli and D. paradisiaca Tus proteins for these Ter sites. Surprisingly, while Ter1-3 were functional, no significant Tus binding was observed for Ter4 even in low salt conditions, which is in stark contrast with the significant non-specific protein-DNA interactions that occur with E. coli Tus. Even more surprising was the finding that D. paradisiaca Tus has a relatively moderate binding affinity to double-stranded Ter while retaining an extremely high affinity to Ter-lock sequences. Our data revealed major differences in the salt resistance and stability between the D. paradisiaca and E. coli Tus protein complexes, suggesting that while Tus protein evolution can be quite flexible regarding the initial Ter binding step, it requires a highly stringent purifying selection for its final locked complex formation.

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

IgG-detection devices for the Tus-Ter-lock immuno-PCR diagnostic platform.

The number of new Immuno-PCR technologies and applications is steadily growing as a result of a general need for more sensitive immunoassays for early detection of diseases. Although Immuno-PCR has been demonstrated to be superior to its immunoassay counterpart, it is still regarded as a challenging technology due to various problems arising from its increased detection power, such as high background noise as well as substantial batch-to-batch reproducibility issues. Current efforts have intensified to produce homogeneous universal protein-DNA conjugates to simplify this technology and render it more robust. We have recently developed a new quantitative Immuno-PCR (qIPCR) technology using the Tus-Ter-lock (TT-lock) interaction to produce homogeneous protein-DNA conjugates that can detect very small numbers of disease-related antibodies. We now report the further development of the TT-lock Immuno-PCR platform for the quasi universal quantitative detection of antigens and mammalian IgG. For this, Tus was fused to various IgG-binding proteins--i.e. protein G, protein L and their LG chimera--and self-assembled to the TT-lock-T template. These detection devices were then evaluated and applied in various direct and indirect Immuno-PCR formats. The direct TT-lock qIPCR could detect goat anti-GFP IgG at concentrations as low as 0.3 pM and total human IgG in serum samples with great sensitivity. Further indirect TT-lock qIPCR systems were developed that could detect 1 pM of GFP and 10 pM of measles nucleoprotein. In all cases, the superiority of the TT-lock Immuno-PCR was demonstrated in terms of sensitivity over an analogous Protein G-Peroxidase ELISA.

High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins.

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

Electrophoretic Mobility Shift Assays with GFP-Tagged Proteins (GFP-EMSA).

The electrophoretic mobility shift assay (EMSA) is commonly used for the study of nucleic acid-binding proteins. The technique can be used to demonstrate that a protein is binding to RNA or DNA through visualization of a shift in electrophoretic mobility of the nucleic acid band. A major disadvantage of the EMSA is that it does not always provide an absolute certitude that the band shift is due to the protein under scrutiny, as contaminants in the sample could also cause the band shift. Here we describe a variation of the standard EMSA allowing to visualize with added certitude, the co-localized band shifts of a GFP-tagged protein binding to its cognate nucleic acid target sequence stained with an intercalator, such as GelRed. Herein, we present an illustrative protocol of this useful technique called GFP-EMSA along with specific notes on its advantages and limitations.

Site-specific covalent attachment of DNA to proteins using a photoactivatable Tus-Ter complex.

Investigations into the photocrosslinking kinetics of the protein Tus with various bromodeoxyuridine-substituted Ter DNA variants highlight the potential use of this complex as a photoactivatable connector between proteins of interest and specific DNA sequences.

Defining specific allergens for improved component-resolved diagnosis of shrimp allergy in adults.

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy.

Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis.

The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.