Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

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.

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.

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.

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.

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.

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.

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase.

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔTm=+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (Kobs=7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns.

Functional characterisation of Burkholderia pseudomallei biotin protein ligase: A toolkit for anti-melioidosis drug development.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopenicillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets. BirA within the Burkholderia genus have not yet been investigated. We present for the first time the cloning, expression, purification and functional characterisation of the putative Bp BirA and orthologous B. thailandensis (Bt) biotin carboxyl carrier protein (BCCP) substrate. A GFP-tagged Bp BirA was produced and applied for the development of a high-throughput (HT) assay based on our differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) principle as well as an electrophoretic mobility shift assay. Our biochemical data in combination with the new HT DSF-GTP and biotinylation activity assay could facilitate future drug screening efforts against this drug-resistant organism.

The Impact of Prior Flavivirus Infections on the Development of Type 2 Diabetes Among the Indigenous Australians.

It is estimated that 5% of Australians over the age of 18 have diabetes, with the number of new cases increasing every year. Type 2 diabetes (T2D) also represents a significant disease burden in the Australian indigenous population, where prevalence is three times greater than that of non-indigenous Australians. Prevalence of T2D has been found to be higher in rural and remote indigenous Australian populations compared with urban indigenous Australian populations. Several studies have also found that body mass index and waist circumference are not appropriate for the prediction of T2D risk in indigenous Australians. Regional and remote areas of Australia are endemic for a variety of mosquito-borne flaviviruses. Studies that have investigated seroprevalence of flaviviruses in remote aboriginal communities have found high proportions of seroconversion. The family Flaviviridae comprises several genera of viruses with non-segmented single-stranded positive sense RNA genomes, and includes the flaviviruses and hepaciviruses. Hepatitis C virus (HCV) has been shown to be associated with insulin resistance and subsequent development of T2D. Flaviviruses and HCV possess conserved proteins and subgenomic RNA structures that may play similar roles in the development of insulin resistance. Although dietary and lifestyle factors are associated with increased risk of developing T2D, the impact of infectious diseases such as arboviruses has not been assessed. Flaviviruses circulating in indigenous Australian communities may play a significant role in inducing glucose intolerance and exacerbating T2D.

Rapid diagnostics for melioidosis: a comparative study of a novel lateral flow antigen detection assay.

The rapid diagnosis of septicaemic melioidosis will have an impact on reduction of mortality. Currently, this relies almost exclusively upon culture of the causative agent Burkholderia pseudomallei from clinical samples. In acute sepsis, blood is the preferred specimen for culture and therefore should be the target for a rapid diagnostic tool. A lateral flow immunoassay (LFI) for the detection of B. pseudomallei antigen has been developed. This was compared with molecular detection using the targets T3SS1 and IpxO. Forty-five clinical samples of EDTA blood, which were culture-positive, were tested using both modalities. The LFI had a sensitivity of 40 %, whilst molecular detection had a sensitivity of 20 %. The poor performance of molecular detection has been described previously and is largely related to the use of whole-blood specimens collected into blood tubes containing EDTA. Whilst suboptimal, the LFI would be an adjunct in the rapid diagnosis of melioidosis.

Improved diagnosis of melioidosis using a 2-dimensional immunoarray.

Melioidosis is caused by the Gram negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days obtaining a result, hindering successful treatment of acute disease. The existing indirect haemagglutination assay (IHA) has several disadvantages, in that approximately half of patients later confirmed culture positive are not diagnosed at presentation and a subset of patients are persistently seronegative. We have developed 2 serological assays, an enzyme-linked immunosorbent assay (ELISA), and a 2-dimensional immunoarray (2DIA), capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The 2DIA format can distinguish between different LPS serotypes. Currently, the 2DIA has a sensitivity and specificity of 100% and 87.1%, respectively, with 100% of culture-positive, IHA-negative samples detected. The ELISA has a sensitivity and specificity of 86.2% and 93.5%, respectively, detecting 67% of culture-positive, IHA-negative samples. The ELISA and 2DIA tests described here are more rapid and reliable for serological testing compared to the existing IHA.