Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Bioluminescence, the light produced by biochemical reactions involving luciferases in living organisms, has been extensively investigated for various applications. It has attracted particular interest as an internal light source for theranostic applications due to its safe and efficient characteristics that overcome the limited penetration of conventional external light sources. Recent advancements in protein engineering technologies and protein delivery platforms have expanded the application of bioluminescence to a wide range of theranostic areas, including bioimaging, biosensing, photodynamic therapy, and optogenetics. This comprehensive review presents the fundamental concepts of bioluminescence and explores its recent applications across diverse fields. Moreover, it discusses future research directions based on the current status of bioluminescent systems for further expansion of their potential.

Quorum sensing (QS) allows bacteria to coordinate their activities by producing and detecting low-molecular-weight signal molecules based on population density, thereby controlling the infectivity of bacteria through various virulence factors. Quorum-sensing inhibition is a promising approach to tackle bacterial communication. Cyclodextrins (CDs) are a class of cyclic oligosaccharides that reversibly encapsulate the acyl chain of the signal molecules, thereby preventing their binding to receptors and interrupting bacterial communication. This results in the inhibition of the expression of various properties, including different virulence factors. To examine the potential quorum-quenching (QQ) ability of newly prepared cyclodextrin derivatives, we conducted short-term tests using Aliivibrio fischeri, a heterotrophic marine bacterium capable of bioluminescence controlled by quorum sensing. α- and β-cyclodextrins monosubstituted with alkylthio moieties and further derivatized with quaternary ammonium groups were used as the test agents. The effect of these cyclodextrins on the quorum-sensing system of A. fischeri was investigated by adding them to an exponential growth phase of the culture and then measuring bioluminescence intensity, population growth, and cell viability. Our results demonstrate that the tested cyclodextrins have an inhibitory effect on the quorum-sensing system of A. fischeri. The inhibitory effect varies based on the length of the alkyl chain, with alkylthio substitution enhancing it and the presence of quaternary ammonium groups decreasing it. Our findings suggest that cyclodextrins can be a promising therapeutic agent for the treatment of bacterial infections.

BACKGROUND: The light organs of the splitfin flashlight fish Anomalops katoptron are necessary for schooling behavior, to determine nearest neighbor distance, and to feed on zooplankton under dim light conditions. Each behavior is coupled to context-dependent blink frequencies and can be regulated via mechanical occlusion of light organs. During shoaling in the laboratory individuals show moderate blink frequencies around 100 blinks per minute. In this study, we correlated bioluminescent blinks with the spatio-temporal dynamics of swimming profiles in three dimensions, using a stereoscopic, infrared camera system.
RESULTS: Groups of flashlight fish showed intermediate levels of polarization and distances to the group centroid. Individuals showed higher swimming speeds and curved swimming profiles during light organ occlusion. The largest changes in swimming direction occurred when darkening the light organs. Before A. katoptron exposed light organs again, they adapted a nearly straight movement direction.
CONCLUSIONS: We conclude that a change in movement direction coupled to light organ occlusion in A. katoptron is an important behavioral trait in shoaling of flashlight fish.

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function, but remains a challenging pursuit. Existing methods are mostly limited to static readouts or difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2- specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernable in both 2D and 3D formats with single cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.

The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype-the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.

Bioluminescence is light chemically produced by an organism. It is widespread across all major marine phyla and has evolved multiple times, resulting in a high diversity of spectral properties and first flash kinetic parameters (FFKP). The bioluminescence of a system is often a good proxy for planktonic biomass. The species-specific parameters of bioluminescent displays can be measured to identify species in situ and describe planktonic biodiversity. Most bioluminescent organisms will flash when mechanically stimulated i.e., when subjected to supra-threshold levels of shear stress. Here we compare first flash kinetic parameters such as flash duration, peak intensity, rise time, decay time, first-flash mechanically stimulated light and e-folding time obtained with the commercially available Underwater Bioluminescence Assessment Tool (UBAT). We provide descriptions of the first flash kinetic parameters of several species of dinoflagellates Pyrocystis fusiformis, Pyrocystis noctiluca, Pyrodinium bahamense, Lingulodinium polyedra, Alexandrium monilatum and two zooplankton (the ctenophore Mnemiopsis leidyi and the larvacean Oikopleura sp.). FFKPs are then compared and discussed using non-parametric analyses of variance (ANOVAs), hierarchical clustering and a linear discriminant analysis to assess the ability to use bioluminescence signatures for identification. Once the first flash kinetic parameters of a bioluminescent species have been described, it is possible to detect its presence using emissions collected by in situ bathyphotometers. Assessing abundance and diversity of bioluminescent species may therefore be possible.

BACKGROUND: Plants are known to be infected by a wide range of pathogenic microbes. To study plant diseases caused by microbes, it is imperative to be able to monitor disease symptoms and microbial colonization in a quantitative and objective manner. In contrast to more traditional measures that use manual assignments of disease categories, image processing provides a more accurate and objective quantification of plant disease symptoms. Besides monitoring disease symptoms, computational image processing provides additional information on the spatial localization of pathogenic microbes in different plant tissues.
RESULTS: Here we report on an image analysis tool called ScAnalyzer to monitor disease symptoms and bacterial spread in Arabidopsis thaliana leaves. Thereto, detached leaves are assembled in a grid and scanned, which enables automated separation of individual samples. A pixel color threshold is used to segment healthy (green) from chlorotic (yellow) leaf areas. The spread of luminescence-tagged bacteria is monitored via light-sensitive films, which are processed in a similar manner as the leaf scans. We show that this tool is able to capture previously identified differences in susceptibility of the model plant A. thaliana to the bacterial pathogen Xanthomonas campestris pv. campestris. Moreover, we show that the ScAnalyzer pipeline provides a more detailed assessment of bacterial spread within plant leaves than previously used methods. Finally, by combining the disease symptom values with bacterial spread values from the same leaves, we show that bacterial spread precedes visual disease symptoms.
CONCLUSIONS: Taken together, we present an automated script to monitor plant disease symptoms and microbial spread in A. thaliana leaves. The freely available software ( https://github.com/MolPlantPathology/ScAnalyzer ) has the potential to standardize the analysis of disease assays between different groups.

Quorum sensing inhibitors (QSIs), as a kind of ideal antibiotic substitutes, have been recommended to be used in combination with traditional antibiotics in medical and aquaculture fields. Due to the co-existence of QSIs and antibiotics in environmental media, it is necessary to evaluate their joint risk. However, there is little information about the acute toxicity of mixtures for QSIs and antibiotics. In this study, 10 QSIs and 3 sulfonamides (SAs, as the representatives for traditional antibiotics) were selected as the test chemicals, and their acute toxic effects were determined using the bioluminescence of Aliivibrio fischeri (A. fischeri) as the endpoint. The results indicated that SAs and QSIs all induced S-shaped dose-responses in A. fischeri bioluminescence. Furthermore, SAs possessed greater acute toxicity than QSIs, and luciferase (Luc) might be the target protein of test chemicals. Based on the median effective concentration (EC50) for each test chemical, QSI-SA mixtures were designed according to equitoxic (EC50(QSI):EC50(SA) = 1:1) and non-equitoxic ratios (EC50(QSI):EC50(SA) = 1:10, 1:5, 1:0.2, and 1:0.1). It could be observed that with the increase of QSI proportion, the acute toxicity of QSI-SA mixtures enhanced while the corresponding TU values decreased. Furthermore, QSIs contributed more to the acute toxicity of test binary mixtures. The joint toxic actions of QSIs and SAs were synergism for 23 mixtures, antagonism for 12 mixtures, and addition for 1 mixture. Quantitative structure-activity relationship (QSAR) models for the acute toxicity QSIs, SAs, and their binary mixtures were then constructed based on the lowest CDOCKER interaction energy (Ebind-Luc) between Luc and each chemical and the component proportion in the mixture. These models exhibited good robustness and predictive ability in evaluating the toxicity data and joint toxic actions of QSIs and SAs. This study provides reference data and applicable QSAR models for the environmental risk assessment of QSIs, and gives a new perspective for exploring the joint effects of QSI-antibiotic mixtures.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.