Maki SHOJIRO (牧 昌次郎)

Abstract

Electronic absorption and fluorescence properties of a red‐emission firefly luciferin analog, seMpai, which is water‐soluble and shows a neutral pH, were revealed by quantitative spectroscopic measurements in aqueous solutions of pH 2–10. They were analyzed by density functional theory (DFT) calculations, time‐dependent DFT calculations, and vibrational analyses using the neutral form of seMpai and its conjugate acids and bases. In a pH 8 solution, which is commonly used for firefly bioluminescence, seMpai showed an absorption maximum at 380 nm. From the theoretical absorption spectra and pH dependence of the normalized concentrations of the neutral form of seMpai and its conjugate acids and bases, it was found that the most abundant chemical species at pH 8 is the carboxylate anion, and the 380‐nm absorption band was assigned as the ‐* transition of this anion. The fluorescence spectrum of seMpai in the pH 8 aqueous solution showed an emission maximum at 534 nm for 380‐nm excitation. According to the theoretical pH dependence of the normalized concentrations and theoretical absorption and fluorescence energies of the chemical forms of seMpai, the most reasonable fluorescence pathway at pH 8 is the emission from the first excited singlet state S₁ of the carboxylate anion through its excitation.

Coelenterazine (CTZ) is a common substrate of marine luciferases upon emission of bioluminescence (BL) in living organisms. Because CTZ works as a “luminophore” in the process of BL emission, the chemical modification has been centered for improving the optical properties of BL. In this review, we showcase recent advances in CTZ designs with unique functionalities. We first elucidate the light-emitting mechanisms of CTZ, and then focus on how the rational modification of CTZ analogs developed in recent years are connected to the development of unique functionalities even without luciferases, which include color tunability covering the visible region, specificity to various proteins (e.g., luciferase, albumin, and virus protein), and activatability to ions or reactive oxygen species (ROS) and anticancer drugs. This review provides new insights into the broad utilities of CTZ analogs with designed functionalities in bioassays and molecular imaging.

ABSTRACT

In vivo imaging of bacterial infection models enables noninvasive and temporal analysis of individuals, enhancing our understanding of infectious disease pathogenesis. Conventional in vivo imaging methods for bacterial infection models involve the insertion of the bacterial luciferase LuxCDABE into the bacterial genome, followed by imaging using an expensive ultrasensitive charge-coupled device (CCD) camera. However, issues such as limited light penetration into the body and lack of versatility have been encountered. We focused on near-infrared (NIR) light, which penetrates the body effectively, and attempted to establish an in vivo imaging method to evaluate the number of lung-colonizing bacteria during the course of bacterial pneumonia. This was achieved by employing a novel versatile system that combines plasmid-expressing firefly luciferase bacteria, NIR substrate, and an inexpensive, scientific complementary metal-oxide semiconductor (sCMOS) camera. The D-luciferin derivative “TokeOni,” capable of emitting NIR bioluminescence, was utilized in a mouse lung infection model of Acinetobacter baumannii , an opportunistic pathogen that causes pneumonia and is a concern due to drug resistance. TokeOni exhibited the highest sensitivity in detecting bacteria colonizing the mouse lungs compared with other detection systems such as LuxCDABE, enabling the monitoring of changes in bacterial numbers over time and the assessment of antimicrobial agent efficacy. Additionally, it was effective in detecting A. baumannii clinical isolates and Klebsiella pneumoniae . The results of this study are expected to be used in the analysis of animal models of infectious diseases for assessing the efficacy of therapeutic agents and understanding disease pathogenesis.

IMPORTANCE

Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although in vivo imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile in vivo pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.