Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice

Abstract

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 ± 2 × 10 2 colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10 4 CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus CalmetteGuérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.infection | pulmonary | Mycobacterium | fluorogenic | beta-lactamase

Bacteria-Targeting Conjugates Based on Antimicrobial Peptide for Bacteria Diagnosis and Therapy

“…Determination of Minimum InhibitoryConcentrations of CAP-UBI[29][30][31][32][33][34][35][36][37][38][39][40][41] …”

An antimycobacterial pleuromutilin analogue effective against dormant bacilli

“…Murine macrophage cells (J774A.1) infected by the transformant Mtb CDC1551 containing tdTomato (MOI = 10) were treated with 1 (at 2xMIC). 35 After 24, 48, 72, and 96 h of incubation, the relative intensity of the fluorescence was measured (emission wavelength (581 nm)) via fluorescence spectroscopy (Figure 5a). Alternatively, the lysates were ten-fold serially diluted in 7H10-S broth and inoculated on 7H11-S plates to determine the number of viable Mtb cells to confirm the bactericidal effect of 1 in 0-72h (Figure 5b).…”

Genes and regulatory networks involved in persistence of Mycobacterium tuberculosis

“…Reporter enzyme fluorescence (REF) allows real-time imaging of pulmonary infections and rapid quantification of M. tuberculosis in living animals. This technology is based on near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by M. tuberculosis, but not by their eukaryotic hosts [71]. Gene regulatory network study is an emerging area for which various models and computational tools are now available.…”