Mtb-Timer: a fluorescent reporter to visualizereplication and antibiotic responses.

UNLABELLED: Despite the importance of monitoring(Mtb) replication and antibiotic responses at the single-cell level, available tools to accurately monitor single Mtb replication are limited. Here, we report the development of Mtb-Timer, a fluorescent reporter in Mtb that exploits the intrinsic green-to-red photoconversion of the Timer protein as a proxy for Mtb replication. We generated an Mtb H37Rv strain expressing Timer (Timer) and monitoredMtb replication at the single-cell level by imaging and flow cytometry. Moreover, we show that exposure to the first-line anti-tuberculosis antibiotics isoniazid, rifampicin, ethambutol, and pyrazinamide resulted in a dose-dependent green-to-red photoconversion by imaging and flow cytometry. When we tested Mtb-Timerusing primary human monocyte-derived macrophages, we observed that intracellular Mtb populations displayed a striking phenotypic heterogeneity. Single-cell Mtb-Timer green-to-red ratios analyzed by both fixed and live imaging revealed intracellular Mtb populations with high green-to-red ratios. In contrast, treatment with antibiotics showed a decrease in green-to-red ratios in a dose-dependent manner by imaging and flow cytometry. Collectively, these results establish Mtb-Timer as a robust tool for monitoring Mtb replication and antibiotic responses, bothand, by imaging and flow cytometry.

IMPORTANCE: Tuberculosis treatment is challenging in part because(Mtb) populations are phenotypically heterogeneous. Within the same infection, bacterial cells can exist in different physiological states, from actively replicating to slow-growing or non-replicating forms. To better study these processes, we developed and validated Mtb-Timer, a fluorescent reporter that enables visualization of bacterial growth dynamics. The system is based on a fluorescent protein that changes color over time: newly synthesized protein emits green fluorescence (GFP) and gradually shifts to red (DsRed) as it matures. The green-to-red fluorescence ratio, therefore, reflects bacterial replication activity. Using microscopy and flow cytometry in bothcultures and infected macrophages (), Mtb-Timer enables the quantification of distinct bacterial subpopulations and the real-time monitoring of the green-to-red fluorescence ratio and bacterial burden during treatment with first-line antibiotics.