Ultrasensitive detection of Mycobacterium tuberculosis infection

PROJECT SUMMARY Tuberculosis (TB) afflicts nearly one-third of the world’s population, but progress in development of interventions is hamstrung by the slow growth rate of Mycobacterium tuberculosis (Mtb), the causative agent, and the need to evaluate viability by colony forming units (cfu). We plan to overcome this roadblock through development and validation of imaging technologies that can be broadly applied to all TB research. Our studies have produced new imaging technologies for TB, with the most sensitive having a threshold of ~103 cfu during pulmonary infection. Imaging allows therapeutic evaluation within hours, rather than weeks, both in vitro and in animals, but improved sensitivity would have a transformative effect on the TB field. We have demonstrated the ability of imaging to quantify and track infection in nearly any tissue if the bacteria are at high enough levels, but the thresholds of detection have not been low enough to ensure relevant infectious doses can be detected in deep tissues. The proposed project would significantly increase sensitivity, offering the unique opportunity to track infection, vaccines, therapeutics and challenge doses for Mtb in real-time at physiologically relevant bacterial numbers. We will build upon our earlier studies by improving and validating the ability of REF imaging to sensitively track the bacteria, evaluate efficacy of therapeutics, determine bacterial loads in nearly any tissue and the ability to rapidly determine vaccine efficacy. Specifically, we propose to: 1) Develop new imaging technologies for Mtb. Our working hypothesis is that optimized imaging technologies can allow detection of single bacteria in vitro and in vivo. Our preliminary data demonstrated that we can improve imaging sensitivity and specificity through modifications of REF and SREL substrates and application of microendoscopy. In this aim we will examine novel substrates and modified microendoscopy systems for improving the threshold of detection and monitoring Mtb in vitro and in vivo. 2) Improve imaging technologies to transform Mtb pathogenesis studies. Our working hypothesis is that the ability of imaging to track Mtb in all organs during infections will give new insight into pathogenesis. Our preliminary data suggest imaging with new reporters (NanoLuc/NanoBRET) could allow more sensitive localization during dissemination from the lungs. In this aim we will validate these observations by quantifying bacteria in lungs and other organs by imaging, validate dissemination and analysis of our existing mutants. 3) Facilitate therapeutic analysis and vaccine efficacy using imaging. Our working hypothesis is that imaging will allow us to evaluate TB therapeutics and vaccines more rapidly. Our preliminary studies suggest that new probes, markers and microendoscopy systems can be used to follow vaccines, challenge doses and impact of therapeutics by imaging, providing immediate feedback on efficacy of new treatments. In this aim we will utilize reporter vaccines and Mtb strains to analyze vaccines and therapeutics in mice to more rapidly evaluate efficacy and compare the data obtained to conventional methods.