Past TRAC Grant Recipients

In TB disease, the shortest duration of currently available standard therapies is 4-6 months, and even with this duration of therapy there remains a 5% risk of relapse. For the bacillus, causing relapse requires survival upon exposure to antibiotics, followed by replication to a degree that causes disease. Understanding how one bacillus may be able to survive and replicate while another cannot, despite being genetically identical, can reveal bacterial vulnerabilities and lead to new therapies. Mtb is well known to enter diverse phenotypic states that impact drug susceptibility, growth rate and cultivability requirements. However, the dynamic changes among these heterogeneous phenotypes within a bulk population after exposure to stress has not been fully characterized, in part due to the technical challenges in studying distinct subpopulations within a bulk culture. This proposal overcomes barriers to observing the transcriptional signatures of subpopulations over time by applying a method of single-cell RNA-sequencing (sc-RNAseq) to mycobacteria that has been under development in my lab with the support of, and in collaboration with, the Kyu Rhee and Carl Nathan labs. The technology can interrogate the individual transcriptomes of 10,000 Mtb simultaneously without the need for single cell isolation, allowing for unbiased discovery and characterization of minority phenotypes present in a bulk culture. To our knowledge, this approach has not been attempted in mycobacteria. To monitor transcripts that are associated with antibiotic survival and replication dynamics, we will apply the sc- RNAseq method to Mtb containing a plasmid that serves as a marker of replication. The plasmid, which encodes a kanamycin resistance marker, is unstable and steadily lost as the bacilli divide—therefore plasmid loss is proportional to growth rate. We will expose Mtb with this replication clock to growth restrictive concentrations of the first-line TB drugs rifampin and isoniazid, and then wash out the drug and place the survivors in either fresh liquid or solid media with and without kanamycin. Samples will be processed for sc- RNAseq before, during, and after antibiotic exposure. The study will define which transcriptomes are enriched upon antibiotic exposure as well as the complexity and composition of the population structure as compared with non-antibiotic exposed cells. It will then examine the resultant heterogeneity in growth dynamics of antibiotic-surviving subpopulations by analyzing transcripts that correlate with retention or loss of the replication clock plasmid over time. We will also track whether transcriptomic signatures enriched among survivor groups persist over generations, as semi-heritable drug resistance has been described in Mtb.

TB/HIV co-infection remains an underestimated epidemic, spreading faster than our ability to detect and manage it, and tuberculosis (TB) remains the leading cause of death in people living with HIV/AIDS (PLWHA) 1,2. Yet approximately half of TB cases in this population remain undiagnosed. The challenges to diagnosing TB in PLWHA are many, including atypical pulmonary presentations, higher incidence rates of extra-pulmonary TB, and higher rates of sputum smear negativity by microscopy and culture due to the immunosuppression caused by HIV. We propose to remedy this situation by applying an innovative metabolomic approach using high- performance liquid chromatography coupled with mass spectrometry (LC-MS) to discover urinary metabolites specifically diagnostic of active TB infection in this population. Urine-based biomarkers have recently begun to emerge as clinically useful diagnostic markers of infectious disease and prognostic markers of treatment efficacy. Yet, there are a surprisingly small number of metabolomic studies of TB biomarkers and an even smaller number that utilize urine, which is an ideal biofluid for point-of-care tests due to its generally abundant and non-invasive nature. We propose to analyze an already existing biobank of urine samples prospectively collected from patients included in the SDART-TB trial ("A Trial of Same-Day Testing and Treatment to Improve Outcomes Among Symptomatic Patients Newly Diagnosed With HIV", NCT03154320). This study was conducted by Serena Patricia Koenig, Brigham and Women's Hospital, in partnership with the Center for Global Health, WCM, and other collaborators in Haiti. It included 500 newly diagnosed patients with HIV and symptoms of TB recruited between 2017 and 2019, of which 460 had urine collected at the time of enrollment and were shipped to the Belfer Research Building, WCM, where they are currently stored at -80°C. We propose to work with 2/3 (304) of the original cohort, of which approximately 1⁄4 (76) were diagnosed with pulmonary TB, to achieve a 3:1 ratio of age- and gendered-matched controls for every case. Previous work from our group identified and validated biomarkers that are increased in pulmonary drug-susceptible TB cases in HIV-negative patients and decreased with effective anti-TB treatment3,4. We now propose to build on these results by validating these previously identified biomarkers in PLHWA co-infected with pulmonary TB or potentially discovering additional biomarkers specific to the HIV+ population. Moreover, we believe we will be able to identify biomarkers that distinguish patients with TB in PLWHA from patients with non-TB pulmonary illness in PLWHA. PLWHA are disproportionally affected by TB and thus urgently need new diagnostic tools.