Role of redox-sensitive transcription factors in Mtb pathogenesis: Mycobacterium tuberculosis (Mtb) is an extremely successful pathogen due to its ability to persist, and to latently infect more than two billion people worldwide. How Mtb can persist in human tissues for decades without replicating in a state of “drug unresponsiveness” wherein the bacilli is resistant to current antimycobacterial agents is a fundamental question in the TB field. Redox signals such as nitric oxide (NO) and oxygen (O2) have been proposed to be main signals that induce a change in the metabolism of Mtb to facilitate its entry into and emergence from dormancy. However, the identity of a sensor that precisely monitors Mtb growth, metabolism and cell division in response to redox stress remains unknown, and would be a significant contribution to the TB field. Based on our preliminary data, we hypothesized that Mtb WhiB4 is a redox-dependent transcription factor that facilitates physiological adaptation of Mtb required for its entry, maintenance, and emergence from a dormant state. From the past few years we have obtained significant insight on the function of WhiB4. We showed that WhiB4 is a main regulator of oxidative stress responsive pathways in Mtb. Using multiple analytical techniques such as atomic force microscopy (AFM) and confocal microscopy we demonstrated that WhiB4 regulates expression by binding non-specifically to the DNA and may function as a nucleoid associated protein in Mtb. Experiments using animal models of TB (e.g mice and guinea pigs) are ongoing to decipher the role of WhiB4 in the pathogenesis of Mtb.
Understanding drug tolerance mechanisms in Mtb: Why do we need such prolonged therapy for TB? It is believed that long term multiple drug therapy is required to eliminate a small sub-population of Mtb which is refractory to current anti-TB drugs. Such noncompliant bacteria are referred to as “persisters” and the phenomenon is known as “persistence”. Persisters are genetically similar to their drug susceptible counterparts but are able to survive the lethal effects of antibiotics, indicating that they might be physiologically different. Therefore, understanding the physiological state of drug-tolerant persisters is one of the foremost challenges in shortening current drug regimens, and developing new drugs or diagnostics against TB. Despite their clinical importance, research in this field is hampered due to the lack of innovative technologies to capture phenotypic and/or physiological diversity within Mtb population during infection. This represents a major technological gap in our understanding of TB disease and drug-resistance.
To figure out the fundamental physiological differences between individual Mtb cells in a population during infection, our team has created mycobacteria engineered with a genetic sequence encoding modified redox sensitive green fluorescent protein (Mrx1-roGFP2) that senses changes in the levels and reduction-oxidation state of mycobacteria-specific antioxidant, mycothiol. The Mrx1-roGFP2 biosensor was constructed by genetically coupling an Mtb mycothiol-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2). The redox circuit thus created allowed continuous equilibration of the biosensor with the mycothiol redox couple (MSH/MSSM) in vitro and inside Mtb. Any changes in the mycothiol status inside mycobacterial cells will be rapidly captured by simply measuring the ratiometric fluorescence of Mrx1-roGFP2 at 405 and 488 nm excitation wavelengths (405/488) and a fixed emission at 510 nm. Using this tool, we show that Mtb cells that are genetically identical differ physiologically at the level of mycothiol inside macrophages. To investigate if heterogeneity in mycothiol redox potential of Mtb at a single cell level contributes to emergence of drug tolerance during infection, we first analyzed the effect of anti-TB drugs on Mtb antioxidant levels. Interestingly, we discovered that the mode of action of current anti-TB drugs differ if bacteria are grown in test tubes than inside macrophages. While most of the frontline anti-TB drugs deplete antioxidant mycothiol to kill Mtb specifically inside macrophages, in test tubes these drugs do not influence mycothiol levels and have entirely different mechanisms of killing. Our results suggest that macrophage environment cooperates with antibiotics to efficiently kill Mtb by depleting mycothiol levels during the natural course of chemotherapy. Lastly, we show that a small subset of bacteria with higher antioxidant levels survives upon exposure to antibiotics. For the first time, we were able to differentiate drug-sensitive Mtb cells from drug-resistant persisters by providing a unique antioxidant barcode to each bacterial cell. The implications of our findings are immense ranging from development of redox-based TB diagnostics/biomarkers to innovative screening approaches for identifying redox-oriented anti-mycobacterial compounds. This bio-tool is currently being used in the lab to investigate various facets of mycobacterial redox behavior during infection.
Mechanisms of HIV-1 latency and reactivation: One of the unique features of the AIDS virus, HIV-1, is that it can exist inside human cells for years without causing any harm. It then reactivates to cause infection when conditions are suitable. We have exploited a non-invasive biosensor that can measure what is going on within HIV-1 infected cells in real-time. This technology led us to carefully manipulate antioxidant levels (glutathione) of HIV-1 infected cell to either keep virus in a sleeping mode or trigger its reactivation. This may allow researchers to adopt a “shock-and-kill” strategy in which virus could be reactivated by mild oxidants and subsequently flushed by current anti-HIV drugs. Taken together, we use unconventional approaches to understand the molecular basis of human diseases and we hope that our work will provide new insights on host-pathogen interactions.