2024 Recipients
View Research Summary
Prabhat Ranjan Singh, Ph.D.
Examining the function of Mycobacterium tuberculosis genes to understand the survival mechanism in macrophages using a lung environment model
One of the key reasons for Mycobacterium tuberculosis (Mtb) success as a pathogen is its numerous virulence factors. These factors modulate the host immune response, allowing the bacteria to evade host phagocytes, delay the onset of adaptive immunity, and cause the chronic inflammation and immunopathology characteristic of tuberculosis. Considerable research has been conducted to understand the early interactions between macrophages and Mtb, typically using log- phase Mtb, which does not replicate the sequential stress model of transmission. In our recently developed in vitro model, which mimics the sequential stress conditions of transmission (unpublished), we introduced Model Aerosol Fluid (MAF) and Model Alveolar Lining Fluid (MALF). These represent initial steps in recreating the composition of caseous necrotic material within cavities and the fluid present in alveoli, respectively. Our preliminary CRISPRi screen data from this sequential model identified 35 gene hits relevant to the aerosolization-to-inhalation stage. We are now well-positioned to examine these hits in early-phase interactions with macrophages.To further enhance this model, I propose adding an additional stage to simulate the early phase of Mtb infection using an air-liquid interface co-culture system that mimics the lung environment. This system will incorporate the bronchial epithelial cell line NuLi-1 and the alveolar macrophage cell line AMJ2-C11. I will examine these hits in early-phase interactions by evaluating the survival phenotypes of individual Mtb gene knockout/knockdown strains in a lung environment model and assessing the macrophage response triggered by these Mtb strains.We expect to discover previously unnoticed mechanisms, given that existing models do not faithfully depict the unexplored physiological state of Mtb within its host.
View Research Summary
Valerie Vinette, Ph.D.
Identification of host TNF-α-dependent pathways involved in tuberculosis relapse
Infection with Mtb most frequently results in latent tuberculosis infection (LTBI) due to a healthy immune system that restricts bacterial replication and prevents disease. Macrophages are the predominant host reservoir of Mtb and as such, elucidating the mechanisms by which they are activated and modulated to impede Mtb growth and survival is vital in understanding the course of TB reactivation and relapse. It has been shown that interstitial macrophages (IMs) residing in the lung are important in restricting Mtb in an acute model of TB infection. TNF-ɑ is a pro-inflammatory cytokine that is a major activator of macrophage recruitment and is required for full microbicidal activity of mycobacteria-infected macrophages. It is known to be important in the protection against infection and reactivation of TB in humans, non-human primates and mice. However, its role in modulating the response of macrophages to Mtb during LTBI has yet to be elucidated. We have recently shown in a chronic mouse model of LTBI that IMs are essential in hindering the reactivation of latent Mtb to prevent disease. Knowing the importance of TNF-ɑ signaling in macrophage activation and TB disease, my goal is to use our paucibacillary mouse model of chronic LTBI to investigate its role on TB reactivation. I aim to first establish the role of TNF-ɑ on tuberculosis relapse (Aim 1). I will then perform single-cell RNA sequencing upon depletion of TNF-ɑ during latency to establish the immune cell landscape and immunometabolic reprogramming upon TNF-ɑ depletion (Aim 2), and to identify a TNF-ɑ-driven molecular signature that is associated with TB relapse (Aim 3). The proposed research program strives to understand these processes in an effort to reduce the reactivation of latent tuberculosis disease in patients undergoing anti-TNF-ɑ chemotherapy.
View Research Summary
Ekaterina Vinogradova, Ph.D.
Characterization of MtB protein landscapes using advanced proteomic platforms
Tuberculosis is the global leading cause of death by infectious disease. The causative bacterium, Mycobacterium tuberculosis (Mtb), is intrinsically resistant to many antibiotics and rapidly develops resistance to new drugs. Drug discovery efforts are hampered by our limited understanding of Mtb biology, with much of its proteome still poorly characterized. Here, we aim to leverage advanced chemical proteomic platforms developed in our lab to address this problem in collaboration with the Rock lab at the Rockefeller University. This approach profiles proteome-wide cysteine reactivity and ligandability by small-molecule electrophiles and has proven powerful in mammalian immunology. By adapting the platform to Mtb for the first time, we can both improve our understanding of mycobacterial proteomics and identify novel putative druggable targets.
View Research Summary
Vijay Soni, Ph.D.
Study of peptidoglycan salvage and recycling pathways and their role in the evolution of antimicrobial resistance in Mycobacterium tuberculosis
Mycobacterium tuberculosis (Mtb) has co-evolved with humans for over 70,000 years, equipped with unique adaptations that facilitate its persistence and virulence. The Mtb cell wall represents a promising target for tuberculosis (TB) treatment. Mtb Peptidoglycan (PG) requires continuous maintenance even when the de novo biosynthesis pathways are inactive such as during host-related stresses or treatments, which occur frequently throughout the infection cycle. Therefore, under such conditions, Mtb needs to switch on salvage pathways to recycle essential components of PG. It remains unexplored how Mtb recycles the amino sugar component of the PG and utilizes them to persist in the infection and treatment cycles. Using metabolomics and isotope labeling methods, I have identified that Mtb recycles a significant portion of the precursors of PG biosynthesis, revealing a previously unknown aspect of Mtb's metabolic plasticity and survival strategy. Further, metabolic supplementation experiments also suggest that these pathways play a role in drug tolerance. These preliminary results provide a basis for the core hypothesis that “Mtb can recycle different components of PG to sustain antimicrobial treatment”. To test this hypothesis, I propose using isotopically labeled metabolic supplementation to trace and unequivocally determine the full metabolic steps of PG recycling under various antibiotic treatments, and host-relevant stresses. Furthermore, I will use CRISPRi-based gene silencing to validate these findings. The same strains will also be used in adaptive evolutionary experiments involving various antibiotic treatments to elucidate the precise role of PG recycling in the development of antimicrobial resistance.
View Research Summary
Jonah Kreniske, M.D.
HIV and the Post-Tuberculosis Cardiopulmonary Syndrome
One of every 50 people currently alive is a survivor of TB disease. More than half of TB survivors experience post-treatment chronic lung disease, and TB survivors carry a six-fold increased risk of pulmonary hypertension. The presentation of acute TB disease is dramatically modulated by HIV status, and HIV itself is a cause of pulmonary hypertension and accelerated chronic lung disease. These colliding epidemics are felt most heavily in sub-Saharan Africa, where 29% of TB patients are people with HIV. However, the cardiopulmonary outcomes of pulmonary TB among PWH have been primarily assessed at treatment completion, and the long term impact on chronic lung disease and persistent pulmonary hypertension are unknown. At present, there are no biomarkers or disease-specific therapies for post-TB chronic lung disease. Our study will include 90 pulmonary TB survivors (75 people with HIV and 15 HIV-uninfected adults) and 180 controls matched for HIV status, age and sex in Mwanza, Tanzania. We hypothesize that a history of pulmonary TB in Tanzania will be a driver of underdiagnosed (Aim 1) chronic lung disease as evidenced by impaired spirometry, and (Aim 2) pulmonary hypertension with right heart dysfunction as evidenced by echocardiography. We further hypothesize that chronic lung disease after pulmonary TB will be associated with persistently elevated plasma levels of Matrix Metalloproteinase-9, as demonstrated in HIV-associated lung disease and highly active COPD, thus opening the door for the use of Matrix Metalloproteinase inhibitors such as doxycycline to mitigate chronic lung disease after pulmonary TB.
2023 Recipients
View Research Summary
Amrita Singh, Ph.D.
Delineating conformational flexibility in Mtb Phosphopantetheinyl transferase
Tuberculosis (TB) remains a major global health problem. Mtb Phosphopantetheinyl Transferase (PptT), is an essential enzyme for Mtb growth and survival, making it an attractive candidate for inhibitor design. However, inhibitor design is complicated by our lack of understanding of PptT’s structural biology. PptT exhibits significant structural heterogeneity. While crystal structures of PptT with Coenzyme A (CoA) bound exist, researchers have been unable to crystallize the protein in its apo state, potentially suggesting a different conformational ensemble. Moreover, our preliminary molecular dynamics (MD) simulations have shown considerable domain motions. These findings suggest the existence of multiple conformational states that could influence inhibitor binding, either by acting as new targets for rational inhibitor design or by affecting the entropic contribution to the free energy of inhibitor binding.
We will perform a detailed investigation into the conformational heterogeneity of PptT. Using MD simulations, we can observe possible conformations and predict their relative free energies. First, we will study domain motion in the N-terminal domain of PptT, with a specific focus on the phosphoadenosine phosphate (PAP) binding loop. Second, we will study conformational change in the phosphopantetheine (ppt) binding tunnel as well as the energetics of ppt binding and unbinding. To quantitatively characterize the free energies associated with the PptT’s conformational changes, we will employ an enhanced sampling algorithm known as Replica Exchange Umbrella Sampling (REUS). We will then confirm predictions from simulation using in vitro studies of rPptT, as well as experiments comparing wild-type and mutant Mtb strains. Together, this research will deepen our understanding of the chemical biology of PptT and provide a foundation for future drug discovery efforts targeting Mtb.
View Research Summary
Erik Thiede, Ph.D.
Delineating conformational flexibility in Mtb Phosphopantetheinyl transferase
Tuberculosis (TB) remains a major global health problem. Mtb Phosphopantetheinyl Transferase (PptT), is an essential enzyme for Mtb growth and survival, making it an attractive candidate for inhibitor design. However, inhibitor design is complicated by our lack of understanding of PptT’s structural biology. PptT exhibits significant structural heterogeneity. While crystal structures of PptT with Coenzyme A (CoA) bound exist, researchers have been unable to crystallize the protein in its apo state, potentially suggesting a different conformational ensemble. Moreover, our preliminary molecular dynamics (MD) simulations have shown considerable domain motions. These findings suggest the existence of multiple conformational states that could influence inhibitor binding, either by acting as new targets for rational inhibitor design or by affecting the entropic contribution to the free energy of inhibitor binding.
We will perform a detailed investigation into the conformational heterogeneity of PptT. Using MD simulations, we can observe possible conformations and predict their relative free energies. First, we will study domain motion in the N-terminal domain of PptT, with a specific focus on the phosphoadenosine phosphate (PAP) binding loop. Second, we will study conformational change in the phosphopantetheine (ppt) binding tunnel as well as the energetics of ppt binding and unbinding. To quantitatively characterize the free energies associated with the PptT’s conformational changes, we will employ an enhanced sampling algorithm known as Replica Exchange Umbrella Sampling (REUS). We will then confirm predictions from simulation using in vitro studies of rPptT, as well as experiments comparing wild-type and mutant Mtb strains. Together, this research will deepen our understanding of the chemical biology of PptT and provide a foundation for future drug discovery efforts targeting Mtb.
View Research Summary
Saurabh Mishra, Ph.D.
Deciphering host macrophage response against phenotypically drug-tolerant cryptic Mycobacterium tuberculosis (Mtb) sub-populations
My proposal aims to study the response of human macrophages induced by Mycobacterium tuberculosis (Mtb) sub-populations which are physiologically relevant, as demonstrated by their presence in TB patients and in-vitro models that mimic stages of transmission both inside and outside the host. These two different kinds of persister populations are: (1) Differentially detectable (DD) Mtb and (2) Mtb present inside cavities:
1. Differentially detectable (DD) Mtb: In my recent work, we have studied the presence of a Mtb subpopulation that remains unculturable under standard growth media as colony forming units (CFU). However, under limiting dilution in liquid nutrient-rich media, this population becomes detectable, thus named "differentially detectable" (DD) Mtb.
2. Mtb present in caseum/cavities: We have recently developed a model to simulate conditions that Mtb experiences while residing in cavities in hypoxic, necrotic lesions. We incubate Mtb in a medium that mimics the composition of the caseum present inside the cavities as it might be fluidized by admixture with bronchial secretions and supports the generation of aerosols. Therefore, this fluid is named "Model Aerosol Fluid" (MAF).
Heterogeneity in mycobacterial culture has been shown to induce a distinct host immune response towards non-replicating state bacteria as compared to replicating ones. However, our recent studies report that non-replicating Mtb itself is quite heterogeneous by exhibiting a subpopulation that remains undetectable as CFU. Therefore, the strength of my proposed work is that it will delineate the host response to this subpopulation which may be responsible for TB reactivation or relapse. Further, my proposal also explores how Mtb present in cavities, which are sites of TB reactivation, can disseminate to other sites, and start new niduses of infection. To address this question, we will investigate how the macrophage response to these sub-populations differs from that of both replicating and other previously reported non-replicating Mtb.
View Research Summary
Shuibing Chen, Ph.D.
Isogenic Human Pluripotent Stem Cell-derived Macrophages to Study Genetic Variants Associated with Tuberculosis
Tuberculosis (TB) remains a major global health concern, with alveolar macrophages (AMs) playing a critical role as the first line of defense against Mycobacterium tuberculosis (Mtb). Early Mtb clearance is strongly linked to robust innate immune responses in AMs, underscoring the need for a reliable human AM platform for TB research. Interestingly, only 5% of Mtb-infected individuals develop active TB, suggesting a significant influence of host genetics. Recent studies identified TYK2 P1104A homozygosity as a key genetic variant associated with TB susceptibility. While previous work links this variant to impaired IL-23-dependent IFN-γ immunity in CD4+ T cells, its effects on AMs remain largely unexplored.
Leveraging human pluripotent stem cell (hPSC)-derived AMs and organoid models, this study will investigate the impact of TYK2 P1104A homozygosity in Mtb infection. Aim 1 focuses on assessing the infection responses of isogenic TYK2 P1104A and TYK2 P1104P hPSC-derived AMs, using CRISPR-edited lines. Aim 2 will explore molecular mechanisms via RNA sequencing of WT, TYK2-/-, and variant AMs under Mtb infection.
This research aims to create a robust hPSC-derived AM platform for studying genetic impacts on TB, offering a valuable tool for the TB research community and advancing understanding of host-pathogen interactions in Mtb infection.
View Research Summary
Nate Cira, Ph.D.
Fluidic tools for high-throughput Mycobacterium tuberculosis (Mtb) experiments
The Cira Lab has developed a fluidic platform, termed “Surface Patterned Omniphobic Tiles” (SPOTs), with excellent liquid handling capabilities. SPOTs allow researchers to quickly, cheaply, and precisely meter and combine liquids in thousands of independent sub-microliter volumes. These attributes make the SPOTs platform a powerful tool for the large set of microbiology experiments that require parallel liquid manipulations (e.g. obtaining dose-response curves for a treatment or assessing the impact of different medias on phenotype and genotype). Initial work suggests that these capabilities should be enabling for TB research, but also that additional work is required to get the platform ready for experiments involving Mycobacterium tuberculosis (Mtb), including optimizing robustness for BSL-3 requirements and validating the platform for common assays with Mtb. In this work we propose to carry out several refinements of the platform targeted toward BSL-3 use and conduct two experiments – involving viability assays and molecular profiling – that demonstrate the platform’s versatility for TB research. After refining the platform, viability assays and molecular profiling validation experiments will be used to generate insights into organism biology in droplets and screen for new antibiotics. This work will establish the SPOTs platform as a useful tool for TB research, and the resulting preliminary data will contribute new fundamental knowledge on transmission and treatment.