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 In our recently developed in vitro model, which mimics the sequential stress conditions of transmission (unpublished), we introduced Model Aerosol Fluid (MAF) and Model Alveolar Lining Fluid (MALF).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.