A Model for Studying Mycobacterial Nutrient Uptake

Overview

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the deadliest infectious disease, responsible for approximately 1.25 million deaths and 10 million new cases in 2023. A major factor contributing to mycobacterial resilience is its highly impermeable outer membrane, which serves as a protective barrier against hostile environments, including antibiotics and immune responses. However, despite this protective "armor," mycobacteria must still acquire essential nutrients for survival. They achieve this through two distinct pathways: (I) the ESX-5 type VII secretion system (T7SS), used by pathogenic species, or (II) MspA porins, found in non-pathogenic species that allow general diffusion. Due to the difficulty of working with pathogenic mycobacteria, we aimed to establish a non-pathogenic model to study ESX-5’s role in nutrient uptake. Using a non-pathogenic species engineered to express the ESX-5 system, we analyzed its membrane permeability and antibiotic resistance compared to control strains. Interestingly, we found that ESX-5 expression reduced membrane permeability, suggesting that this system contributes to a more restrictive cell envelope. To determine whether ESX-5 enables nutrient uptake in this new host, we attempted to delete the native nutrient uptake systems, the MspA porins, using CRISPR-Cas. This proved unsuccessful, likely due to the absence of ESX-5 substrates fulfilling a nutrient transport role. A genetic and structural analysis of known ESX-5 substrates identified potential substrate candidates based on published work. Future research will focus on introducing these substrates alongside ESX-5 to assess their impact on membrane permeability and nutrient uptake, providing deeper insight into the role of ESX in pathogenic mycobacteria.