The Effect of Mutagenesis on the MCE4 Permease, in Cholesterol-Based Mycobacterial Growth

MCE4, a protein complex found in Mycobacterium tuberculosis, is hypothesized to transport cholesterol into the pathogenic bacterium. This project’s goal is to understand how a part of the MCE4 complex, the permease proteins YrbE4A and YrbE4B, binds substrate and transports cholesterol across the cell envelope. The genome of M. tuberculosis encodes 4 distinct MCE transporters dedicated to the uptake of nutrients from outside the M. tuberculosis cell for energy acquisition and cell envelope biogenesis. For this experiment, based on our current model of MCE4, we introduced 11 strategic point mutations into the YrbE4AB permease to assess whether these mutants can rescue the yrbE4AB knockout strain. We then monitored how these mutations affect cholesterol uptake in Mycobacterium smegmatis by monitoring the cell growth over the course of 4-5 days. Relative to the yrbE4AB strain complemented with a plasmid expressing WT YrbE4AB, two point mutants showed a significant decrease in cell growth (I73N and L106N). One mutation (L114N), showed no cell growth, indicating almost no cholesterol uptake. These reductions/inhibitions, suggest that these residues may play a key role in cholesterol binding and/or transport.