Elucidation of the structure and function of the Mycosins, a family of essential subtilisin-like serine proteases of Mycobacterium tuberculosis

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis is an ancient pathogen, which has been infecting humans for millennia. It remains globally spread, infecting one third of the world's population and causing around two million fatal cases per year. The treatment of this infectious disease remains complex, time consuming, resource demanding and costly. In the absence of adherence, good quality drugs and appropriate treatment regimens, the pathogen is highly likely to develop resistance to the antibiotics used during treatment. Searching for new drug targets and developing new drugs are constantly in progress to address these issues.Type VII secretion system (T7SS) is a signature protein secretion system in mycobacteria, associated with virulence and nutrient acquisition. There are five copies of the T7SS in M. tuberculosis, namely ESX-1 to -5, with ESX-3 being essential for the bacterial growth in vitro. The importance of ESX-3 is supported by the fact that it influences two major iron uptake pathways in mycobacteria, namely mycobactin-mediated iron acquisition and heme acquisition. Mycosin-3 is the only membrane core component of ESX-3 that has a subtilisin-like serine protease signature and plays an essential role in ESX-3 secretion. Mycosin-3 has a unique catalytic specificity and its substrates are unknown. Elucidating the protein structure and determining the function of mycosin-3 will help the design of effective inhibitors to abolish the protease function providing for a potential therapeutic option.In this study, the mycosin-3 gene from the M. tuberculosis genome was cloned and expressed in Escherichia coli and the protein was purified in vitro, with the aim of conducting structural studies. However, the amount of soluble and stable mycosin-3 was insufficient to progress to X-ray crystallography for protein structure determination. According to the literature, this technical difficulty is not uncommon for M. tuberculosis genes because the gene transcription and protein production machineries in mycobacteria are distinct from the conventional protein production host E. coli. In vitro expression analysis suggested that mycosin-3 possibly exerts a toxic effect on the expression hosts: E. coli and Mycobacterium smegmatis. To overcome these complexities, M. smegmatis was used as a model organism for functional studies; the mycP3 gene was deleted from the genome, and the proteomes of the wild type and mycosin-3 deletion mutant were compared. No major phenotypic differences were observed between the wild type and mutant possibly because the model organism has an alternative exochelin-mediated iron acquisition pathway. Interestingly, in the absence of mycosin-3, one component of the mycobactin export system, MmpL5, was not detected in the whole cell lysate (containing both cytosolic and membrane fractions) by mass spectrometry although the mmpL5 gene was transcribed, suggesting rapid protein degradation. We hypothesize that mycosin-3 may plays a role in maintaining the integrity of the membrane protein MmpL5 prior to its secretion and in facilitating its localization on the membrane. The direct involvement of mycosin-3 in the posttranslational modification of MmpL5 is currently under investigation. This study provides evidence that mycosin-3 may be an attractive drug target - abolishing mycosin-3 could disable mycobactin export, with ensuring toxicity from intracellular mycobactin accumulation thereby eliminating M. tuberculosis.

[发布机构] Stellenbosch University