Like many others, one of the main reasons I wanted to become a scientist was to help solve some of life’s unanswered questions. Remarkably, despite being the leading cause of death worldwide for an infectious agent, there is much about the cell biology of M. tuberculosis that we do not understand. In this, my first primary author paper, recently published in Nature Communications, we identified the first glycoside hydrolase family 76 enzyme from a human pathogen and decipher its role in the mycobacterial cell envelope. This was the main work from my PhD and is a story I am continuing to explore in my current role as a post-doctoral research fellow.
Paper Summary
Capsular arabinomannan is an important structure used by mycobacteria to interact with the host immune system. It has long been believed to be a derivative of the periplasmic lipoarabinomannan, but no mechanism for its release and transport had ever been described. We hypothesised that the carbohydrate domain of LAM was being released from the lipid anchor by an unknown enzyme and then trafficked to the capsule. In this study, we identified the mystery enzyme as LamH. Using bioinformatic tools we scoured the M. tuberculosis genome for enzymes that may have this activity. This led to the discovery of LamH/Rv0365c and in subsequent biochemical assays, we were able to demonstrate that it is highly specific for LAM. Through a range of techniques, we showed that loss of LamH activity in the model organism M. bovis BCG results in an accumulation of LAM and its precursor LM and that no capsular AM can be detected. We were also able to show, with the help of Dr Nicholas Scott, that loss of LamH activity and subsequent increase in LAM causes the cell to actively downregulate production of the enzymes that synthesise LAM. We were really excited to discover this, as it suggested that the cells were actively responding to changes in LAM levels to maintain a glycolipid homeostasis. We were also surprised to discover that mycobacteria respond to AM levels, where this carbohydrate triggers exit from lag-phase growth.
Challenges of the Project
One of the biggest struggles of this project was starting my PhD during the COVID-19 pandemic. Restricted lab working hours, socially distanced training, and learning how to purify proteins over Zoom was definitely challenging! Additionally, to make things worse, LamH was a particularly unhappy protein to work with. My preps would consistently result in poor yields, or protein unexpectedly crashing out on me. Fortunately, I think this experience taught me how important persistence in science is, and so after 12 months of optimisation, I was finally able to purify active protein confidently.
However, the most frustrating aspect of this project was working with a mutant with such a strong growth defect. It would routinely take over two weeks before I could detect any growth in our LamH mutant, and nearly a month before the culture would reach mid-log phase. M. bovis BCG is already a ‘slow growing’ species, but this extended lag phase phenotype was extremely annoying to work with when I was desperate to collect new data. However, it turned out that this growth defect was actually a really important part of LamH’s biology. As we investigated this phenotype further, we found that the capsular AM was an important molecule for growth of mycobacteria. This was a really cool finding and is something I am currently exploring further!
One of the most rewarding aspects of this paper was the collaborative efforts of everyone involved. After learning that another group was also working on this enzyme, my supervisor, Dr Patrick Moynihan, chose collaboration over competition. This decision to work with Marcelo and Rafa allowed us to learn from their efforts to work with this protein and include macrophage infection studies in our paper, significantly broadening the scope of our study.
Significance of the Paper
Despite answering some questions, this paper raises many more about the role of lipoarabinomannan in M. tuberculosis. For a long time, LAM was primarily thought to play a role in pathogenesis. However, this paper and recent work from the Morita group [1] suggest that LAM may have many more roles in the cell beyond virulence. Many mycobacteria are not considered pathogenic, yet they all produce LAM. This suggests that LAM is a considerably more important molecule than we may realise for mycobacteria, and much of its role in the cell envelope remains unknown.
Over the next few years, I am planning to further study some of the results discussed in this paper and I hope to provide many more answers regarding the role LAM, AM, and alpha-mannan degrading enzymes play in M. tuberculosis.
References
[1] Sparks, I. L. et al. Lipoarabinomannan mediates localized cell wall integrity during division in mycobacteria. Nat. Commun. 15, 2191 (2024).
The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth
