Meet Smegmatis, TB's Least Threatening Sibling

Creating liquid overnight cultures of mc²155 and Jucho cells for later use.

Cells are kind of like humans. If they recognize each other as family, they’re not going to exchange DNA. If they aren’t family, they will exchange DNA. That’s probably not something you want to think about, but it’s exactly how Mycobacteria, on a baseline level, choose to interact with one another.

Let me back up. If you’ve been reading my blog posts, you know that I worked for six weeks in the Gray Lab this summer, which is a part of the Department of Genetics which is a part of the Wadsworth Center which is a part of the New York Department of Health. I’ve been working closely with my mentor, but probably closer with someone else: M. smegmatis. If that name sounds like a good target for bullying, you’re not wrong. Thankfully, the organism named that lacks sentience, and operates solely on basic biological processes. Hopefully you’ve guessed by now: M. smegmatis is a type of mycobacteria, and actually one of the heroes of the Gray Lab. Unfortunately, the bacterium posing a large global health risk that the DOH cares about (Mycobacterium tuberculosis) is a nightmare to work with directly. TB has a thick, waxy cell envelope that makes it stubborn against antibiotics, it doubles maybe once a day (compare that to E. coli, which can double in twenty minutes), and it requires a BSL-3 lab, which is a level of biosafety clearance that any juvenile researcher won’t have. Thankfully, M. smegmatis has 70% genetic overlap with M. tuberculosis. It also grows six times faster, is completely non-pathogenic, and only needs a BSL-2 lab. Therefore, although the work my lab does is a step removed from working directly with contagious pathogens, the high genetic similarity means that if we understand the role of a gene in smegmatis, there's a very good chance the same machinery is doing something analogous in TB.

Both M. tuberculosis and M. smegmatis have ESX-1 secretion systems; this system plays a different role in the respective bacteriums. In TB, this system is responsible for exporting virulence proteins that help TB break the compartment our immune cells use to trap diseases and continue spreading. In M. smegmatis, this system is crucial for a newly discovered method of DNA transfer: Distributive Conjugal Transfer, or DCT in researcher slang. 

DCT is a form of horizontal gene transfer, meaning bacteria swapping DNA with each other outside of normal reproduction. In classic conjugation, a donor cell pushes a copy of a plasmid (a circular, non-chromosomal piece of DNA) through a pilus (a small “tube” between the cells) into a recipient cell. DCT breaks basically every rule of that. Instead of being plasmid-driven, the DNA exchanged is entirely chromosomal. Both the donor and recipient strain are active participants. And the transconjugants that are produced are described as mosaics, with small and large chunks of up to donor DNA from across the donor genome added to recipient DNA. Transconjugants can have up to 25% of the donor genome. My mentor described it to me as closer to eukaryotic sexual reproduction (what humans do) than to asexual reproduction (the main way bacteria replicate). DCT demonstrates bacteria’s ability to make large evolutionary leaps, passing on genes through generations quickly. This risks a faster spread of antibiotic-resistant genes and an outpacing of standard antibiotics. Antimicrobial resistance is already projected to cause an estimated 10 million deaths a year by 2050, so DCT has high public health relevance. 

I’d like to note that not every strain of M. smegmatis can perform DCT with one another. Take mc²155 and Jucho. They happen to share an identical stretch of the ESX-1 genes called the mid-locus, which the system uses to tell kin from non-kin. Because they recognize each other as family, mc²155 can't donate to Jucho at all. These are the two strains that I worked with during my time at Wadsworth. Way before I even got there, my lab confirmed which strains could do DCT and which ones couldn’t. After figuring this out, they turned to investigating specific genes and found three main genes in the ESX-1 mid-locus responsible for kin recognition. When one of them (MSMEG_70) is mutated, a variety of genes located beside the ESX-1 genes are upregulated, meaning their expression dramatically increases. My mentor gave me three of these genes to look at and see if they play a role in DCT: MSMEG_2508, MSMEG_61-69, and MSMEG_61-71. 

My job this summer has been trying to knock each of those genes out, one at a time, and seeing what happens to DCT mating when I do. I tried two completely different ways of doing that, and I’ll get into that in my next blog post. You’re probably wondering: okay, Anushka, so you figure out if these genes play a role in DCT. How is that at all related to M. tuberculosis? Well, avid reader, the genes governing cell-to-cell contact in M. smegmatis are the same genes helping TB evade and manipulate our immune system. Therefore, characterizing the roles of the genes may inform future non-antibiotic therapies to target M. tuberculosis, which are needed in a time where bacteria is evolving much faster than the rate at which companies produce new antibiotics.