Ok, so I had a patient. The actual history isn't terribly important because this sort of thing happens relatively frequently, but to give you a quick one-liner: he was an older male with rheumatoid arthritis admitted for Staph bacteremia. In cases of blood infections, we order tests called "clearance cultures" to track and confirm that the organism we're fighting disappears with treatment. In this case, 1 out of 4 of these samples tested positive for a potential Bacillus species—the genus to which anthrax belongs. That being said, completely inert species of Bacillus are common contaminants in this setting, and the fact that only 1 out of 4 samples tested positive definitely makes you think this is such a case of contamination.
However, we treat it as if it were anthrax until we're completely certain it isn't. It's Schrödinger's anthrax! After all, you don’t want to be the lab that missed anthrax.
Bacillus anthracis Identification
Colonies of B. anthracis appear non-hemolytic, consist of gram-variable rods with spore forms, and are non-motile. In other words, when grown on sheep's blood agar, they do not break down hemoglobin (a feature many microorganisms possess), appear elongated and purple or pink under a microscope after staining (gram-variable), produce spores (a survival mechanism), and lack motility (i.e., they don’t move via structures like flagella). We use these properties to rule out B. anthracis. While mass spectrometry is the gold standard for organism identification in modern microbiology, when it comes to potential anthrax, we revert to basic microbiological methods for safety reasons (which we can discuss more in the comments if you're interested).
Bacillus anthracis: What Sets It Apart?
Bacillus anthracis, the causative agent of anthrax, is a zoonotic disease, meaning it can be transmitted to humans through the handling or consumption of contaminated animal products. Due to its potential use as a bioweapon, B. anthracis is classified as a Tier I Category A agent by the CDC. Even though infection is rare in the United States, the micro lab remains vigilant in identifying this organism due to its serious implications.
Plasmids and Virulence Factors
What makes B. anthracis particularly dangerous are its virulence plasmids, pXO1 and pXO2, which carry the genes responsible for toxin production and capsule formation, respectively. These plasmids play a crucial role in the organism’s ability to cause disease, enabling it to evade the immune system and produce lethal toxins.
But what exactly is a plasmid?
What is a Plasmid?
A plasmid is a small, circular piece of DNA that exists independently of the bacterial chromosome. Unlike the bacterial genome, which contains essential genes for the organism’s survival, plasmids often carry genes that provide advantages under certain conditions—such as antibiotic resistance or, in the case of B. anthracis, virulence factors.
Plasmids are particularly interesting biologically and evolutionarily because they can be transferred between bacteria via a process called horizontal gene transfer. This means bacteria can acquire new traits, such as antibiotic resistance or enhanced pathogenicity, from other bacteria without evolving them slowly over generations. In essence, plasmids allow bacteria to adapt quickly to new challenges, making them highly versatile and resilient organisms. From an evolutionary standpoint, plasmids accelerate genetic diversity and adaptability, giving certain bacteria a survival edge in hostile environments.
Think of it this way: plasmids let bacteria "plug and play" abilities. Imagine if I could transfer my height, immune system, or ability to play the ocarina just by touching you... now you're getting it. Because of these abilities plasmids are, in many ways, the cornerstone of modern biomedical tech. We will definitely be talking about them again.
What is Bacillus cereus biovar anthracis and why use it to intro plasmids?
Now, why bring up plasmids in this way? Because I can. Stories are nice. Anyway, plasmids are key to understanding another entity: Bacillus cereus biovar anthracis. This variant of B. cereus (the contaminant in our story) has acquired plasmids nearly identical to those found in B. anthracis, meaning it can cause anthrax-like diseases, particularly in animals. While B. cereus is more commonly known for causing food poisoning or being a random contaminant, its biovar anthracis variant is a real concern due to its ability to acquire these plasmids, making it capable of causing serious infections similar to anthrax. Mother nature is getting scarier!
In 2016, this variant was added to the CDC’s select agent list, emphasizing the significance of monitoring its presence, especially in cases involving animals. Though not as common in humans, its existence underscores the evolutionary importance of plasmids in spreading virulence factors across bacterial species.
Conclusion
To wrap it up: Plasmids are fascinating, highly relevant to the changing landscape of infectious diseases, and, as will be discussed later, they might even change what it means to be human.
My suspicion is that a big part of this discussion revolves around integrating vs. non-integrating gene therapies, so let’s start there.
At a high level, viral gene therapies use a viral vector (the capsid or container) to deliver a genetic payload into a cell. That payload can then integrate into the host’s genome if the lysogenic machinery is part of the payload—but that doesn’t necessarily have to be the case.
Plasmid therapies, on the other hand, involve non-chromosomal DNA that stays outside the host’s genome but can still express the proteins it encodes independently. In most cases, plasmids don’t come with the machinery that promotes integration with the host genome, but it’s not an absolute safeguard against integration either. Additionally, a plasmid cargo still needs a vector to gain access into the cell.
In the follistatin gene therapy paper, the authors use a plasmid (the genetic cargo) to encode instructions for follistatin. They deliver this via polyethyleneimine (PEI), a cationic polymer that helps get the plasmid into cells—so PEI acts as the vector here, instead of a virus.
Now, the superiority of either approach really depends on the use case:
Integrating Gene Therapies (more commonly viral-based, since many naturally have integrating machinery that can be included as part of the cargo) are ideal when you want a one-time, permanent fix—for example, in conditions like sickle cell anemia, where a single gene mutation needs to be corrected. In this case, you’d want the therapeutic gene to integrate into the genome for long-term expression and potentially a cure with just one treatment.
Non-integrating Therapies (more commonly plasmid + non-viral vector based) are ‘better’ when you want temporary gene expression. For example, if you're priming the body to fight a new pathogen or delivering a protein with a temporary therapeutic effect, plasmid-based therapies are argued to be more practical. These are also great for delivering proteins that need short-term action but shouldn’t stick around indefinitely, especially if there’s a risk of side effects from prolonged exposure.
That said, I don’t see why viral/non-plasmid strategies couldn’t do these things as well. In fact, many such strategies are in development.
Other Considerations for Viral vs. Plasmid-Based Therapies: Viral Vectors: These also come with higher risks like immune responses, insertional mutagenesis (which can potentially lead to cancers), and limited payload sizes. There are some neat solutions to these in the research sector that we should chat about in the future.
Plasmid Vectors: Generally less immunogenic, but they offer shorter-lived expression, meaning you might need repeated doses to maintain effects. The big benefit in my opinion is they deliver a much larger payload when compared to viruses. Not relevant if you are aiming for a single gene therapeutic but I feel it's the big draw.
Now, About the Follistatin Paper... I’ll hold back some of my critiques of the paper that are beyond the scope of your question, but let me address the safety aspects they mention:
Inherently Transient Expression: This is generally true for plasmids since they don’t integrate into the genome. However, I’m cautious about saying this is 100% guaranteed. There’s always a small risk of integration, even with non-integrating strategies, although the probability is low.
Drug-Inducible Reversibility: The paper mentions this, but it’s not clear how exactly they plan to achieve it. They didn’t include details about the plasmid construct or any antibiotic kill switch, which would be crucial to back up their claim. If such a switch were tied to any potential integrations, in theory, it could allow them to kill off any cells where integration occurred—but more details are needed here. This strategy also isn’t 100% effective, by the way.
Excision of Transfected Tissue: This one made me laugh a bit—“Oops, we made a tumor—CUT IT OUT!” Brilliant and novel, guys. Thanks for mentioning it. While theoretically possible, it doesn’t seem like a reasonable safety net for a clinical approach. Given that cancer development is one of the big concerns with these therapies, and cancer is notoriously slippery, this doesn’t offer much reassurance.
In my opinion, the advantages of plasmids mentioned in the paper could also apply to viral vectors.
So, Where Do I Stand? Both viral and plasmid approaches have their place, and the choice really depends on the situation and how the technology evolves. I suspect that in the long term, viral vectors will be the better choice, despite their risks. There’s a lot of work going into custom capsid design, which will allow for specific targeting and immune evasion. I think the idea that plasmid-based therapies are "safer" may be leading to a false sense of security.
That said, I’m definitely flirting with both. Can you ask me again in 5 years? Maybe 10?
What are your thoughts?