This story is brought to you by Azitra, a preclinical stage biotechnology company combining the power of the microbiome with cutting-edge genetic engineering to treat skin disease.With all of the talk about CRISPR and genome editing these days, it may seem as though scientists have genome editing in the bag. Surely if we can modify complex human cells, much simpler microorganisms like bacteria must be a breeze. Indeed, E. coli has been used as a model organism for decades, undergoing transformations that allow it to produce foreign proteins from a wide variety of organisms, from jellyfish (green fluorescent protein) to human (insulin).But as an increasing number of research groups seek to improve human health by genetically modifying an organism that makes the human body its home, new requirements surface. Now, the organisms we modify are returned to the human body in their modified state, where they produce just the right amount of protein, and produce it until no longer necessary (hopefully). Genetic modification of bacteria is moving beyond the realm of mass protein production in the laboratory into fine-tuned protein production in living beings. This requires the introduction of more complex DNA combinations than have been used traditionally.And the process isn’t so simple.The model organism, E. coli, is an intestinal microbe usually found at very low percentages in the gut, and not found in other areas of the human body, such as the mouth, vagina, or skin. Other organisms must be used if we want to target these areas of the human body. And many of these organisms prove to be much more resistant to genetic modification than the E. coli that scientists have cut their teeth on.

Plasmids: the lynchpin of bacterial transformation

Plasmids -- small, circular, extrachromosomal DNA molecules -- are ubiquitous in nature, and bacteria can pick them up from the environment or from other bacterial cells. Usually they benefit the bacterium in some way -- making it resistant to an antibiotic, for example, or enabling it to digest toxic substances. This is a key characteristic of plasmids: replicating them uses up energy, so to avoid being “kicked out” of the bacterial cell, plasmids ensure their survival by providing advantages to their bacterial hosts.Plasmids also have their own “origin of replication,” which means that they don’t have to wait for the cell to copy the chromosomal DNA right before the cell divides in two -- they can copy their DNA whenever they want. Because of this, a bacterial cell can contain many, many copies of the same plasmid.Leveraging these characteristics, scientists have long been building their own plasmids in the laboratory. Starting with a parent plasmid, they cut open the plasmid using restriction enzymes, and insert the DNA sequence of their gene of interest, “taping” the DNA back together at the broken spots. The plasmid is then introduced into bacterial cells, which copy the plasmid and produce the protein encoded by the gene of interest, which can then be purified. Several important molecules have been produced at large-scale in this way, including insulin for diabetics.

Not all bacteria are created equal

Because E. coli grows so quickly (doubling time of 20 minutes) and is so well characterized genetically, bacterial transformation has been optimized for this organism. Using other species isn’t always straightforward -- some have undiscovered characteristics that prevent successful transformation.Staphyloccous epidermidis, a skin commensal, and its close relative Staph aureus (widely known as the cause of MRSA), are a case in point.For years, scientists struggled to manipulate these organisms genetically, severely impeding their ability to perform functional genomic analyses of these species. Only a few strains would successfully uptake foreign DNA. In 2012, we finally figured out why.In S. aureus and S.epidermidis, two restriction endonucleases (bacterial enzymes that cleave DNA molecules at specific sequence motifs) prevent the bacterial cells from taking up foreign DNA, with the major barrier to transformation presented by the type IV restriction system, which recognizes cytosine-methylated DNA. In 2012, Timothy Foster and colleagues from Trinity College Dublin and biotech corporation Genentech reported the successful transformation of previously impervious S. aureus and S. epidermidis strains.Critical to the success of their system were an improved electroporation protocol and an optimized host E. coli strain from which the plasmid was derived. Bypassing the problematic type IV restriction system, the group was able to induce transformation-resistant S. aureus and S. epidermidis strains to take up a foreign plasmid for the first time.

Modifying the unmodifiable to improve skin health

The potential benefits are clear: an improved understanding of S. aureus genetics, permissible now by successful plasmid-based genetic manipulation, could help us better fight, or even prevent, severe skin infections caused by this opportunistic pathogen. But others are focused on leveraging and building upon the beneficial characteristic of S. epidermidis.S. epidermidis is a commensal skin bacterial species that is one of the first colonizers of human skin. It plays an important role in cutaneous immunity and maintaining a healthy skin microbiome. Several research groups have attempted to leverage S. epidermidis’ inherent beneficial properties to improve skin health. In Japan, topical application of S. epidermidis increased lipid content of the skin, suppressed water evaporation, and improved skin moisture retention. Others have shown that S. epidermidis produces antimicrobial peptides (AMPs) that selectively target S. aureus, which is increased on the skin of those with eczema. Adding S. epidermidis and a related species, S. hominis, to the skin of people with atopic dermatitis caused levels of S. aureus on the skin to decrease significantly.But what if you could genetically manipulate the organism to optimize its benefits to human skin? That is exactly the question that biotech company Azitra is trying to answer.Azitra is focused on using bioengineered S. epidermidis to not only target skin conditions such as eczema but also to strengthen and maintain healthy skin. They are using a sophisticated plasmid, introduced into S. epidermidis, to deliver the human protein filaggrin to the skin. Filaggrin maintains a strong skin barrier, and is often damaged in people suffering from eczema. Essentially, the Azitra team have taken an already beneficial microbe and upped the ante on its protective effects by giving it the means to produce a human protein essential for healthy skin.

Azitra’s modular plasmid design. Image credit Azitra Inc.The plasmid that Azitra has designed contains several important components as well. Because they want to use their plasmid system to target several skin conditions, they’ve facilitated easy switch in and out of different components by making the system modular. Different target genes and specific promoters can be easily added to the plasmid scaffold. The Azitra team is utilizing biosensing promoters that permit the bioengineered S. epidermidis to recognize S. aureus, essentially creating a fine-tuned system that produces needed proteins when damage-inducing S. aureus is present.The plasmid also includes an export signal, permitting protein to be extruded from the bacterial cell and into the environment where it is needed (i.e., the skin). And, important for safety, the Azitra plasmid system could include a kill switch, permitting fine-tuned control of when the desired protein is (and isn’t) being produced.E. coli has been the historic light-bearer for bacterial genetic manipulation. But as advances in synthetic biology are allowing an ever-growing repertoire of bacteria (and other microorganisms) to be genetically modified to benefit humankind, it won’t be long until E. coli is simply a single tool among many. S. epidermidis is a prime example. Building upon the organism’s inherent beneficial properties, the Azitra team is enhancing the organism’s ability to promote skin health, opening the door to a variety of novel therapeutics -- and truly harnessing the power of the skin microbiome.